-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs -cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature -cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.">
功能成熟和新生儿猪胰岛细胞单层分化不久体外共培养的加速度与微囊化支持细胞</Ť一世Ť升e>
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体积<!-- -->
<!-- -->2010<!-- -->
<span class="sc-dEoRIm gusDuS">|</span>文章的ID<!-- -->
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<span class="sc-dEoRIm gusDuS">|</span>
<!-- -->14<!-- -->
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<span class="sc-dEoRIm gusDuS">|</span>
<a class="sc-htpNat bUhGXt link sc-jtggT iUDBGe" href="https://doi.org/10.4061/2010/587213" aria-label="Doi-link">https://doi.org/10.4061/2010/587213</一个>
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<h1 class="sc-jAaTju cxkaTM sc-bmyXtO lmNMHJ article_title">功能成熟和新生儿猪胰岛细胞单层分化不久体外共培养的加速度与微囊化支持细胞</h1>
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<div class="sc-jeCdPy bpzNtR article_authors">
<hr class="sc-likbZx PHYWq hr__author">
<span class="">弗朗西斯·曼库索<!-- -->,<sup>1</sup></span>
<span class="">马里奥Calvitti<!-- -->,<sup>2</sup></span>
<span class="">乔凡尼卢卡<!-- -->,<sup>1</sup></span>
<span class="">克劳迪奥·Nastruzzi<!-- -->,<sup>3.</sup></span>
<span class="">Tiziano布洛尼<!-- -->,<sup>2</sup></span>
<span class="">普玛兹泰利<!-- -->,<sup>3.</sup></span>
<span class="">埃尼奥Becchetti所在<!-- -->,<sup>2</sup></span>
<span class="">伊娃阿拉托<!-- -->,<sup>1</sup></span>
<span class="">卡罗·博塞利<!-- -->,<sup>4</sup></span>
<span class="">Monique D. Ngo Nselel<!-- -->,<sup>1</sup>
<!-- -->和</span>
<span class=""><b>里卡多。Calafiore</b><a class="sc-htpNat bUhGXt link" href="mailto:" aria-label="Mail to Author Option"><img alt="" class="sc-EHOje cPuqxU sc-jtRlXQ jIaDFG" title="" role="presentation" src="data:image/svg+xml;base64,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" height="16"></a><sup>1</sup></span>
<div class="sc-ePZHVD eecoTM isHide">
<p><sup>1</sup><span>内科和内分泌代谢学，内科，佩鲁贾大学，06126佩鲁贾，意大利的科</span></p>
<p><sup>2</sup><span>佩鲁贾大学实验医学与生化科学系，06126佩鲁贾，意大利</span></p>
<p><sup>3.</sup><span>佩鲁贾大学药学院药物化学与技术系，佩鲁贾06126，意大利</span></p>
<p><sup>4</sup><span>外科，佩鲁贾大学，06126佩鲁贾，意大利</span></p>
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<span class="articleContent__PublicationField-sc-2yl9jy-6 cIribm">修改后的</span>
<span class="articleContent__PublicationValue-sc-2yl9jy-7 khHUcu">2009年7月20日</span>
</div>
<div class="articleContent__PublcationCol-sc-2yl9jy-5 hgKieC notPublished">
<span class="articleContent__PublicationField-sc-2yl9jy-6 cIribm">接受</span>
<span class="articleContent__PublicationValue-sc-2yl9jy-7 khHUcu">2009年8月27日</span>
</div>
<div class="articleContent__PublcationCol-sc-2yl9jy-5 hgKieC isPublished">
<span class="articleContent__PublicationField-sc-2yl9jy-6 cIribm">发布时间</span>
<span class="articleContent__PublicationValue-sc-2yl9jy-7 khHUcu">2009年11月19日</span>
</div>
</div>
</div>
<article class="sc-kZmsYB iukjRu article_body ">
<div>
<div class="xml-content">
<h4 class="header" id="abstract">抽象</h4>
<p>尸体供体胰腺的有限可用性和异体胰岛埃德蒙顿协议的不完全成功，促使寻找新的胰岛素来源生产细胞替代细胞治疗胰岛素依赖性糖尿病(T1DM)。从分离的新生儿猪胰岛(NPIs)开始，我们获得细胞单层暴露于微囊化单单层支持细胞(ESCs)不同时间段(7、14、21天)。为了评估共培养细胞单分子层的发展，我们研究了内分泌细胞表型分化标记物或c-kit，一种造血干细胞标记物，最近参与的生长和分化<svg height="13.425" id="M1" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</Ťspan>
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</svg>-细胞亚群在人类和啮齿动物模型。ESC对npi的成熟和分化有促进作用<svg height="13.425" id="M2" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</Ťspan>
</text>
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</svg>-细胞表型或鉴定胰岛细胞亚群标记阳性的c-kit。胰岛素/c-kit阳性细胞可能是一种新的功能不成熟的细胞<svg height="13.425" id="M3" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</svg>-猪胰腺中的细胞样成分。我们的NPI细胞单层成熟和分化的加速可能产生一个潜在的新机会，开发生产胰岛素的细胞，可能为T1DM的细胞治疗试验提供支持。</p>
<span class="end-abs"></span>
<h4 id="introduction">1.简介</h4>
<p>通过外源性胰岛素高血糖的修正可能会延迟或减弱，但从来没有消除，对T1DM [时间过程中发生继发并发症的风险<一个href="#B1">1</一个>]。此外，最近有报道称，长期使用胰岛素类似物治疗的糖尿病患者可能存在恶性肿瘤风险[<一个href="#B2">2</一个>]。移植产生胰岛素的组织的（TX），是否被包括整个胰腺或分离的胰岛细胞，可能会完全恢复正常血糖，因此避免了对每日外源性胰岛素补充的需求。此外，血糖控制改善了稳定性，由于胰岛TX实现，可以抑制发病和疾病相关的慢性并发症的严重程度两者。然而，在完全免疫抑制治疗1型糖尿病人的胰岛移植已被证明是十分有限的，在取得成功结果的条件[<一个href="#B3">3.</一个>]。大多数长期移植的胰岛不能完全发挥功能并维持暂时恢复的血糖[<一个href="#B3">3.</一个>]。此外，人供体的可用性通常胰岛非常有限由于低器官捐赠速率不令寻找像新生儿猪胰岛细胞的替代来源（非营利机构），如人类组织代用品[<一个href="#B4">4</一个>- - - - - -<一个href="#B7">7</一个>]。</p>
<p>事实上，同时它是很难获得和文化维护，因为其内在的脆弱性和短暂的功能寿命的成年猪胰岛，相比之下，非营利机构的采购是很容易的，首先，非营利机构，比成年猪胰岛细胞生长和分化更容易，有很长的功能寿命，和CD8 +攻击的保护[<一个href="#B8">8</一个>]。</p>
<p>目前，将NPI用于异种移植的一个主要障碍是糖尿病患者移植后血糖正常化的延迟。事实上，Korbutt等人报道[<一个href="#B9">9</一个>]和Korbutt等。[<一个href="#B4">4</一个>]，NPI可能需要4-10周或更长时间以达到足够的，分化<svg height="13.425" id="M4" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</svg>-细胞数量，以逆转糖尿病鼠TX后的高血糖。</p>
<p>这种预TX时间滞后造成两个主要问题：（A）收件人需要用外源性胰岛素进行治疗，直到移植物变功能;（b）该植入胰岛暴露于环境，尽可能有害，慢性高血糖症。</p>
<p>为了加快NPI在体内和体外漫长的成熟过程，人们做了很多努力，但不幸的是，这些步骤与相关的细胞质量损失有关。</p>
<p>新分离的NPI细胞群通常由少数的<svg height="13.425" id="M5" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</svg>大部分细胞角蛋白-7+ (Ck7，导管细胞标志物)细胞。其余的细胞共同表达胰岛素或上皮细胞标记物[<一个href="#B5">5</一个>]或胰十二指肠同源盒基因-1（PDX-1），其调节葡萄糖刺激的胰岛素基因表达<一个href="#B9">9</一个>]。在这方面，我们以前所示NPI与塞尔托利细胞（SC），该共同培养诱导的未成熟的NPI的快速和显著成熟和分化成在功能上主管，成熟<svg height="13.425" id="M6" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</text>
</g>
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</svg>-细胞，通过促进分化/发育过程的加速[<一个href="#B5">5</一个>]。</p>
<p>假设SC将提供营养素、免疫调节和营养因子，可能改善NPI的生存和发育以及功能能力[<一个href="#B5">5</一个>,<一个href="#B10">10</一个>]。对NPI改善SC影响，我们已经开发出一种培养过程以获得NPI单层（在体外保持最多90天）具有高电平的葡萄糖和胰高血糖素样肽1处理（GLP-1）[<一个href="#B11">11</一个>]。</p>
<p>为了确定sc驱动加速NPI细胞单层分化的机制，我们用ESC孵育NPI不同时间段(7、14、21天)。在起始时间，细胞单层免疫表型与分离的原代NPI相似。为了监测培养细胞分化的进展，我们检测了内分泌细胞表型分化标记或c-kit，一种造血干细胞标记，最近参与生长和分化<svg height="13.425" id="M7" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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</svg>β细胞亚群在人类和啮齿类动物。事实上，酪氨酸激酶蛋白参与在不同细胞类型的生长和分化。在这样的蛋白质，典型的造血干细胞标记物[<一个href="#B13">12</一个>- - - - - -<一个href="#B15">14</一个>，其配体是干细胞因子(stem cell factor, SCF)，使人们对其在胰岛细胞发育生物学中可能发挥的作用越来越抱有希望[<一个href="#B15">14</一个>]。此外，在a中发现了RNA转录本形式的c-kit<svg height="13.425" id="M8" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
<g transform="translate(72,-61.26)">
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<tspan style="font-size: 12.50px; " x="0" y="0">
</Ťspan>
</text>
</g>
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</svg>-细胞亚群，提示成熟<svg height="13.425" id="M9" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</Ťspan>
</text>
</g>
</g>
</svg>-细胞可能来源于c-kit阳性细胞，存在于细胞再生和新生途径中[<一个href="#B16">15</一个>,<一个href="#B17">16</一个>]。即使的c-kit已被证明在啮齿动物和也在Li等人。[<一个href="#B18">17</一个>，据我们所知，我们的数据是第一个在猪身上观察到的数据，这是一种有潜力的用于T1DM细胞治疗的异种细胞来源，因为尸体供体胰腺的可用性有限。</p>
<p>综上所述，本文描述了SC诱导NPI细胞快速、显著地成熟和分化为功能胜任细胞的可能机制<svg height="13.425" id="M10" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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</svg>肽。</p>
<h4 id="materials-and-methods">2。材料和方法</h4>
<h5 id="sec2.1">2.1。新生儿猪胰岛的分离</h5>
<p>npi是按照先前确定的方法分离出来的[<一个href="#B3">3.</一个>,<一个href="#B4">4</一个>]从新生儿“大白”猪（出生时间范围：1-3天）。B[R一世efly, the piglets were anesthetized with 0.1 mg/Kg azaperon (Stresnil, 40 mg/mL, Janssen, Bruxelles, Belgium) and 15 mg/Kg ketamine (Imalgene, 100 mg/mL, Gellini Farmaceutici, Aprilia, Italy) coadministered intramuscularly. The piglets underwent total laparotomy, by midline incision, in order to carefully remove the pancreas. To prevent bacterial contamination, particular care was taken to avoid bowel nicking. Upon transportation to the laboratory in Eurocollins (SALF, Bergamo, Italy) on ice, the pancreas was cut into small pieces (1–3 <svg height="13.775" id="M11" style="vertical-align:-0.0pt;width:30.5375px;" version="1.1" viewbox="0 0 30.5375 13.775" width="30.5375" xmlns="http://www.w3.org/2000/svg">
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米</Ťspan>
<tspan style="font-size: 12.50px; " x="9.727334" y="0">
米</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-52.5,66.03)">
<tspan style="font-size: 8.75px; " x="0" y="0">
3.</Ťspan>
</text>
</g>
</g>
</svg>)和水洗汉克的平衡盐溶液(HBSS) (Sigma化学公司，圣路易斯，美国密苏里州)，根据之前报道的方法。将组织切碎，然后在胶原酶溶液(胶原酶P，罗氏公司，米兰，意大利)中振荡，随后在HBSS (Sigma Chemical Co)中洗涤两次，HBSS中补充100u /mL青霉素+0.1 mg/mL链霉素(Sigma Chemical Co)。最后，用含0.5%牛血清白蛋白，分数V (BSA) (Sigma Chemical Co)， 50的HAM-F12 (EuroClone, Wetherby, UK)重悬组织<svg height="9.6750002" id="M12" style="vertical-align:-2.29482pt;width:9.6374998px;" version="1.1" viewbox="0 0 9.6374998 9.6750002" width="9.6374998" xmlns="http://www.w3.org/2000/svg">
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</svg>m3 -异丁基-1甲基黄嘌呤(IBMX) (Sigma Chemical Co)， 10mm烟酰胺(Sigma Chemical Co)， 2mm l-谷氨酰胺(Sigma Chemical Co)，青霉素+0.1 mg/mL链霉素(Sigma Chemical Co)，并镀入<svg height="11.2625" id="M13" style="vertical-align:-0.3135pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.2625" width="56.150002" xmlns="http://www.w3.org/2000/svg">
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1</Ťspan>
<tspan style="font-size: 12.50px; " x="38.559254" y="0">
5</Ťspan>
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</g>
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</svg>mm培养皿(Becton Dickinson Labware，林肯公园，纽约，美国)(10000 NPCC/板)。</p>
<h5 id="sec2.2">2.2。新生儿猪胰岛细胞单层培养维护</h5>
<p>在分离的第4天，通过溴化乙腈(EB) (Sigma Chemical Co)和荧光素-二醋酸盐(FDA) (Sigma Chemical Co)对制剂进行染色，评估NPI活性，如前所述[<一个href="#B19">18</一个>]。NPI比山肩点击的介质(西格玛化工有限公司)补充10%胎牛血清(EuroClone, Wetherby,英国),0.5%的牛血清白蛋白,分数V (BSA)(西格玛化工有限公司),10毫米烟酰胺(西格玛化工有限公司),2毫米谷酰胺(西格玛化工有限公司)和青霉素+ 0.1毫克/毫升链霉素(西格玛化工有限公司)使用T25组织粘附细胞增长的烧瓶(他一一Bio-one、Frickenhausen、德国)的浓度30 NPI /瓶。粘在烧瓶上后，NPI失去了正常的三维结构，形成了细胞单层。</p>
<h5 id="sec2.3">2.3。睾丸支持细胞的分离</h5>
<p>旺从新生儿青春期“大白”猪，老化7-10天，根据先前建立的方法，稍微在我们的实验室修改为独立<一个href="#B5">5</一个>,<一个href="#B20">19</一个>]。</p>
<p>短暂地，麻醉后，在进行逐步的酶消化前，睾丸被取出并切碎:第一步用胶原酶P(罗氏诊断公司，S.p.A)。(意大利蒙扎)在HBSS (Sigma化学Co)解离精管;第二步胰蛋白酶+DNAse I在HBSS中的作用。组织被消化，用甘氨酸重悬，以清除残留的间质细胞和管周细胞[<一个href="#B21">20.</一个>，在含0.166 nM维甲酸(Sigma Chemical Co)和5 ml/500 ml胰岛素转化硒(ITS)培养基(Becton Dickinson, NJ, NJ)的HAM F12中培养，95%的空气/ Co<sub>2</sub>在<svg height="11.0375" id="M14" style="vertical-align:-0.17555pt;width:30.625px;" version="1.1" viewbox="0 0 30.625 11.0375" width="30.625" xmlns="http://www.w3.org/2000/svg">
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7</Ťspan>
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</svg>。体外培养维持3天后，按照文献报道，将SCs与10 mM羟甲基氨基甲基盐酸盐缓冲液(tris) (Sigma Chemical Co)孵育，以清除残留的生发细胞[<一个href="#B22">21</一个>]。</p>
<h5 id="sec2.4">2.4。基于海藻酸钠微囊内支持细胞的封装</h5>
<p>SC是按照我们之前公布的方法[封装在基于藻酸盐的微胶囊<一个href="#B23">22</一个>- - - - - -<一个href="#B25">24</一个>]。简而言之，融合SC单层膜(<svg height="14.4625" id="M15" style="vertical-align:-0.3135pt;width:54.424999px;" version="1.1" viewbox="0 0 54.424999 14.4625" width="54.424999" xmlns="http://www.w3.org/2000/svg">
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2</Ťspan>
<tspan style="font-size: 12.50px; " x="6.2515001" y="0">
0</Ťspan>
<tspan style="font-size: 12.50px; " x="15.278666" y="0">
×</Ťspan>
<tspan style="font-size: 12.50px; " x="26.056252" y="0">
1</Ťspan>
<tspan style="font-size: 12.50px; " x="32.307751" y="0">
0</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-33.38,65.96)">
<tspan style="font-size: 8.75px; " x="0" y="0">
6</Ťspan>
</text>
</g>
</g>
</svg>0.05%胰蛋白酶/乙二胺四乙酸(EDTA) (Gibco, Grandisland)孵育2分钟后，刮除培养板上的细胞(cell /T75 flask)，然后洗涤，用血细胞计计数，检测细胞活力。SCs悬浮于1 mL内部高纯度海藻酸钠(Na-AG)的1.8%水溶液中(Stern Italia, Milano, Italy)。用蠕动泵以12-14 mL/min的流速连续吸入AG/SC悬液，在无菌条件下通过单喷气装置(空气流速为5 l/min)挤压。将藻酸盐悬浮液连续搅拌以防止细胞结块，这可能导致微胶囊内SC分布不均匀。将形成的微滴收集在a上<svg height="14.7125" id="M16" style="vertical-align:-3.13504pt;width:38.349998px;" version="1.1" viewbox="0 0 38.349998 14.7125" width="38.349998" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,14.7125)">
<g transform="translate(72,-60.23)">
<text transform="matrix(1,0,0,-1,-71.95,63.41)">
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B</Ťspan>
<tspan style="font-size: 12.50px; " x="8.3395014" y="0">
一个</Ťspan>
<tspan style="font-size: 12.50px; " x="13.890833" y="0">
C</Ťspan>
<tspan style="font-size: 12.50px; " x="22.230333" y="0">
升</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-46.24,60.28)">
<tspan style="font-size: 8.75px; " x="0" y="0">
2</Ťspan>
</text>
</g>
</g>
</svg>bath (1.2%， w/v)立即将其转化为凝胶微球，盐水冲洗两次，体外培养维持24小时。微胶囊化前后，在荧光显微镜下用溴化乙二胺(EB) (Sigma Chemical Co)和荧光素-二醋酸盐(FDA) (Sigma Chemical Co)对制剂进行染色，评估SC存活率，如前所述[<一个href="#B19">18</一个>]。</p>
<h5 id="sec2.5">2.5。NPI单层的分泌胰岛素的模式</h5>
<p>NPI获得细胞单层(来自30 NPI / T25瓶)cocultured 7、14、21天在点击microencapsulated SC的介质(西格玛化工有限公司)补充10%胎牛血清(EuroClone, Wetherby,英国),0.5%的牛血清白蛋白,分数V (BSA)(西格玛化工有限公司),10毫米烟酰胺(西格玛化工有限公司),2毫米谷酰胺(西格玛化工有限公司)和青霉素+ 0.1毫克/毫升链霉素(西格玛化工有限公司)。这项研究重复了三次。从烧瓶中收集培养基，在培养维持期间，以300 g离心5分钟。细胞上清保存于<svg height="11.0375" id="M17" style="vertical-align:-0.17555pt;width:41.337502px;" version="1.1" viewbox="0 0 41.337502 11.0375" width="41.337502" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,11.0375)">
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</g>
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</svg>在接受RIA(米兰，意大利)胰岛素检测之前。葡萄糖刺激胰岛素释放(GSIR)是在组织暴露于50 mg/dL (2.66 mM) - 300 mg/dL (16.7 mM) - 50 mg/dL (2.66 mM) d -葡萄糖中90分钟后进行的<svg height="11.0375" id="M18" style="vertical-align:-0.17555pt;width:30.625px;" version="1.1" viewbox="0 0 30.625 11.0375" width="30.625" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,11.0375)">
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</g>
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</svg>，在95％空气/<svg height="14.6" id="M19" style="vertical-align:-3.13504pt;width:27.799999px;" version="1.1" viewbox="0 0 27.799999 14.6" width="27.799999" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,14.6)">
<g transform="translate(72,-60.32)">
<text transform="matrix(1,0,0,-1,-71.95,63.5)">
<tspan style="font-size: 12.50px; " x="0" y="0">
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<tspan style="font-size: 12.50px; " x="8.2394772" y="0">
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</text>
<text transform="matrix(1,0,0,-1,-54.68,60.37)">
<tspan style="font-size: 8.75px; " x="0" y="0">
2</Ťspan>
</text>
</g>
</g>
</svg>。所获得的总胰岛素然后通过总细胞数和/或胰岛素+细胞数目归一化。总细胞数通过染色细胞核用结晶紫，增溶剂被吸附的染料成的Triton X-100的溶液和评估光学密度用分光光度计测定[<一个href="#B26">25</一个>]。胰岛素+细胞数由免疫细胞化学测定(见章节)<一个href="#sec2.6">2.6</一个>)。此外，胰岛素含量与细胞总蛋白质含量之比(<svg height="9.6750002" id="M20" style="vertical-align:-2.29482pt;width:9.6374998px;" version="1.1" viewbox="0 0 9.6374998 9.6750002" width="9.6374998" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,9.675)">
<g transform="translate(72,-64.26)">
<text transform="matrix(1,0,0,-1,-71.95,66.6)">
<tspan style="font-size: 12.50px; " x="0" y="0">
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</text>
</g>
</g>
</svg>测定了单独培养或ESC存在下的NPI细胞单层蛋白(U/mg蛋白)。简言之，全细胞提取物，准备用于western-blotting分析(见章节)<一个href="#sec2.7">2.7</一个>)， RIA法测定胰岛素，Bradford法测定总蛋白[<一个href="#B27">26</一个>]。数据以平均3个独立实验。</p>
<h5 id="sec2.6">2.6。免疫细胞化学</h5>
<p>对照和ESC共培养的NPI细胞单层沉积在玻璃杯盖上，用PBS洗涤，4%多聚甲醛固定在Dulbecco's磷酸盐缓冲液(PBS)中(EuroClone, Wetherby, UK)。用PBS/1% BSA洗涤细胞三次，然后用10%山羊血清PBS/1% BSA孵育30分钟，以阻止非特异性IgG结合。在稀释PBS / 1% BSA和转移到玻璃盖玻片,具体主要抗体,即1:200只兔子anti-mouse /大鼠胰腺和十二指肠同源框基因1蛋白(PDX-1) (Chemicon国际,泰梅库拉,CA), 1: 150年兔antiglucotransporter-2 (Glut-2)(美国加州Chemicon国际,泰梅库拉),1:200年豚鼠反胰岛素(Linco,圣路易斯,密苏里州,美国),1:50单克隆鼠标反细胞角蛋白7 (Ck7) (Dako Cytomation,斯特鲁普,丹麦),1:50只兔抗人葡萄糖激酶(GK)多克隆抗体(Santa Cruz Biotechnology Inc.， Santa Cruz, Calif, Calif, USA)和小鼠抗猪c-kit (Dominguez博士的礼物，西班牙马德里)在<svg height="11.0375" id="M21" style="vertical-align:-0.17554pt;width:22.8125px;" version="1.1" viewbox="0 0 22.8125 11.0375" width="22.8125" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,11.0375)">
<g transform="translate(72,-63.17)">
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4</Ťspan>
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C</Ťspan>
</text>
</g>
</g>
</svg>在一个潮湿的房间里。用PBS/1%BSA洗涤3次，5分钟后与二抗(AlexaFluor 488山羊抗兔、AlexaFluor 488山羊抗小鼠)孵育<b>,</b>工作溶液1：500）（Molecular Probes公司，卡尔斯巴德，加利福尼亚州，美国），（Cy3标记的驴抗小鼠，Cy3的驴抗兔工作溶液1：500，Amersham公司国际奥特芬根，瑞士），（兔抗豚鼠TRITC工作solution 1 : 320, (Sigma Chemical Co). The coverslips were washed thrice with PBS/1%BSA, then mounted and examined under fluorescence microscopy (Nikon Eclipse TE2000-U) at 492 ± 15 nm for AlexaFluor 488 and 552 ± 15 nm for Cy3. The image processing and percentage of immunopositive cells (%) were determined, by using the software Nikon Eclipse EZ C1, version 2.30, by two independent investigators. For each experimental condition, 20 different images, containing at least 300 cells in total, were counted. In every image, all the cells were counted and subdivided into different groups if labeled for green fluorescence or red fluorescence, or both (yellow fluorescence). The experiment was repeated in triplicate.</p>
<h5 id="sec2.7">2.7。免疫印迹分析</h5>
<p>考虑到ESC对NPI单层细胞存活率和功能能力的重要影响，通过免疫组化方法研究ESC对NPI分化标志物的影响。</p>
<p>检查各种分化标志物（见表<一个href="//www.newsama.com/journals/sci/2010/587213/tab1/" target="_blank">1</一个>)。</p>
<div class="floats-partial-section partial-other-wrapper" data-section="tab1">
<div class="floats-partial-content fig-content-tab1">
<table class="table-group" id="tab1">
<tbody>
<tr>
<td>
<table class="table">
<tbody>
<tr>
<td class="thead-hr" colspan="4">
<hr></td>
</tr>
<tr class="thead">
<td align="left">的名字</Ťd>
<td align="center">缩写</Ťd>
<td align="center">细胞定位</Ťd>
<td align="center">细胞类型标记</Ťd>
</tr>
<tr>
<td class="thead-hr" colspan="4">
<hr></td>
</tr>
<tr>
<td align="left">干细胞因子受体</Ťd>
<td align="center">c - kit / SCFR</Ťd>
<td align="center">跨膜</Ťd>
<td align="center">造血干细胞标志物;生长和分化<米在h id="M22" xmlns="http://www.w3.org/1998/Math/MathML">
<mrow>
<mi>
β</米一世>
</mrow>
</math>细胞亚类标记</Ťd>
</tr>
<tr>
<td align="left">细胞角蛋白7</Ťd>
<td align="center">CK7</Ťd>
<td align="center">cytoplasmatic</Ťd>
<td align="center">导管细胞标记</Ťd>
</tr>
<tr>
<td align="left">胰岛素</Ťd>
<td align="center">插件</Ťd>
<td align="center">cytoplasmatic</Ťd>
<td align="center">
<math id="M23" xmlns="http://www.w3.org/1998/Math/MathML">
<mrow>
<mi>
β</米一世>
</mrow>
</math>细胞标记</Ťd>
</tr>
<tr>
<td align="left">胰十二指肠同源框基因1蛋白</Ťd>
<td align="center">PDX-1</Ťd>
<td align="center">核与细胞质</Ťd>
<td align="center">
<math id="M24" xmlns="http://www.w3.org/1998/Math/MathML">
<mrow>
<mi>
β</米一世>
</mrow>
</math>细胞标记</Ťd>
</tr>
<tr class="table-tr">
<td colspan="4">
<hr class="tbody-hr"></td>
</tr>
</tbody>
</table></td>
</tr>
</tbody>
</table>
</div>
<div class="floats-partial-footer">
<div class="floats-partial-caption">
<span class="caption-text">表1</span>
<a aria-label="full view" class="caption-partial-url" href="//www.newsama.com/journals/sci/2010/587213/tab1/" title="" target="_blank"></a>
</div>
<div class="floats-partial-caption-text">
用免疫细胞化学法分析单独培养或体外培养的NPI细胞单层细胞类型标记物。</div>
</div>
</div>
<p>制备全细胞提取物:将NPI单层提取的细胞重悬于100℃<svg height="9.6750002" id="M25" style="vertical-align:-2.29482pt;width:9.6374998px;" version="1.1" viewbox="0 0 9.6374998 9.6750002" width="9.6374998" xmlns="http://www.w3.org/2000/svg">
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</text>
</g>
</g>
</svg>pH 7.4时10 mM三碱(Sigma Chemical Co)， 150 mM NaCl, 1 mM EDTA, 1 mM乙二醇二(氨基乙基醚)四乙酸(EGTA)， 1% v/v Triton X-100 (Sigma Chemical Co)， 0.5% Nonidet P-40 (Sigma Chemical Co)， 1 M NaF (Sigma Chemical Co)， 0.2 M<svg height="14.75" id="M26" style="vertical-align:-3.25793pt;width:46.724998px;" version="1.1" viewbox="0 0 46.724998 14.75" width="46.724998" xmlns="http://www.w3.org/2000/svg">
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N</Ťspan>
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</text>
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</svg>(Sigma Chemical Co)和0.2 M苯甲磺氟(Sigma Chemical Co)。以1000 g (Mikro 200, Hettich zentrifugen, Tuttlingen, Germany)旋转10分钟，收集上清，Bradford法测定总蛋白含量[<一个href="#B27">26</一个>]。小样本样本储存在<svg height="11.0375" id="M27" style="vertical-align:-0.17555pt;width:41.337502px;" version="1.1" viewbox="0 0 41.337502 11.0375" width="41.337502" xmlns="http://www.w3.org/2000/svg">
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</svg>用于Western blotting分析。</p>
<p>用4-12%硫酸十二烷基钠聚丙烯酰胺凝胶电泳(SDS-PAGE)， 50分离细胞提取物<svg height="9.6750002" id="M28" style="vertical-align:-2.29482pt;width:9.6374998px;" version="1.1" viewbox="0 0 9.6374998 9.6750002" width="9.6374998" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,9.675)">
<g transform="translate(72,-64.26)">
<text transform="matrix(1,0,0,-1,-71.95,66.6)">
<tspan style="font-size: 12.50px; " x="0" y="0">
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</text>
</g>
</g>
</svg>g蛋白/巷,涂抹在硝化纤维膜(美国加州Biorad,大力神)和孵化在缓冲区包含三10毫米,0.5 M氯化钠,1% (v / v)渐变20(西格玛化工有限公司),1:1000年anti-PDX-1 Ab (Chemicon国际,泰梅库拉,CA), 1: 200年anti-GK Ab(圣克鲁斯生物技术公司,圣克鲁斯,加州,美国),1:2500年anti-Glut-2 Ab (Chemicon国际),1:1000年anti-actin Ab(西格玛化工有限公司)。然后将膜在含有1:5000马萝卜过氧化物酶标记抗兔IgG次级Ab (Sigma Chemical Co)的缓冲液中孵育60分钟，检测Ag-Ab复合物。特异性条带通过ECL(增强化学发光)检测。</p>
<h5 id="sec2.8">2.8。RT-PCR</h5>
<p>利用Invisorb Spin-cell RNA微型试剂盒(Invitek GmbH, Berlin, Germany)从NPI细胞单层中提取总RNA。RT由<svg height="14.1875" id="M29" style="vertical-align:-2.72116pt;width:38.637501px;" version="1.1" viewbox="0 0 38.637501 14.1875" width="38.637501" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,14.1875)">
<g transform="translate(72,-60.65)">
<text transform="matrix(1,0,0,-1,-71.95,63.41)">
<tspan style="font-size: 12.50px; " x="0" y="0">
年代</Ťspan>
<tspan style="font-size: 12.50px; " x="6.9516678" y="0">
p</Ťspan>
<tspan style="font-size: 12.50px; " x="13.203168" y="0">
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<tspan style="font-size: 12.50px; " x="17.591721" y="0">
一世</Ťspan>
<tspan style="font-size: 12.50px; " x="21.067554" y="0">
ñ</Ťspan>
<tspan style="font-size: 12.50px; " x="27.319056" y="0">
Ť</Ťspan>
</text>
</g>
</g>
</svg>功率<svg height="14.1875" id="M30" style="vertical-align:-2.72116pt;width:37.737499px;" version="1.1" viewbox="0 0 37.737499 14.1875" width="37.737499" xmlns="http://www.w3.org/2000/svg">
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</text>
</g>
</g>
</svg>PrePrimed SingleShots kit (Clontech, Palo Alto，加利福尼亚州，美国)。所使用的寡核苷酸序列依次为前向序列和反向序列，<svg height="13.175" id="M31" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
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5</Ťspan>
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</g>
</g>
</svg>来<svg height="12.9875" id="M32" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
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</text>
</g>
</g>
</svg>:<svg height="13.425" id="M33" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
<g transform="translate(72,-61.26)">
<text transform="matrix(1,0,0,-1,-71.95,63.6)">
<tspan style="font-size: 12.50px; " x="0" y="0">
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</text>
</g>
</g>
</svg><i>肌动蛋白</一世>
<svg height="13.175" id="M34" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 13.175" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-67.78,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-61.53,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-ATGGTGGGTATGGGTCAGAA -<svg height="12.9875" id="M35" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
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</text>
</g>
</g>
</svg>和<svg height="13.175" id="M36" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
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</text>
</g>
</g>
</svg>-CTTCTCCATGTCGTCCCAGT-<svg height="12.9875" id="M37" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>放大123bp的产品，<一世>NKX6.1</一世>
<svg height="13.175" id="M38" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 13.175" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-67.78,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-61.53,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-AGGATCCATTTTGTTGGACA -<svg height="12.9875" id="M39" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
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</text>
</g>
</g>
</svg>和<svg height="13.175" id="M40" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-CGCCAAGTATTTCGTTTGTT -<svg height="12.9875" id="M41" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 12.9875" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>一个米p升一世fy a product of 111 bp,<一世>PDX-1</一世>
<svg height="13.175" id="M42" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 13.175" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-67.78,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-61.53,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-AGAGCCCGAGGAGAACAAG -<svg height="12.9875" id="M43" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>和<svg height="13.175" id="M44" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-GCGGCCTAGAGATGTATTTG-<svg height="12.9875" id="M45" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>放大100个bp的产品，<一世>Glut-2</一世>
<svg height="13.175" id="M46" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 13.175" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-67.78,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-61.53,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-CCGAGTTTTTCAGTCAAGGA -<svg height="12.9875" id="M47" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 12.9875" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
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</text>
</g>
</g>
</svg>和<svg height="13.175" id="M48" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-AGTCCGCAATGTACTGGAAG -<svg height="12.9875" id="M49" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>一个米p升一世fy a product of 109 bp,<一世>GK</一世>
<svg height="13.175" id="M50" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 13.175" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-67.78,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
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<text transform="matrix(1,0,0,-1,-61.53,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-TAGAGCAGATCCTGGCAGAG -<svg height="12.9875" id="M51" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
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</text>
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</g>
</svg>和<svg height="13.175" id="M52" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-GTAGGTGGGCAGCATCTTC -<svg height="12.9875" id="M53" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>放大99个bp的产品，<一世>NeuroD的/ Beta2中</一世>
<svg height="13.175" id="M54" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 13.175" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-67.78,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-61.53,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-CCTGTGCACCCCTACTCTTA-<svg height="12.9875" id="M55" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>和<svg height="13.175" id="M56" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-TGCAGGATAGTGCATGGTAA -<svg height="12.9875" id="M57" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>放大272个bp的产品，<一世>胰岛素</一世>
<svg height="13.175" id="M58" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 13.175" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-67.78,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
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<text transform="matrix(1,0,0,-1,-61.53,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-CTTCTTCTACACGCCCAAGG -<svg height="12.9875" id="M59" style="vertical-align:-0.17555pt;width:17.487499px;" version="1.1" viewbox="0 0 17.487499 12.9875" width="17.487499" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
</text>
<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>和<svg height="13.175" id="M60" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 13.175" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.175)">
<g transform="translate(72,-61.46)">
<text transform="matrix(1,0,0,-1,-71.95,61.69)">
<tspan style="font-size: 12.50px; " x="0" y="0">
5</Ťspan>
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<text transform="matrix(1,0,0,-1,-65.7,67.01)">
<tspan style="font-size: 8.75px; " x="0" y="0">
</Ťspan>
</text>
</g>
</g>
</svg>-CGGCCTAGTTGCAGTAGTTC -<svg height="12.9875" id="M61" style="vertical-align:-0.17555pt;width:12.2875px;" version="1.1" viewbox="0 0 12.2875 12.9875" width="12.2875" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,12.9875)">
<g transform="translate(72,-61.61)">
<text transform="matrix(1,0,0,-1,-71.95,61.84)">
<tspan style="font-size: 12.50px; " x="0" y="0">
3.</Ťspan>
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<p>RT-PCR分析使用Mx3000P仪器(Stratagene, La Jolla, Calif, USA)，总容量为20<svg height="9.6750002" id="M66" style="vertical-align:-2.29482pt;width:9.6374998px;" version="1.1" viewbox="0 0 9.6374998 9.6750002" width="9.6374998" xmlns="http://www.w3.org/2000/svg">
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</svg>升反应混合物，按照制造商的建议，使用亮的SYBR Green QPCR主混合物2×（Stratagene公司）和10 <svg height="9.6750002" id="M67" style="vertical-align:-2.29482pt;width:9.6374998px;" version="1.1" viewbox="0 0 9.6374998 9.6750002" width="9.6374998" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,9.675)">
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</text>
</g>
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</svg>使用离解协议的各引物的M个。阴性对照含有水代替第一链cDNA的。每个样品归其看家基因的基础（上<svg height="13.425" id="M68" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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</svg>肌动蛋白)。相对基因表达水平归一化到校准器中，选择该校准器作为对照样本(未处理)。用相对表达式计算最终结果<svg height="10.625" id="M69" style="vertical-align:-0.12538pt;width:43.462502px;" version="1.1" viewbox="0 0 43.462502 10.625" width="43.462502" xmlns="http://www.w3.org/2000/svg">
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<tspan style="font-size: 12.50px; " x="11.115167" y="0">
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</g>
</g>
</svg>软件（Stratagene公司）。</p>
<h5 id="sec2.9">2.9。统计分析</h5>
<p>All data were expressed as mean ± SD of three independent experiments. Statistical analysis was conducted by ANOVA.<svg height="10.325" id="M70" style="vertical-align:-0.0pt;width:11.75px;" version="1.1" viewbox="0 0 11.75 10.325" width="11.75" xmlns="http://www.w3.org/2000/svg">
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</svg>- < .05的值被认为是显著的。</p>
<h4 id="results">3.结果</h4>
<h5 id="sec3.1">3.1。形态和胰岛素分泌模式：上NPI细胞单层封装的支持细胞的影响</h5>
<p>如数字所报<一个href="//www.newsama.com/journals/sci/2010/587213/fig1/" target="_blank">1(一)</一个>和<一个href="//www.newsama.com/journals/sci/2010/587213/fig1/" target="_blank">1（d）</一个>，新产品导入的粘接过程从第一天开始，7-10天完全粘接。在21天内，单独培养的NPI单分子层(数据未显示)和与ESC共培养的NPI单分子层之间没有明显的形态学差异。未观察到共培养的NPI细胞单层和单独的NPI细胞单层之间的显著差异(数据未显示)。如图所示<一个href="//www.newsama.com/journals/sci/2010/587213/fig1/" target="_blank">1（d）</一个>，与NPI单层共培养的ESC在第21天表现出良好的形态和细胞组织，细胞存活率测试高于80%。</p>
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<strong>(一)</strong>
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<strong>(b)</strong>
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<strong>(d)</strong>
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<button class="partial-carousel-dot" aria-label="(e)"><img alt="(e)" name="587213.fig.001e" src="https://static-01.hindawi.com/articles/sci/volume-2010/587213/figures/587213.fig.001e.jpg"><br><strong>(e)</strong></button>
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(a) - (c) NPI在T25组织瓶上培养1 (a)、6 (b)和10 (c)天后的显微照片。(d)含SC的Ba-AG微胶囊的光场(左)和荧光(右)显微照片，用EB+FDA染色后获得荧光显微照片，评估SC活性。(e)对照NPI细胞单层单独培养14天(开条)或NPI细胞单层与微胶囊SC共培养7天(灰色条)、14天(孵化条)和21天(充满条)的胰岛素分泌模式。在静态孵育期间，用指示浓度的葡萄糖处理细胞。数据为3次独立实验的平均值;每次胰岛素测定均以3个重复±SD进行。</Ť一个ble>
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<p>ESC强烈影响NPI的胰岛素分泌模式(图)<一个href="//www.newsama.com/journals/sci/2010/587213/fig1/" target="_blank">1（e）中</一个>)。事实上，对于培养时间的整个持续时间（长达21天）的NPI细胞单层培养单独，结果显示，只有有限的胰岛素分泌（<0.7 <svg height="9.6750002" id="M71" style="vertical-align:-2.29482pt;width:9.6374998px;" version="1.1" viewbox="0 0 9.6374998 9.6750002" width="9.6374998" xmlns="http://www.w3.org/2000/svg">
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</svg>而对照组的NPI显示明显缺乏胰岛素对葡萄糖的反应(50-300-50 mg/dL)。与之形成鲜明对比的是，与ESC共培养的NPI显示的GSIR逐渐增强。在第21天，共培养的npi对葡萄糖完全反应，最重要的是，对葡萄糖水平的变化高度反应(<svg height="11.25" id="M72" style="vertical-align:-0.30096pt;width:48.337502px;" version="1.1" viewbox="0 0 48.337502 11.25" width="48.337502" xmlns="http://www.w3.org/2000/svg">
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<</Ťspan>
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。</Ťspan>
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0</Ťspan>
<tspan style="font-size: 12.50px; " x="32.307751" y="0">
5</Ťspan>
</text>
</g>
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</svg>)。如数字所报<一个href="//www.newsama.com/journals/sci/2010/587213/fig2/" target="_blank">2</一个>和<一个href="//www.newsama.com/journals/sci/2010/587213/fig3/" target="_blank">3.</一个>与单独培养的NPI细胞相比，与ESC共培养21天的NPI细胞单层中胰岛素/总细胞数和/或胰岛素+细胞数的比值下降，可能是因为胰岛素释放增加反映了处理后的细胞单层中有更多的胰岛素表达细胞。</p>
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<strong>(一)</strong>
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<strong>(b)</strong>
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胰岛素/总细胞数与对照NPI细胞单分子膜(开条形)或与微胶囊SC共培养的NPI细胞单分子膜(灰色条形)7 (a)、14 (b)和21天(c)的比值。</Ť一个ble>
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<strong>(一)</strong>
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<strong>(b)</strong>
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<strong>(c)</strong>
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<button class="partial-carousel-dot" aria-label="(c)"><img alt="(c)" name="587213.fig.003c" src="https://static-01.hindawi.com/articles/sci/volume-2010/587213/figures/587213.fig.003c.jpg"><br><strong>(c)</strong></button>
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比胰岛素/单独（空心条）控制NPI细胞单层，或者NPI细胞单层胰岛素+细胞数与微胶囊化的SC（灰色柱）中共培养7的（a），图14（b），和21天（C）。数据以平均值±标准差表示。</Ť一个ble>
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<p>比胰岛素含量/ mg总蛋白，指示所述NPI细胞单层和总细胞蛋白含量指示组织的量的分化/成熟的胰岛素含量，表明在共培养的NPI细胞单层的内源性胰岛素的合成显著逐步增加与ESC长达21天相比NPI细胞单层单独培养如（图<一个href="//www.newsama.com/journals/sci/2010/587213/fig8/" target="_blank">图8（c）</一个>)。</p>
<h5 id="sec3.2">3.2。囊化的支持细胞对NPI细胞单层的影响:免疫组织化学评价分化标记物</h5>
<p>不同样本的对照(未处理)NPI细胞单层和ESC共培养NPI采用不同的一抗对进行双重免疫染色，即Ck7/胰岛素(图)<一个href="//www.newsama.com/journals/sci/2010/587213/fig4/" target="_blank">4</一个>(图),PDX-1 /胰岛素<一个href="//www.newsama.com/journals/sci/2010/587213/fig5/" target="_blank">5</一个>),PDX-1 / c - kit(图<一个href="//www.newsama.com/journals/sci/2010/587213/fig6/" target="_blank">6</一个>),胰岛素+ / c - kit(图<一个href="//www.newsama.com/journals/sci/2010/587213/fig7/" target="_blank">7</一个>)。</p>
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<strong></strong>
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<math id="M73" xmlns="http://www.w3.org/1998/Math/MathML">
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双荧光免疫标记(绿色信号:anti-CK-7 Mo-Ab;红信号抗胰岛素多克隆抗体在共聚焦激光显微镜下培养了7 (a)， (d)， 14 (b)， (e)和21 (c)， (f)天，单独(a) - (c)或SC (d) - (f)。酒吧= 10米。</Ť一个ble>
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双荧光免疫标记（绿色信号：抗PDX-1抗体;红色信号抗胰岛素抗体）下培养7的（a），（d），图14（b）NPI细胞单层的激光共聚焦显微镜，（e）和如图21（c），（F）天，单独的（a） - （c）或与SC（d） - （f）所示。酒吧= 10米。</Ť一个ble>
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双荧光免疫标记(绿信号:anti-PDX-1 Ab;红信号抗c-kit Ab)在共聚焦激光显微镜下培养7 (a)， (d)， 14 (b)， (e)和21 (c)， (f)天，单独(a) - (c)或SC (d) - (f)。酒吧= 10米。</Ť一个ble>
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双荧光免疫标记（绿色信号：抗c-kit的抗体;红色信号抗胰岛素抗体）下培养7的（a），（d），图14（b）NPI细胞单层的激光共聚焦显微镜，（e）和如图21（c），（F）天，单独的（a） - （c）或与SC（d） - （f）所示。酒吧= 10米。</Ť一个ble>
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<strong>(c)</strong>
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<span class="caption-text">图8</span>
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<math id="M77" xmlns="http://www.w3.org/1998/Math/MathML">
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<table>
(a) Western blot分析对照NPI细胞单层(C)或与ESC (+ESC)共培养的NPI细胞单层(+ESC)所示蛋白。(b)条带密度分析确定的PDX-1(开条)、Glut-2(阴影条)和GK(填充条)水平的相对强度。条带强度的比率表示为相对于未处理的对照NPI细胞单层的百分比。数据以平均值±标准差表示。*两组在各时间点有显著性差异()。（C）比率胰岛素含量的单独控制NPI细胞单层（空心条），或者NPI细胞单层，用微胶囊化的SC（灰色柱）共培养/ mg总蛋白，7，14，和21天的培养。数据以平均值±标准差表示。</Ť一个ble>
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<p>原始分离的NPI在7天的免疫表型与NPI细胞单层模式相似(数据未显示)。</p>
<p>荧光显微照片报告在表<一个href="//www.newsama.com/journals/sci/2010/587213/tab2/" target="_blank">2</一个>结果显示阳性细胞都CK7和胰岛素，未成熟细胞表型的是（A）的百分比，这很可能暗示的导管起源<svg height="13.425" id="M78" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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</svg>肽(图<一个href="//www.newsama.com/journals/sci/2010/587213/fig4/" target="_blank">4</一个>）与ESC共培养的NPI细胞单层逐渐下降，相比于未处理的NPI细胞单层然而，差别仅是为在21天处理的NPI统计学显著（<svg height="11.25" id="M79" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg">
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<svg height="13.425" id="M80" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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</svg>-cell表型（图<一个href="//www.newsama.com/journals/sci/2010/587213/fig5/" target="_blank">5</一个>和胰岛素/c-kit，一种与功能不成熟相关的细胞表型<svg height="13.425" id="M81" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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<tspan style="font-size: 12.50px; " x="0" y="0">
</Ťspan>
</text>
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</svg>-细胞亚群，在第14天和第21天显著增加(<svg height="11.25" id="M82" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg">
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0</Ťspan>
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<tspan style="font-size: 12.50px; " x="38.559254" y="0">
0</Ťspan>
</text>
</g>
</g>
</svg>）（图<一个href="//www.newsama.com/journals/sci/2010/587213/fig6/" target="_blank">6</一个>），最后（c）中的细胞阳性PDX-1 / C-KIT的百分比（图<一个href="//www.newsama.com/journals/sci/2010/587213/fig7/" target="_blank">7</一个>)，但只在第21天。未处理的NPI与esc处理的NPI差异有统计学意义(<svg height="11.25" id="M83" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg">
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0</Ťspan>
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5</Ťspan>
<tspan style="font-size: 12.50px; " x="38.559254" y="0">
0</Ťspan>
</text>
</g>
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</svg>)。</p>
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<td class="thead-hr" colspan="5">
<hr></td>
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<tr class="thead">
<td align="left">细胞(天)</Ťd>
<td align="center">Ck7 + /胰岛素+ (%)</Ťd>
<td align="center">PDX-1 + /胰岛素+ (%)</Ťd>
<td align="center">胰岛素+ / c - kit + (%)</Ťd>
<td align="center">PDX-1 / C-KIT +（％）</Ťd>
</tr>
<tr>
<td class="thead-hr" colspan="5">
<hr></td>
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<tr>
<td align="left">C (7)</Ťd>
<td align="center">58.5±1.4</Ťd>
<td align="center">14.8±3.9</Ťd>
<td align="center">10.0±1.7</Ťd>
<td align="center">13.1±2.3</Ťd>
</tr>
<tr>
<td align="left">+ ESC（7）</Ťd>
<td align="center">55.3±5.5</Ťd>
<td align="center">14.3±3.0</Ťd>
<td align="center">9.7±4.0</Ťd>
<td align="center">14.3±9.7</Ťd>
</tr>
<tr>
<td align="left">C (14)</Ťd>
<td align="center">50.0±3.8</Ťd>
<td align="center">20.7±3.1</Ťd>
<td align="center">14.0±3.7</Ťd>
<td align="center">25.7±10.0</Ťd>
</tr>
<tr>
<td align="left">+ ESC（14）</Ťd>
<td align="center">40.2±2.4</Ťd>
<td align="center">33.3±<米在h id="M84" xmlns="http://www.w3.org/1998/Math/MathML">
<mi>
</mi>
<msup>
<mrow>
<mn>
5.1</米ñ>
</mrow>
<mrow>
<mi>
*</米一世>
</mrow>
</msup>
</math></td>
<td align="center">26.0±<米在h id="M85" xmlns="http://www.w3.org/1998/Math/MathML">
<mi>
</mi>
<msup>
<mrow>
<mn>
0.9</米ñ>
</mrow>
<mrow>
<mi>
*</米一世>
</mrow>
</msup>
</math></td>
<td align="center">31.1±13.5</Ťd>
</tr>
<tr>
<td align="left">C (21)</Ťd>
<td align="center">45.6±4.7</Ťd>
<td align="center">29.5±4.9</Ťd>
<td align="center">15.8±2.9</Ťd>
<td align="center">45.1±11.0</Ťd>
</tr>
<tr>
<td align="left">+ ESC (21)</Ťd>
<td align="center">32.9±<米在h id="M86" xmlns="http://www.w3.org/1998/Math/MathML">
<mi>
</mi>
<msup>
<mrow>
<mn>
4.1</米ñ>
</mrow>
<mrow>
<mi>
*</米一世>
</mrow>
</msup>
</math></td>
<td align="center">74.8±<米在h id="M87" xmlns="http://www.w3.org/1998/Math/MathML">
<mi>
</mi>
<msup>
<mrow>
<mn>
4.9</米ñ>
</mrow>
<mrow>
<mi>
*</米一世>
</mrow>
</msup>
</math></td>
<td align="center">38.3±<米在h id="M88" xmlns="http://www.w3.org/1998/Math/MathML">
<mi>
</mi>
<msup>
<mrow>
<mn>
5.2</米ñ>
</mrow>
<mrow>
<mi>
*</米一世>
</mrow>
</msup>
</math></td>
<td align="center">71.8±<米在h id="M89" xmlns="http://www.w3.org/1998/Math/MathML">
<mi>
</mi>
<msup>
<mrow>
<mn>
10.8</米ñ>
</mrow>
<mrow>
<mi>
*</米一世>
</mrow>
</msup>
</math></td>
</tr>
<tr class="table-tr">
<td colspan="5">
<hr class="tbody-hr"></td>
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<td>C：对照的未处理NPI单层。<br>+ ESC：NPI单层包封塞尔托利细胞的存在共培养。<br>数据表示阳性细胞的百分比，以平均值±标准差表示(<米在h id="M90" xmlns="http://www.w3.org/1998/Math/MathML">
<mrow>
<mi>
ñ</米一世>
</mrow>
</math>= 3)。<br><i>* P</一世>< .050。<br></td>
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<span class="caption-text">表2</span>
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NPI单层的分化标记物的免疫组织化学分析培养单独或ESC的存在。</div>
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</div>
<h5 id="sec3.3">3.3。囊化支持细胞对NPI细胞单层的影响:Western Blot分析分化标志物的评价</h5>
<p>为了证实免疫细胞化学得到的数据，我们计划利用互补的western blotting技术进行一套新的实验。实验结果如图所示<一个href="//www.newsama.com/journals/sci/2010/587213/fig8/" target="_blank">8</一个>其中硝基纤维素膜的照片和密度分析的吊柱显示。Western blots证实，在ESC存在的情况下，对照NPI与对照NPI之间存在显著差异。值得注意的是，与对照组相比，共培养的NPI中PDX-1、葡萄糖激酶(GK)和GLUT-2的表达显著增加(<svg height="11.25" id="M91" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg">
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<tspan style="font-size: 12.50px; " x="22.930502" y="0">
。</Ťspan>
<tspan style="font-size: 12.50px; " x="26.056252" y="0">
0</Ťspan>
<tspan style="font-size: 12.50px; " x="32.307751" y="0">
5</Ťspan>
<tspan style="font-size: 12.50px; " x="38.559254" y="0">
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</svg>)。</p>
<h5 id="sec3.4">3.4。在NPI细胞单层封装睾丸支持细胞的影响：通过实时PCR分化指标的评估（定量PCR）</h5>
<p>qPCR分析，图报道<一个href="//www.newsama.com/journals/sci/2010/587213/fig9/" target="_blank">9</一个>显示出在许多处理和未处理的NPI之间的基因的表达具有统计学显著差异。在第21天，共培养NPI显示出PDX-1，NKX6.1，胰岛素的表达显着增加，和c-kit基因（<svg height="11.25" id="M92" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg">
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</Ťspan>
<tspan style="font-size: 12.50px; " x="10.890113" y="0">
<</Ťspan>
<tspan style="font-size: 12.50px; " x="22.930502" y="0">
。</Ťspan>
<tspan style="font-size: 12.50px; " x="26.056252" y="0">
0</Ťspan>
<tspan style="font-size: 12.50px; " x="32.307751" y="0">
5</Ťspan>
<tspan style="font-size: 12.50px; " x="38.559254" y="0">
0</Ťspan>
</text>
</g>
</g>
</svg>)。在第7天和第21天，共培养的NPI在Neuro D和glut2方面与对照组相比有统计学差异(<svg height="11.25" id="M93" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,11.25)">
<g transform="translate(72,-63)">
<text transform="matrix(1,0,0,-1,-71.95,63.35)">
<tspan style="font-size: 12.50px; " x="0" y="0">
</Ťspan>
<tspan style="font-size: 12.50px; " x="10.890113" y="0">
<</Ťspan>
<tspan style="font-size: 12.50px; " x="22.930502" y="0">
。</Ťspan>
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0</Ťspan>
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5</Ťspan>
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<g transform="matrix(1.25,0,0,-1.25,0,11.25)">
<g transform="translate(72,-63)">
<text transform="matrix(1,0,0,-1,-71.95,63.35)">
<tspan style="font-size: 12.50px; " x="0" y="0">
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<tspan style="font-size: 12.50px; " x="10.890113" y="0">
<</Ťspan>
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。</Ťspan>
<tspan style="font-size: 12.50px; " x="26.056252" y="0">
0</Ťspan>
<tspan style="font-size: 12.50px; " x="32.307751" y="0">
5</Ťspan>
<tspan style="font-size: 12.50px; " x="38.559254" y="0">
0</Ťspan>
</text>
</g>
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</svg>)。Gk无统计学差异(数据未显示)。</p>
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从NPI细胞单层所指示基因的qPCR分析在ESC的存在所指示的时间段共培养。（a）中PDX-1（空心条），NeuroD的（灰色柱），NKX6.1（阴影条），和c-kit（实心柱）。（B）胰岛素（空心柱），GLUT-2（阴影条），和GK（实心柱）。报告的结果是用百分比表示了未经处理的对照NPI细胞单层的。Data are shown as means ± SD from 3 samples.</Ť一个ble>
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<h4 id="discussion">4。讨论</h4>
<p>尸体人供体胰腺的与免疫耐受网络（ITN）对人胰岛细胞移植的多中心临床试验的效果差一起使用时的有限的可及[<一个href="#B3">3.</一个>已经大大降低了人类胰岛移植对TIDM可能治愈的影响。此外，最近有报道称，用人胰岛素或胰岛素类似物治疗的糖尿病患者可能存在恶性肿瘤风险[<一个href="#B2">2</一个>]。由于这些原因，产生胰岛素的细胞的新来源正在积极寻求[<一个href="#B4">4</一个>,<一个href="#B5">5</一个>]。在这方面，我们之前证明了SCs能够诱导成年大鼠胰岛细胞的有丝分裂活性[<一个href="#B28">27</一个>或新分离的NPI迅速而显著地成熟和分化为功能胜任的<svg height="13.425" id="M95" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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</svg>-细胞 [<一个href="#B5">5</一个>]。</p>
<p>新分离的NPI细胞群通常由少数的<svg height="13.425" id="M96" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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<tspan style="font-size: 12.50px; " x="0" y="0">
</Ťspan>
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</svg>-细胞，大部分CK7+细胞，而其余的细胞群共同表达胰岛素和上皮细胞标记物。事实上，正如Trivedi等人报道的那样[<一个href="#B7">7</一个>]和Korbutt等。[<一个href="#B4">4</一个>]，NPI可能需要4-10周或更长时间以达到足够的，分化<svg height="13.425" id="M97" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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<tspan style="font-size: 12.50px; " x="0" y="0">
</Ťspan>
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</svg>-细胞数量，以逆转糖尿病鼠TX后的高血糖。体外暴露于SC后，双荧光免疫标记清楚地显示，抗ck7染色的上皮细胞最初占总细胞总数的60%以上，变成了胰岛素阳性细胞(74%，对照NPI为6%)。通过9天的SC共培养，可以加速胰岛细胞的成熟，并得到胰岛素功能数据的证实[<一个href="#B5">5</一个>]。这一过程受到实验过程中NPI质量损失的限制。因此，从NPI开始，我们能够生成持久的NPI细胞单层，无需使用细胞外基质或细胞工程方法[<一个href="#B11">11</一个>]。在这方面，NPI细胞单分子膜可以作为一个实验工具来评估几种生长因子对<svg height="13.425" id="M98" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</svg>-cell分子途径，可能允许检查胰岛细胞谱系承诺和扩大起始细胞材料[<一个href="#B11">11</一个>]。</p>
<p>获得的结果首次显示，NPI细胞单层与c-kit+细胞相关，与之前在小鼠和大鼠的报道一致[<一个href="#B16">15</一个>,<一个href="#B17">16</一个>,<一个href="#B29">28</一个>]。共聚焦显微镜检查显示的c-kit + / PDX-1 +和c-kit + /胰岛素+细胞可能代表祖细胞和possibily<svg height="13.425" id="M99" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</svg>细胞的前体。事实上，在胰腺发育过程中，分化的细胞来自于PDX-1+导管前体细胞，因此c-kit/PDX-1的共定位可能与内分泌相吻合<svg height="13.425" id="M100" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
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</svg>共培养21天时，在胰腺新生模型中，c-kit和PDX-1转录因子上调。NeuroD /<一世></i>
<svg height="13.425" id="M101" style="vertical-align:-2.29482pt;width:8.8500004px;" version="1.1" viewbox="0 0 8.8500004 13.425" width="8.8500004" xmlns="http://www.w3.org/2000/svg">
<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
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</svg>2和NKX6.1启动子的mRNA表明，细胞共培养过程中，细胞朝着更成熟的表型转变。GLUT-2 mRNA表达趋于在共培养的14和21天下降，可能表明蛋白解除管制。不像一些作者[<一个href="#B30">29</一个>]并且根据人[<一个href="#B31">30.</一个>，我们观察到，在我们的细胞单层中，c-kit和胰岛素在培养21天时被因子3-4共定位。WB密度分析显示，与对照单分子膜相比，治疗组(21天具有统计学意义)磷酸化PDX-1升高。已知PDX-1与其他属于螺旋-环-螺旋碱性蛋白家族的因子共同调控胰岛素基因表达。由此产生的异二聚体结合到胰岛素启动子的E2元素上[<一个href="#B11">11</一个>]。这个数据已经通过qPCR表明在21天时有胰岛素mRNA的转录活性在结合最高胰岛素含量/ mg总蛋白的比例证实。然而，并非所有的胰岛素阳性细胞也正标记为的c-kit [<一个href="#B17">16</一个>这表明这些细胞可能包含一个内分泌前体细胞的子集。</p>
<p>其中一个行动的可能机制可能与SCF分泌。SC的产生SCF或c-kit配体结合并激活跨膜酪氨酸激酶受体的c-kit。SCF / c-kit的相互作用在发展，功能朗格汉斯的啮齿动物胰岛的一个非常重要的角色，和生存[<一个href="#B15">14</一个>,<一个href="#B16">15</一个>]。已证明SCF可促进胎鼠胰岛胰岛素输出量的增加[<一个href="#B16">15</一个>]。有趣的是，我们与微囊化SC细胞单层的共培养诱导的PDX-1 + /胰岛素+和c-kit + /胰岛素+细胞百分比的增加，根据通过使用SCF [的先前的观察<一个href="#B32">31</一个>]在任何一个胎儿中[<一个href="#B18">17</一个>]或鼠[<一个href="#B29">28</一个>胰岛的实验设置。同样地，增加PDX-1和c-kit的mRNA也进行了观察。</p>
<p>我们发现内源性胰岛素输出的显著增加，下葡萄糖刺激，从共培养与SC，相比于单独NPI细胞单层的NPI细胞单层。事实上，静态孵化明确记载，ESC处理的细胞单层生理回应血糖的变化在90分钟内，从不同了没有进行比较的成熟模式的控制。的降低率胰岛素/总细胞中NPI细胞单层数目和/或胰岛素+细胞共培养数与ESC，长达21天，与单独相比NPI细胞单层，可能反映了胰岛素表达细胞在经处理的单层的增加。此外，比胰岛素含量/ mg总蛋白逐步增加用于可能指示ECS似乎促进成熟整个培养时间（长达21天）。ESC衍生SCF可以通过不同的机制，包括磷脂酰肌醇-3-激酶（PI3K9），所述Janus家族蛋白酪氨酸激酶诱导胰岛细胞前体分化，Src家族成员，和RAS-RAF-促分裂原活化蛋白（MAP）激酶。这些途径介导几个细胞过程，包括增加基因转录，增殖，分化，存活和代谢平衡[<一个href="#B33">32</一个>,<一个href="#B34">33</一个>]。</p>
<p>总之，我们的数据似乎支持了微囊化SC可能在短期内加速单层猪细胞培养分化的观点。这可能是由于sc诱导的PDX-1+/胰岛素+和c-kit+/胰岛素+细胞质量增加所致。这些观察对成熟猪分化的潜在后果<一世>β</一世>-<一世></i><i></i>细胞可能是糖尿病的异种细胞来源，这一点是显而易见的。</p>
<h4 id="acknowledgments">致谢</h4>
<p>这项工作是由意大利罗马的奥加诺大学间财团支持的。老鼠抗猪c-kit是Dominguez博士的礼物。感谢M. C. Aglietti博士和C. Proietti博士的技术援助。感谢Giuliano Pannacci和Paolo Serra(农场“Barilari”，Gubbio)的技术支持。F. Mancuso, M. Calvitti和G. Luca对这项工作做出了同样的贡献。</p>
</div>
<div class="xml-content">
<h4 class="references" id="references">参考</h4>
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</math>，但不是CD8<米在h id="M105" xmlns="http://www.w3.org/1998/Math/MathML">
<mrow>
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+</米Ø>
</mrow>
</math>，淋巴细胞增殖和抗蚀剂在体外细胞介导的溶细胞损伤，”<一世>糖尿病</一世>卷。48，没有。9，第1713至1719年，1999年。<span class="reflinks">视图:<一个href="https://doi.org/10.2337/diabetes.48.9.1713">出版商的网站</一个><span class="sep">|</span><a href="https://scholar.google.com/scholar_lookup?title=Neonatal%20porcine%20islet%20cells%20induce%20human%20CD4+,%20but%20not%20CD8+,%20lymphocyte%20proliferation%20and%20resist%20cell-mediated%20cytolytic%20injury%20in%20vitro&author=A. G. Murray&author=R. C. Nelson&author=G. R. Rayat&author=J. F. Elliott&author=&author=G. S. Korbutt&publication_year=1999" target="_blank">谷歌学术</一个></span></li>
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<mrow>
<mi>
β</米一世>
</mrow>
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<li id="B18">J.李，K.莱特，F.研究员，C. G. Goodyer和R.王“c-kit和在发展人胰腺及其配体干细胞因子的作用下，”<一世>糖尿病</一世>卷。54，第S39，2005年。<span class="reflinks">视图:<一个href="https://scholar.google.com/scholar_lookup?title=The%20role%20of%20c-kit%20and%20its%20ligand%20stem%20cell%20factor%20in%20the%20development%20human%20pancreas&author=J. Li&author=K. Lyte&author=F. Fellows&author=C. G. Goodyer&author=&author=R. Wang&publication_year=2005" target="_blank">谷歌学术</一个></span></li>
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<mrow>
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</math>激酶依赖性林恩/ TEC / DOK-1复合物的形成在造血细胞，”<一世>血</一世>第96卷第1期2000年，第3406-3413页。<span class="reflinks">视图:<一个href="https://scholar.google.com/scholar_lookup?title=Stem%20cell%20factor%20induces%20phosphatidylinositol%203'-kinase-dependent%20Lyn/Tec/Dok-1%20complex%20formation%20in%20hematopoietic%20cells&author=T. B. Van Dijk&author=E. Van den Akker&author=M. Parren-Van Amelsvoort&author=H. Mano&author=B. Lowenberg&author=&author=M. Von Lindern&publication_year=2000" target="_blank">谷歌学术</一个></span></li>
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<a class="sc-htpNat bUhGXt link sc-cbkKFq dNPgJJ sc-iybRtq jghqNV pdf_desktop" href="//www.newsama.com/downloads/journals/sci/2010/587213.pdf" aria-label="" target="_blank" id=""><span><img alt="" class="sc-EHOje fEOMwM sc-krvtoX isUmzT" title="" role="presentation" src="https://static-01.hindawi.com/next_assets/Lvhw1J_a6cWVqaLmZO6Im/_next/static/node_modules/hindawi-ui/src/icon/svg/pdf.svg" height="24"><span class="text">PDF</span></span><img alt="" class="sc-EHOje ggEPYc sc-fYiAbW eBEQa fileArrow" title="" role="presentation" src="data:image/svg+xml;base64,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" height="20"></a>
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src="data:image/svg+xml;base64,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" height="24"><span class="text">下载文献</span></span><img alt="" class="sc-EHOje ggEPYc sc-fYiAbW eBEQa fileArrow" title="" role="presentation" src="data:image/svg+xml;base64,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" 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src="data:image/svg+xml;base64,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" height="24"><span class="text">引用</span></span><img alt="" class="sc-EHOje ggEPYc sc-fYiAbW eBEQa fileArrow" title="" role="presentation" src="data:image/svg+xml;base64,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" 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<span><img alt="" class="sc-EHOje fEOMwM sc-iomxrj czMNsj" title="" role="presentation" src="data:image/svg+xml;base64,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" height="24"><span class="sc-iFMziU gCiAsp">下载其他格式</span><span class="sc-keVrkP hYjHxB">更多</span></span>
<img alt="" class="sc-EHOje fEOMwM sc-dvCyap bivFoZ" title="" role="presentation" src="data:image/svg+xml;base64,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" height="24">
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<a class="sc-htpNat bUhGXt link sc-eTpRJs eqSlZb open_panel" href="//www.newsama.com/downloads/journals/sci/2010/587213.xml" aria-label="XML" property="background-color" color="#D8D8D8">
<div class="sc-dxZgTM jwQjXi">
XML</div><img alt="" class="sc-EHOje fEOMwM sc-iomxrj czMNsj" title="" role="presentation" src="data:image/svg+xml;base64,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" height="24"></a>
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<li><a class="sc-htpNat bUhGXt link editors" href="//www.newsama.com/publish-research/editors/" aria-label="Editors"><img alt="" class="sc-EHOje jRIjVn" title="" role="presentation" src="data:image/svg+xml;base64,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" height="14">编辑器</一个></li>
<li><a class="sc-htpNat bUhGXt link reviewers" href="//www.newsama.com/publish-research/reviewers/" aria-label="Reviewers"><img alt="" class="sc-EHOje jRIjVn" title="" role="presentation" src="data:image/svg+xml;base64,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" height="14">评论家</一个></li>
</ul>
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