SCI 干细胞国际 1687 - 9678 1687 - 966 x Hindawi 10.1155 / 2018/8478953 8478953 研究文章 背根神经节维护具备干细胞的骨髓间充质干细胞通过增强自噬通过AMPK / mTOR Coculture系统路径 Shuaishuai 1 黄俊钦 1 Huijie 1 1 Pengzhen 1 专业 1 Donglin 2 冀萌当初 3 首歌 1 1 1 纯美少女 1 http://orcid.org/0000 - 0002 - 6498 - 4702 1 http://orcid.org/0000 - 0002 - 5089 - 5012 Guoxian 1 布鲁诺 1 整形外科学系 Xijing医院 第四军医大学 西安710032年 中国 fmmu.edu.cn 2 整形外科学系 解放军第463医院 沈阳110042 中国 3 整形外科学系 解放军第251医院 张家口075000 中国 2018年 30. 9 2018年 2018年 27 03 2018年 10 07年 2018年 14 08年 2018年 30. 9 2018年 2018年 版权©2018 Shuaishuai Zhang et al。 这是一个开放的文章在知识共享归属许可下发布的,它允许无限制的使用,分布和繁殖在任何媒介,提供最初的工作是正确的引用。

我们先前的研究发现,感觉神经束植入组织工程骨(TEB)可能导致更好的骨生成。探讨感觉神经促进骨生成的机制在TEB体外,一个transwell coculture实验设计之间的背根神经节(DRG)细胞和骨髓间充质干细胞(bmsc)。BMSC增殖是由CCK8化验,骨的,细粒,和去评估茜素红、阿尔新蓝,油红染色。我们发现bmsc的增殖和多功能分化都是增强coculture组相比bmsc组。结晶紫染色显示clone-forming coculture组的综合能力也增强和mRNA水平Sox2, Nanog, Oct4显著调节coculture组。此外,自噬水平的bmsc,调节其具备干细胞,被提拔coculture组,由AMPK / mTOR通路。此外,AMPK抑制剂化合物C可以显著下调LC3的蛋白表达和具备干细胞基因的mRNA水平coculture组。最后,我们发现NK1受体拮抗剂,aprepitant,可以一定程度上阻止这种效应,这表明,P物质发挥了重要作用的效果。一起,我们得出结论,DRG可以保持bmsc的具备干细胞通过增强自噬通过AMPK / mTOR通路在transwell coculture系统,这可能有助于解释更好的骨植入后感觉神经TEB。 中国国家自然科学基金 81772377 81430049 1。介绍</tgydF4y2Baitle> <p>骨组织工程提供了一种很有前途的解决大型骨缺损的治疗,但仍有许多问题需要解决(<gydF4y2Baxref ref-type="bibr" rid="B1"> 1</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B3"> 3</gydF4y2Baxref>),如低存活率和可怜的bmsc的成骨分化。以前,我们发现感觉神经束preimplanted在组织工程骨(TEB)可以显著提高成骨tep (<gydF4y2Baxref ref-type="bibr" rid="B4"> 4</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B5"> 5</gydF4y2Baxref>),但这种现象的潜在机制还不太为人所知。</gydF4y2Bap> <p>感觉神经已经被报道在体内骨骼的新陈代谢和再生扮演关键角色(<gydF4y2Baxref ref-type="bibr" rid="B6"> 6</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B9"> 9</gydF4y2Baxref>]。一些研究发现,感觉神经支配导致骨小梁的维护质量和其力学性能通过抑制骨吸收(<gydF4y2Baxref ref-type="bibr" rid="B8"> 8</gydF4y2Baxref>]。此外,感觉神经传出功能在组织他们内向,由发射器释放从周围神经终端,这有助于维护骨小梁完整性(<gydF4y2Baxref ref-type="bibr" rid="B10"> 10</gydF4y2Baxref>]。最近,感觉neuron-derived Sema3A发现负责Sema3A骨量丢失<gydF4y2Basup>−−/</gydF4y2Basup>老鼠,Sema3A调节骨重建间接调制感觉神经支配,而不是直接作用于成骨细胞(<gydF4y2Baxref ref-type="bibr" rid="B7"> 7</gydF4y2Baxref>]。</gydF4y2Bap> <p>体外研究表明,神经肽,如P物质和CGRP怎样<gydF4y2Baxref ref-type="bibr" rid="B11"> 11</gydF4y2Baxref>),可能会影响preosteoblast细胞。例如,SP显著增加扩散bmsc体外剂量依赖性的方式(<gydF4y2Baxref ref-type="bibr" rid="B12"> 12</gydF4y2Baxref>]。和SP可能通过Wnt bmsc /诱导成骨细胞的分化<gydF4y2Baitalic> β</gydF4y2Baitalic>bmsc的连环蛋白通路,促进血管生成能力<gydF4y2Baxref ref-type="bibr" rid="B13"> 13</gydF4y2Baxref>]。此外,据报道,CGRP怎样发挥其合成代谢作用对人类成骨细胞通过刺激规范化Wnt信号和抑制人类成骨细胞凋亡(<gydF4y2Baxref ref-type="bibr" rid="B14"> 14</gydF4y2Baxref>]。</gydF4y2Bap> <p>总的来说,这些发现体内和体外建议感觉神经在骨的新陈代谢和再生起到了至关重要的作用。然而,在细胞水平上的机制研究很少,和一种神经肽可能不能反映整体效果的感觉神经在骨细胞。因此,总体感觉神经的调节骨细胞,其内在的分子机制需要进一步的研究。</gydF4y2Bap> <p>在这项研究中,我们使用一个transwell coculture系统对DRG细胞对bmsc的影响进行调查。我们的研究结果表明DRG可以帮助保持bmsc的具备干细胞通过改善基底自噬水平通过激活AMPK /在这个coculture mTOR信号系统。</gydF4y2Bap> </sec> <sec id="sec2"> <title>2。材料和方法</tgydF4y2Baitle> <sec id="sec2.1"> <title>2.1。隔离和GFP鼠bmsc的表征</tgydF4y2Baitle> <p>bmsc从枚GFP Sprague-Dawley两星期(SD)老鼠收获使用良好的协议与轻微的修改(<gydF4y2Baxref ref-type="bibr" rid="B15"> 15</gydF4y2Baxref>]。短暂,bmsc的主要文化是在无菌条件下获得一枚GFP鼠两星期。过量的老鼠牺牲2%戊巴比妥钠(<gydF4y2Baitalic> w</gydF4y2Baitalic>/<gydF4y2Baitalic> v</gydF4y2Baitalic>)。沉浸在75%乙醇后(<gydF4y2Baitalic> v</gydF4y2Baitalic>/<gydF4y2Baitalic> v</gydF4y2Baitalic>为5分钟),股骨和胫骨被孤立和附加的软组织切除,然后用无菌剪刀松果体被切断。然后,骨髓是刷新骨干注射器和收集的基本培养基(<gydF4y2Baitalic> α</gydF4y2Baitalic>mem包含10%的边后卫和1%的抗生素(青霉素和链霉素))。收集到的介质包含骨髓细胞培养在6-well文化板块2毫升<gydF4y2Baitalic> α</gydF4y2Baitalic>mem包含10%的边后卫。24小时后,不依从细胞被小心地删除。细胞生长介质是改变每2天,和细胞融合亚文化,直到达到80%。通过3 bmsc被播种在6-well文化板2×10的浓度<gydF4y2Basup>3</gydF4y2Basup>与DRG细胞/毫升coculture。bmsc从通道3与流式细胞术和多能分化特征。</gydF4y2Bap> </sec> <sec id="sec2.2"> <title>2.2。流式细胞术</tgydF4y2Baitle> <p>bmsc从通道3使胰蛋白酶化,离心机,悬浮在寒冷的PBS,和细胞数量计算。然后,bmsc与2% BSA被封锁在室温下30分钟。与此同时,抗体与PBS稀释至推荐的浓度。之后,细胞密度调整1×10<gydF4y2Basup>6</gydF4y2Basup>每个管。然后,细胞被孵化了40分钟在4°C PerCP anti-rat CD90抗体(202512年,BioLegend), PE anti-rat CD11b / C抗体(201807年,BioLegend), PE anti-rat CD34抗体(ab187284 Abcam)和PE-Cy7 anti-rat CD45抗体(202214年,BioLegend)。流式细胞术分析了培养细胞(cytomics FC 500年,贝克曼库尔特)使用isotype-identical抗体作为控制。</gydF4y2Bap> </sec> <sec id="sec2.3"> <title>2.3。制备DRG、Coculture bmsc和DRG细胞之间</tgydF4y2Baitle> <p>刚产后SD大鼠被牺牲,和隔离DRG是基于前面的研究(<gydF4y2Baxref ref-type="bibr" rid="B16"> 16</gydF4y2Baxref>]。脊柱暴露,从胸腰椎地区打开。然后,按从腰椎脊髓和切割用冰冷的PBS。结缔组织是小心地删除。DRG汇集在<gydF4y2Baitalic> α</gydF4y2Baitalic>mem培养基在冰上,直到进一步的过程。收集到的DRG为2分钟,然后用剪刀将机械孵化30分钟37°C和0.1% (<gydF4y2Baitalic> v</gydF4y2Baitalic>/<gydF4y2Baitalic> v</gydF4y2Baitalic>)胰蛋白酶。然后,通过100细胞被过滤<gydF4y2Baitalic> μ</gydF4y2Baitalic>m细胞过滤器和离心机,享年180岁<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"> <mml:mi> g</gydF4y2Bamml:mi> </mml:math> </inline-formula>5分钟。收获DRG细胞种植在transwell细胞插入(美国康宁3450多),这已经被放入6-well文化板块,1×10的浓度<gydF4y2Basup>4</gydF4y2Basup>细胞每口井。然后,1毫升细胞的生长培养基添加到transwell插入,这意味着有3毫升细胞生长介质的6井的盘子。bmsc培养的DRG细胞作为对照组。他们培养37°C公司5%<gydF4y2Basub>2</gydF4y2Basub>的气氛。使用transwell背根神经节细胞和细胞插入允许bmsc共享相同的细胞生长介质,但没有直接接触。</gydF4y2Bap> </sec> <sec id="sec2.4"> <title>2.4。BMSC成骨分化和茜素红染色</tgydF4y2Baitle> <p>成骨分化培养基(rasmx - 90021)从Cyagen生物科学公司,购买和接受了手术治疗产品的用户手册。短暂,coculture 8天后,bmsc分离和播种密度的新12-well培养板2×10<gydF4y2Basup>4</gydF4y2Basup>细胞/。成骨的介质包括DMEM和10%的边后卫,50毫克/毫升抗坏血acid-2phosphate, 100海里地塞米松,10毫米<gydF4y2Baitalic> β</gydF4y2Baitalic>甘油磷酸的100 U /毫升青霉素和链霉素100毫克/毫升。细胞汇合的大约60 - 70%时,生长媒介也从每个小心翼翼地吸气和2毫升的成骨分化培养基补充道。成骨诱导媒介是改变每3天。14天后,细胞可以用4%多聚甲醛固定和染色茜素红S (Cyagen Biosciences Inc .) 3 - 5分钟,然后在显微镜下观察细胞。</gydF4y2Bap> </sec> <sec id="sec2.5"> <title>2.5。BMSC Chondrogenic分化和阿尔新蓝染色</tgydF4y2Baitle> <p>chondrogenic分化,0.5毫升bmsc包含2.5×10<gydF4y2Basup>5</gydF4y2Basup>细胞需要形成一个chondrogenic颗粒在15毫升聚丙烯文化管分化诱导介质(美国Cyagen生物科学rasmx - 90041)。管帽的放松为了让气体交换,和细胞孵化37°C在湿润的气氛中5%的有限公司<gydF4y2Basub>2</gydF4y2Basub>。24小时的球没有打扰。chondrogenic感应媒介改变了每2 - 3天在每个管(为了避免吸气吸气时颗粒中,附加无菌1 - 200<gydF4y2Baitalic> μ</gydF4y2Baitalic>l吸管提示吸气吸管的结束)。0.5毫升的刚做好完成chondrogenic介质被添加到每个管。确保颗粒是自由浮动的,管的底部是挥动几次。Chondrogenic丸后20天的文化。8的小球formalin-fixed,冰冻切片<gydF4y2Baitalic> μ</gydF4y2Baitalic>米厚度均获得阿尔新蓝染色分析。</gydF4y2Bap> </sec> <sec id="sec2.6"> <title>2.6。脂肪形成的分化和油红染色</tgydF4y2Baitle> <p>脂肪形成的分化,分化诱导介质(rasmd - 90031, 1<gydF4y2Baitalic> μ</gydF4y2Baitalic>M敏捷,1<gydF4y2Baitalic> μ</gydF4y2Baitalic>g / ml胰岛素和0.5毫米3-isobutyl-1-methylxanthine)和分化基础培养基(谷氨酰胺、胰岛素)准备根据用户的手册。第八天的实验中,伴着受移植者在2×10<gydF4y2Basup>4</gydF4y2Basup>细胞/厘米<gydF4y2Basup>2</gydF4y2Basup>在6-well细胞培养板介质体积的2毫升/。支流或postconfluent到达100%,2毫升的感应介质添加/。三天后,媒介是维护媒介改变了。24小时后,介质改变感应介质。4次重复周期后,维护媒介不断用于4 - 7天。后,细胞分化,细胞被冲洗,用4%多聚甲醛固定。1毫升油红O (Cyagen生物科学有限公司)工作的解决方案是添加(与蒸馏水稀释3:2和过滤滤纸)每30分钟。然后,细胞在显微镜下观察。</gydF4y2Bap> </sec> <sec id="sec2.7"> <title>2.7。细胞增殖实验</tgydF4y2Baitle> <p>如前所述(执行细胞增殖试验<gydF4y2Baxref ref-type="bibr" rid="B17"> 17</gydF4y2Baxref>]。简单地说,细胞被播种6-well板(10<gydF4y2Basup>4</gydF4y2Basup>细胞/)和培养有或没有按表示时间长度。天1,2,4,6,8,10,细胞计数Kit-8 (Dojindo)应用和孵化了2小时,然后甲瓒染料的吸光度由活细胞在450纳米测量标仪(美国BioTek协同H1)。所有实验进行三次。</gydF4y2Bap> </sec> <sec id="sec2.8"> <title>2.8。克隆形成单位(CFU)测定</tgydF4y2Baitle> <p>CFU试验进行了测量bmsc有或没有DRG细胞的自我更新能力。短暂、bmsc与DRG coculture后8天,被播种在50个细胞的数量60毫米板和培养10天,然后用结晶紫染色法染色方案。殖民地含有超过50个细胞在显微镜下计数。单一bmsc被认为控制所有的实验。所有实验进行三次。</gydF4y2Bap> </sec> <sec id="sec2.9"> <title>2.9。实时定量聚合酶链反应</tgydF4y2Baitle> <p>所有程序都是根据产品的指令执行,指的是证据确凿的方法在之前的一项研究[<gydF4y2Baxref ref-type="bibr" rid="B15"> 15</gydF4y2Baxref>]。从bmsc短暂,总RNA提取总RNA工具包使用ω我(美国很多R6834-01ωbio-tek)根据制造商的协议。核糖核酸的浓度和纯度测定通过测量吸光度在TE缓冲(10毫米Tris-HCl、pH值8.0和1毫米EDTA)在260年和280海里。然后,总RNA的互补脱氧核糖核酸合成使用豆类PrimeScript™RT大师混合实时(完美)工具包(很多RR036,豆类,日本)后,供应商的指令。Sox2的mRNA水平,Nanog, Oct4 bmsc实时定量rt - pcr测定使用豆类SYBR绿色我装备根据用户手册(Bio-Rad CFX96,实时系统,美国)。引物的序列如下:Sox2,正向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M2"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-GTCAGCGCCCTGCAGTACAA-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M3"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>和反向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M4"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-GCGAGTAGGACATGCTGTAGGTG-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M5"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>;Oct4、正向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M6"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-GACAACCATCTGCCGCTTC-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M7"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>和反向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M8"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-TCCTCCACCCACTTCTCCA-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M9"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>;Nanog,正向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M10"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-TGGACACTGGCTGAATCCTTC-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M11"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>和反向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M12"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-CGCTGATTAGGCTCCAACCAT-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M13"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>;和GADPH(内部控制),正向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M14"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-ACAGGGCTATCAGGGAGCA-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M15"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>和反向引物5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M16"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-GGAGCGAGATCCCTCCAAAAT-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M17"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ′</gydF4y2Bamml:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>。</gydF4y2Bap> </sec> <sec id="sec2.10"> <title>2.10。免疫印迹分析</tgydF4y2Baitle> <p>免疫印迹分析进行了如前所述[<gydF4y2Baxref ref-type="bibr" rid="B18"> 18</gydF4y2Baxref>]。短暂,bmsc 6-well板洗在cold-buffered PBS和细胞溶解与1毫米PMSF在冰里帕缓冲区。细胞溶解产物离心机(12000 rpm, 10分钟)在4°C,和上层清液的蛋白质转移到新管。蛋白质样品的浓度决定BCA蛋白试验设备(PC0020, Solarbio,北京)。一个20<gydF4y2Baitalic> μ</gydF4y2Baitalic>g总蛋白质样本解决使用12% sds - page和转移到PVDF膜。包含渐变的膜被封锁在Tris-buffered盐水20 5% BSA在室温下2 h。主抗体(LC3A / B, 50<gydF4y2Baitalic> μ</gydF4y2Baitalic>g / 50<gydF4y2Baitalic> μ</gydF4y2Baitalic>l, AF5402, anti-LC3A / B抗体,1:1000年,亲和力,美国;Abcam AMPK ab32047 1: 2500年,美国;Abcam p-AMPK ab133448 1: 5000年,美国;Abcam P-AKT ab81283 1: 7000年,美国;Abcam AKT ab8805 1: 500年,美国;Abcam mTOR ab2732 1: 2000年,美国;Abcam P-mTOR ab137133 1: 5000年,美国;和<gydF4y2Baitalic> β</gydF4y2Baitalic>肌动蛋白,66009 - 1 - lg, 1: 20000年,Proteintech集团一夜之间,美国)被孵化的膜在4°C。膜与辣根孵化peroxidase-conjugated anti-rabbit二级抗体(山羊anti-rabbit免疫球蛋白(合),Abcam, ab6721,美国),和蛋白质被增强化学发光检测(Beyotime、上海、中国)使用Amersham成像仪600(美国通用电气公司)。<gydF4y2Baitalic> β</gydF4y2Baitalic>肌动蛋白作为内部控制规范化装运材料。</gydF4y2Bap> </sec> <sec id="sec2.11"> <title>2.11。免疫荧光</tgydF4y2Baitle> <p>细胞固定化与4%多聚甲醛和permeabilized 1% triton x - 100 10分钟,其次是阻止2%牛血清白蛋白30分钟。主要的抗体,兔多克隆抗体(LC3A / B, 50<gydF4y2Baitalic> μ</gydF4y2Baitalic>g / 50<gydF4y2Baitalic> μ</gydF4y2Baitalic>l, AF5402, anti-LC3A / B抗体,1:100年,亲和力,美国),用于孵化一夜之间在4°C。然后,细胞被孵化二级抗体Alexa萤石647驴anti-rabbit (Abcam ab150075, 1: 200年,美国)远离光1小时。用DAPI之后,细胞被安装(1:1000年,32670 - 5 - mg - f,σ,美国)5分钟。Immunofluorescent图像捕获和分析用共焦显微镜(FV10-ASW3.1奥林巴斯,日本)。</gydF4y2Bap> </sec> <sec id="sec2.12"> <title>2.12。药物治疗</tgydF4y2Baitle> <p>AMPK抑制剂、复合维他命C,购买微孔(美国默克,Billerica的),和复合C的剂量是20<gydF4y2Baitalic> μ</gydF4y2Baitalic>Μ[<gydF4y2Baxref ref-type="bibr" rid="B19"> 19</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B20"> 20.</gydF4y2Baxref>]。C化合物添加到系统24小时后coculture 8天。aprepitant NK1受体拮抗剂,是购自Sigma-Aldrich (SML2215-5MG、上海、中国)。aprepitant的剂量是50<gydF4y2Baitalic> μ</gydF4y2Baitalic>Μ[<gydF4y2Baxref ref-type="bibr" rid="B21"> 21</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B22"> 22</gydF4y2Baxref>]。coculture 8天后,DRG、bmsc aprepitant 48小时处理。然后,bmsc收集进行以下分析。</gydF4y2Bap> </sec> <sec id="sec2.13"> <title>2.13。统计分析</tgydF4y2Baitle> <p>所有数据都表示为意味着±标准差和分析通过SPSS软件(版本13.0)。学生的团体之间的差异比较<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M18"> <mml:mi> t</gydF4y2Bamml:mi> </mml:math> </inline-formula>以及,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M19"> <mml:mi> P</gydF4y2Bamml:mi> </mml:math> </inline-formula>值小于0.05被认为是具有统计学意义。</gydF4y2Bap> </sec> </sec> <sec id="sec3"> <title>3所示。结果</tgydF4y2Baitle> <sec id="sec3.1"> <title>3.1。GFP鼠bmsc的表征</tgydF4y2Baitle> <p>bmsc似乎梭状形态与绿色荧光的古典形式(图<gydF4y2Baxref rid="fig1a" ref-type="fig"> 1(一)</gydF4y2Baxref>)。bmsc的multipotential分化是验证了茜素红染色(图<gydF4y2Baxref rid="fig1b" ref-type="fig"> 1 (b)</gydF4y2Baxref>),油红O染色(图<gydF4y2Baxref rid="fig1c" ref-type="fig"> 1 (c)</gydF4y2Baxref>),阿尔新蓝染色(图<gydF4y2Baxref rid="fig1d" ref-type="fig"> 1 (d)</gydF4y2Baxref>),这表明bmsc用于这个研究可以分化为成骨细胞、脂肪细胞、软骨细胞在诱导条件下。流式细胞仪分析表明,P3细胞用于实验有丰富的表达CD90(99.8%)和缺乏CD34(2.5%)的情况下,CD11b / c(1.5%),和CD45(2.3%)(图<gydF4y2Baxref rid="fig1e" ref-type="fig"> 1 (e)</gydF4y2Baxref>)。在一起,伴在这项研究中有多个分化潜力,bmsc的纯度是很高的,所以P3 bmsc可用于以下实验。</gydF4y2Bap> <fig-group id="fig1"> <label>图1</gydF4y2Balabel> <p>GFP bmsc的表征。(一)P3 GFP bmsc的形态。(b)茜素红染色的bmsc成骨诱导后14天。(c)油红染色后的bmsc脂肪形成的感应了10天。(d)阿尔新蓝染色后的bmsc chondrogenic归纳为20天。(e)代表P3 bmsc的流式细胞仪分析结果表明CD90丰富的表情和没有CD 34, CD11b / c和CD45。</gydF4y2Bap> <fig id="fig1a"> <label>(一)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.001a"></graphic> </fig> <fig id="fig1b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.001b"></graphic> </fig> <fig id="fig1c"> <label>(c)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.001c"></graphic> </fig> <fig id="fig1d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.001d"></graphic> </fig> <fig id="fig1e"> <label>(e)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.001e"></graphic> </fig> </fig-group> </sec> <sec id="sec3.2"> <title>3.2。Coculturing DRG推广扩散和增强Multipotential bmsc的分化</tgydF4y2Baitle> <p>在这里,我们分析的影响与DRG coculturing BMSC multipotential分化和增殖。我们发现coculturing DRG显著提升BMSC扩散和提高其成骨的脂肪形成的,chondrogenic分化。老鼠GFP-BMSCs coculture组显示类似成纤维细胞的形态与对照组(图<gydF4y2Baxref rid="fig2a" ref-type="fig"> 2(一个)</gydF4y2Baxref>)。然而,bmsc的密度在coculture组比对照组在不同的时间点(第三天,第五天,天8)(图<gydF4y2Baxref rid="fig2a" ref-type="fig"> 2(一个)</gydF4y2Baxref>)。评估两组的扩散,CCK8分析进行到第十天,和BMSC扩散曲线表明,BMSC coculturing DRG扩散更重要的是在每个时间点,特别是在每天6和8,而控制BMSC组(图<gydF4y2Baxref rid="fig2b" ref-type="fig"> 2 (b)</gydF4y2Baxref>)。所以我们选择bmsc coculture第八天分析诊断相关的影响在多个bmsc的分化。茜素红的结果,油红O,成骨的阿尔新蓝染色显示,脂肪形成的,和chondrogenic求同存异BMSC都增强DRG + BMSC组,而BMSC组(图<gydF4y2Baxref rid="fig2c" ref-type="fig"> 2 (c)</gydF4y2Baxref>)。这些发现表明coculture DRG不仅可以增强扩散但也促进bmsc的多个分化,这暗示coculture bmsc的DRG具备干细胞可能有影响。</gydF4y2Bap> <fig-group id="fig2"> <label>图2</gydF4y2Balabel> <p>Coculturing DRG推广扩散和增强multipotential bmsc的分化。(a)形态和密度的GFP bmsc天3,5,8。(b)增殖曲线后的bmsc coculture CCK8诊断相关的试验。(c)成骨、脂肪形成的和chondrogenic bmsc的分化有显著提升与DRG coculture后8天。</gydF4y2Bap> <fig id="fig2a"> <label>(一)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.002a"></graphic> </fig> <fig id="fig2b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.002b"></graphic> </fig> <fig id="fig2c"> <label>(c)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.002c"></graphic> </fig> </fig-group> </sec> <sec id="sec3.3"> <title>3.3。Coculturing DRG提升自我更新能力和bmsc的干细胞相关基因表达</tgydF4y2Baitle> <p>具备干细胞研究的影响与DRG coculturing bmsc, CFU化验,发现bmsc的自我更新能力,。bmsc在第8天使用和培养了10天。与BMSC组相比,更多的殖民地形成和殖民地地区大得多的DRG + BMSC集团(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M20"> <mml:mi> P</gydF4y2Bamml:mi> <mml:mo> <</gydF4y2Bamml:mo> <mml:mn> 0.05</gydF4y2Bamml:mn> </mml:math> </inline-formula>,图<gydF4y2Baxref rid="fig3a" ref-type="fig"> 3(一个)</gydF4y2Baxref>)。验证DRG对BMSC具备干细胞的影响,我们发现干细胞相关基因的表达,Nanog, Oct4、Sox2,和所有人都显著调节coculture 3后,5日和8天,尤其是在天5和8 (<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M21"> <mml:mi> P</gydF4y2Bamml:mi> <mml:mo> <</gydF4y2Bamml:mo> <mml:mn> 0.05</gydF4y2Bamml:mn> </mml:math> </inline-formula>,图<gydF4y2Baxref rid="fig3b" ref-type="fig"> 3 (b)</gydF4y2Baxref>),这符合BMSC扩散的结果。这些发现表明,coculture bmsc的DRG具备干细胞可以维持。</gydF4y2Bap> <fig-group id="fig3"> <label>图3</gydF4y2Balabel> <p>Coculturing DRG提升自我更新能力和bmsc的干细胞相关基因表达。(a) CFU bmsc的化验和bmsc +诊断相关组。紫色的点是指细胞殖民地。(b)干细胞相关基因表达在coculture 3, 5, 8天。<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M22"> <mml:mo> ∗</gydF4y2Bamml:mo> </mml:math> </inline-formula>表示之间的差异是显著bmsc + DRG、bmsc组。</gydF4y2Bap> <fig id="fig3a"> <label>(一)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.003a"></graphic> </fig> <fig id="fig3b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.003b"></graphic> </fig> </fig-group> </sec> <sec id="sec3.4"> <title>3.4。Coculturing DRG增强BMSC自噬</tgydF4y2Baitle> <p>被越来越多的证据表明,自噬中起关键作用的具备干细胞维护干细胞。验证自噬是否具备干细胞参与的过程维护的bmsc coculture集团,我们发现自噬标记的蛋白表达LC3II和LC3I通过免疫印迹和免疫荧光在第八天。bmsc coculture组显示强LC3荧光强度比对照组的bmsc(图<gydF4y2Baxref ref-type="fig" rid="fig4a"> 4(一)</gydF4y2Baxref>)。转换的可溶性LC3I lipid-bound LC3II自噬体形成的是一个指示器,所以LC3II / LC3I比率意味着自噬的激活水平。免疫印迹结果表明有更多LC3II coculture组蛋白表达和LC3II / LC3I比率为主要coculture组比对照组(图<gydF4y2Baxref ref-type="fig" rid="fig4b"> 4 (b)</gydF4y2Baxref>)。这些结果表明,自噬过程中被激活在bmsc coculture打钻。</gydF4y2Bap> <fig-group id="fig4"> <label>图4</gydF4y2Balabel> <p>Coculturing DRG增强bmsc自噬。(一)LC3 coculture 8天后免疫荧光图像。(b)的免疫印迹分析LC3II和LC3I LC3II / LC3I coculture 8天后比率分析。国防部:平均光密度。<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M23"> <mml:mo> ∗</gydF4y2Bamml:mo> </mml:math> </inline-formula>表示之间的差异是显著bmsc + DRG、bmsc组。</gydF4y2Bap> <fig id="fig4a"> <label>(一)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.004a"></graphic> </fig> <fig id="fig4b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.004b"></graphic> </fig> </fig-group> </sec> <sec id="sec3.5"> <title>3.5。AMPK Coculture组/ mTOR信号激活</tgydF4y2Baitle> <p>为了确定自噬的机制激活的bmsc coculture集团,我们下了两个经典信号通路参与了自噬的激活,包括AMPK / mTOR和AKT / mTOR信号通路,通过免疫印迹coculture后8天。mTOR的激活可以抑制自噬,磷酸化的AMPK抑制mTOR的激活,激活AMPK会促进自噬的水平。结果表明,蛋白质的表达p-AMPK coculture组显著调节相比,在对照组,两组之间虽然P-AKT保持不变的表情(图<gydF4y2Baxref rid="fig5" ref-type="fig"> 5</gydF4y2Baxref>)。这些结果提供的证据表明,自噬的激活bmsc coculture组可能是介导通过AMPK / mTOR信号而不是通过一种蛋白激酶/ mTOR信号。</gydF4y2Bap> <fig id="fig5"> <label>图5</gydF4y2Balabel> <p>AMPK信号通路,而不是一种蛋白激酶信号通路被激活后在bmsc coculture 8天。</gydF4y2Bap> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.005"></graphic> </fig> </sec> <sec id="sec3.6"> <title>3.6。复合C治疗Coculture组中表达下调自噬和干细胞相关基因</tgydF4y2Baitle> <p>进一步确认是否AMPK / mTOR信号介导的自噬和维护BMSC具备干细胞,我们对待BMSC复合C,一个AMPK-specific抑制剂,coculture组。与复合C治疗后8天,LC3II的蛋白表达和LC3I与coculture组相比明显减少没有复合C (<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M24"> <mml:mi> P</gydF4y2Bamml:mi> <mml:mo> <</gydF4y2Bamml:mo> <mml:mn> 0.05</gydF4y2Bamml:mn> </mml:math> </inline-formula>,数据<gydF4y2Baxref rid="fig6a" ref-type="fig"> 6(一)</gydF4y2Baxref>和<gydF4y2Baxref rid="fig6b" ref-type="fig"> 6 (b)</gydF4y2Baxref>)。这表明,AMPK / mTOR信号介导的自噬coculture bmsc的系统。此外,具备干细胞基因的mRNA水平,Sox2, Oct4, Nanog,主要是表达下调,但仍然高于对照组(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M25"> <mml:mi> P</gydF4y2Bamml:mi> <mml:mo> <</gydF4y2Bamml:mo> <mml:mn> 0.05</gydF4y2Bamml:mn> </mml:math> </inline-formula>,图<gydF4y2Baxref rid="fig6c" ref-type="fig"> 6 (c)</gydF4y2Baxref>)。这些数据表明,自噬由AMPK / mTOR信号具备干细胞积极参加BMSC维护coculture组,虽然不是整个部分,干细胞相关基因的表达在coculture +复合C组仍高于对照组。</gydF4y2Bap> <fig-group id="fig6"> <label>图6</gydF4y2Balabel> <p>复合C治疗coculture组中表达下调自噬和干细胞相关基因。(a)在治疗后免疫印迹分析LC3II和LC3I c化合物(b)分析LC3II / LC3I三组。(c)干细胞相关基因表达与复合治疗后c。<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M26"> <mml:mo> ∗</gydF4y2Bamml:mo> </mml:math> </inline-formula>表示,两组之间差异具有统计学意义。复合C本身没有改变bmsc的自噬水平或具备干细胞基因(无花果<gydF4y2Baxref ref-type="sec" rid="supplementary-material-1"> S1</gydF4y2Baxref>)。</gydF4y2Bap> <fig id="fig6a"> <label>(一)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.006a"></graphic> </fig> <fig id="fig6b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.006b"></graphic> </fig> <fig id="fig6c"> <label>(c)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.006c"></graphic> </fig> </fig-group> <p>确定因素在bmsc DRG-derived细胞的影响,DRG、bmsc cocultured 8天,然后是处理aprepitant, NK1受体拮抗剂,48 h 50的浓度<gydF4y2Baitalic> μ</gydF4y2Baitalic>Μ。收集bmsc, autophagy-related蛋白质LC3I和LC3II具备干细胞基因表达在三组,bmsc + DRG bmsc + DRG + aprepitant, bmsc,被检测到。我们发现autophagy-related蛋白质的表达和LC3II /我的比率下降后添加aprepitant coculture系统(数据<gydF4y2Baxref rid="fig7a" ref-type="fig"> 7(一)</gydF4y2Baxref>和<gydF4y2Baxref rid="fig7b" ref-type="fig"> 7 (b)</gydF4y2Baxref>),但自噬蛋白表达和LC3II /我的比例仍高于对照组(数字<gydF4y2Baxref rid="fig7a" ref-type="fig"> 7(一)</gydF4y2Baxref>和<gydF4y2Baxref rid="fig7b" ref-type="fig"> 7 (b)</gydF4y2Baxref>)。自噬水平相似,治疗后也具备干细胞基因表达下调与aprepitant coculture集团相比,但仍高于对照组(图<gydF4y2Baxref rid="fig7c" ref-type="fig"> 7 (c)</gydF4y2Baxref>)。这些结果表明aprepitant可以一定程度上阻止bmsc DRG-derived细胞的效应,这表明,P物质可能发挥重要作用的影响DRG-derived细胞在bmsc coculture系统。</gydF4y2Bap> <fig-group id="fig7"> <label>图7</gydF4y2Balabel> <p>aprepitant对自噬的影响和具备干细胞基因coculture系统。(一)免疫印迹分析LC3I和LC3II aprepitant治疗后。(b)分析LC3II / LC3I三组。(c)具备干细胞基因表达与aprepitant治疗后。<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M27"> <mml:mo> ∗</gydF4y2Bamml:mo> </mml:math> </inline-formula>表示,两组之间差异具有统计学意义。</gydF4y2Bap> <fig id="fig7a"> <label>(一)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.007a"></graphic> </fig> <fig id="fig7b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.007b"></graphic> </fig> <fig id="fig7c"> <label>(c)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.fig.007c"></graphic> </fig> </fig-group> </sec> </sec> <sec id="sec4"> <title>4所示。讨论</tgydF4y2Baitle> <p>多项研究表明,体内和体外,探讨了不同角色的感觉神经在骨生理学<gydF4y2Baxref ref-type="bibr" rid="B6"> 6</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B10"> 10</gydF4y2Baxref>]。然而,直接影响骨的感觉神经细胞在细胞水平上,其内在的分子机制仍不清楚。为了调查的总体效应bmsc的感觉神经细胞的生物学行为,我们设计了一个transwell coculture DRG、bmsc之间的系统,考虑到之间没有直接接触DRG、bmsc的体内。我们发现按可以促进bmsc的增殖和自我更新能力。更重要的是,multipotential分化的bmsc coculture组更有意义。因为自我更新能力和多分化潜能的干细胞的两个主要特征(<gydF4y2Baxref ref-type="bibr" rid="B23"> 23</gydF4y2Baxref>),我们假设coculture bmsc的DRG具备干细胞可能有助于保持。接下来,我们发现干细胞相关基因,Sox2, Nanog, Oct4、调节coculture组,进一步证实了诊断相关的角色在这个coculture具备干细胞bmsc的维护系统。</gydF4y2Bap> <p>据报道,具备干细胞维持干细胞与自噬(有密切关系<gydF4y2Baxref ref-type="bibr" rid="B24"> 24</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B27"> 27</gydF4y2Baxref>]。自噬是一个自甘堕落的蜂窝组件的过程中,双层膜自噬小体隔离细胞器或部分细胞溶质和与溶酶体融合或液泡破裂的居民水解酶(<gydF4y2Baxref ref-type="bibr" rid="B28"> 28</gydF4y2Baxref>]。自噬的机制具备干细胞维持在许多研究研究。他们认为自噬帮助维持具备干细胞通过清算抑制干细胞新陈代谢活跃,健康的线粒体(<gydF4y2Baxref ref-type="bibr" rid="B25"> 25</gydF4y2Baxref>),清除有毒细胞废物(<gydF4y2Baxref ref-type="bibr" rid="B26"> 26</gydF4y2Baxref>),和防止活性氧(ROS)积累(<gydF4y2Baxref ref-type="bibr" rid="B27"> 27</gydF4y2Baxref>]。在自噬过程中,据报道,LC3是一个著名的自噬小体标记,和LC3II的比例/ LC3I反映自噬的水平(<gydF4y2Baxref ref-type="bibr" rid="B26"> 26</gydF4y2Baxref>]。在我们的研究中,证实自噬是否发挥了作用在维护具备干细胞coculture bmsc的系统,发现LC3的表达和LC3II / LC3I的比例进行了分析。我们的结果证明了基底自噬水平coculture组高于对照组,但我们仍然不能认为自噬水平越高导致bmsc的维护。</gydF4y2Bap> <p>坐骑的证据显示信号调节自噬的<gydF4y2Baxref ref-type="bibr" rid="B29"> 29日</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B30"> 30.</gydF4y2Baxref>),其中AMPK / mTOR和AKT / mTOR研究最多有两个途径(<gydF4y2Baxref ref-type="bibr" rid="B31"> 31日</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B34"> 34</gydF4y2Baxref>]。我们研究了AMPK的蛋白表达,一种蛋白激酶,并在第八天mTOR coculture期间,我们发现,与对照组相比,p-AMPK调节和mTOR表达下调,而AKT持平coculture组。因此,AMPK / mTOR信号介导的自噬增强。更重要的是,化合物C, AMPK抑制剂(<gydF4y2Baxref ref-type="bibr" rid="B35"> 35</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B36"> 36</gydF4y2Baxref>),自噬和干细胞相关基因的表达下调coculture集团具备干细胞的证明自噬维持bmsc coculture组由AMPK / mTOR通路。此外,具备干细胞基因复合C组,尽管低于DRG coculture集团仍高于BMSC单作集团,这可能表明存在其他机制具备干细胞维护coculture组除了增强的自噬。</gydF4y2Bap> <p>为了进一步确定的因素按作用于bmsc,两大感觉神经肽SP和CGRP怎样考虑。然而,根据一些研究CGRP怎样受体不仅存在于bmsc也在DRG、雪旺细胞、和CGRP怎样可以发挥重要作用在雪旺细胞的功能和按<gydF4y2Baxref ref-type="bibr" rid="B37"> 37</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B39"> 39</gydF4y2Baxref>]。因此,coculture系统,如果CGRP怎样添加受体抑制剂,它不仅会阻止bmsc的CGRP怎样受体,但也阻止CGRP怎样雪旺细胞受体和DRG神经元,这将完全改变原来的系统,使结果很难分析。因为我们并没有发现对DRG、雪旺细胞SP有任何影响,我们使用了NK1受体拮抗剂,aprepitant [<gydF4y2Baxref ref-type="bibr" rid="B21"> 21</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B22"> 22</gydF4y2Baxref>),探讨SP在这个coculture系统的作用。</gydF4y2Bap> <p>在几项研究中,coculture系统已被用于研究神经元之间的通信和其他细胞(<gydF4y2Baxref ref-type="bibr" rid="B16"> 16</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B40"> 40</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B43"> 43</gydF4y2Baxref>]。据报道,DRG能促进成骨细胞的增殖分化bmsc和成骨的直接接触条件下的基因表达(<gydF4y2Baxref ref-type="bibr" rid="B40"> 40</gydF4y2Baxref>]。和感觉神经元可以调节MC3T3-E1细胞通过胞外分泌的谷氨酸和P物质,并证实肽能的神经元参与了这一过程(<gydF4y2Baxref ref-type="bibr" rid="B43"> 43</gydF4y2Baxref>]。最近,席尔瓦et al。<gydF4y2Baxref ref-type="bibr" rid="B41"> 41</gydF4y2Baxref>)设计了一个微流控设备,只有从DRG神经元神经突达到了msc对bmsc研究感觉神经元的直接影响。他们发现DRG神经元增强成骨分化的通过激活Wnt / msc<gydF4y2Baitalic> β</gydF4y2Baitalic>连环蛋白信号通路,证明upregulation成骨基因和细胞质的积累和易位的细胞核<gydF4y2Baitalic> β</gydF4y2Baitalic>在bmsc连环蛋白,但DRG具备干细胞被发现有能力维护的bmsc coculture系统在我们的研究中。似乎与我们的结果不同。然而,研究培养的bmsc的地塞米松,抗坏血酸<gydF4y2Baitalic> β</gydF4y2Baitalic>甘油磷酸,从而诱导成骨分化本身没有其他因素(<gydF4y2Baxref ref-type="bibr" rid="B44"> 44</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B45"> 45</gydF4y2Baxref>),而我们在研究中没有添加一个成骨诱导物。也没有差别在成骨细胞标记基因在mono -和coculture团体没有成骨的感应中,这意味着DRG神经元的影响本身并不足以诱导osteoblastogenesis体外。他们得出的结论是,DRG只能提高成骨分化成骨诱导介质的存在,但不能启动它。这一发现是在符合我们的结果。在我们的研究中,促进骨生成的机制是,transwell coculture DRG帮助保持bmsc的具备干细胞;然后,成骨诱导介质将显示一个更重要的对成骨分化的影响。</gydF4y2Bap> <p>据报道,在离体培养bmsc的具备干细胞受损<gydF4y2Baxref ref-type="bibr" rid="B46"> 46</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B47"> 47</gydF4y2Baxref>),导致受损的自我更新和multilineage分化能力,这是不利于他们的作为骨组织工程的种子细胞。此外,BMSC具备干细胞维护机制的增强基底自噬水平在我们的研究中。这表明自噬的温和的增强可能是有益的文化干细胞体外,清除受损细胞的组件和维持细胞内稳态的<gydF4y2Baxref ref-type="bibr" rid="B48"> 48</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B49"> 49</gydF4y2Baxref>]。此外,自噬的强度可以由某些药物,如雷帕霉素(<gydF4y2Baxref ref-type="bibr" rid="B50"> 50</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B51"> 51</gydF4y2Baxref>)和bafilomycin A1 (<gydF4y2Baxref ref-type="bibr" rid="B52"> 52</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B53"> 53</gydF4y2Baxref>]。bmsc以来被广泛应用于骨组织工程种子细胞(<gydF4y2Baxref ref-type="bibr" rid="B54"> 54</gydF4y2Baxref>),我们的研究可能提供一个新的策略来扩大bmsc在体外通过调节自噬强度没有损害他们的自我更新和多潜能分化能力。</gydF4y2Bap> <p>应该注意,DRG细胞感觉神经元和雪旺细胞包含在我们的实验中,尽管DRG外植体被分解和消化。这两个组件是类似于感觉神经在体内,在那里感觉缠绕的雪旺细胞(神经突<gydF4y2Baxref ref-type="bibr" rid="B55"> 55</gydF4y2Baxref>]。因此,具备干细胞增强实验中观察到的现象应该考虑感觉神经元和雪旺细胞的合成效果。在另一项研究中,发现雪旺细胞分泌细胞外囊泡促进和维护人类牙髓细胞的增殖和multipotency (hDPCs),通过蛋白质组学和免疫印迹分析,他们丰富的Oct4浓缩和TGF信号被探测到<gydF4y2Baitalic> β</gydF4y2Baitalic>年代的雪旺细胞衍生细胞外囊泡,这解释了upregulation扩散的干细胞相关基因和加速度hDPCs [<gydF4y2Baxref ref-type="bibr" rid="B17"> 17</gydF4y2Baxref>]。与bmsc hDPCs表现出共同的特征在许多方面,这一结果可能部分解释的bmsc具备干细胞增强coculture系统包含在我们研究雪旺细胞。同时,这也可能解释为什么具备干细胞增强bmsc仍然存在即使阻断自噬与coculture复合C组。</gydF4y2Bap> <p>在我们的研究中,我们使用一个transwell coculture系统探索的影响DRG bmsc,模仿植入感觉神经和bmsc TEB。我们发现DRG可以帮助保持bmsc的具备干细胞体外,在从DRG神经元分泌P物质的过程中发挥了重要作用。基于这一发现,它可能是假设的感觉神经植入组织工程骨的自噬增强bmsc,帮助保持其具备干细胞,从而保持自我更新和multilineage bmsc的分化能力。因此,bmsc更准备增殖并分化成其他类型的细胞,如成骨细胞、软骨细胞、血管内皮细胞、雪旺细胞,诱导条件下(<gydF4y2Baxref ref-type="bibr" rid="B56"> 56</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B58"> 58</gydF4y2Baxref>]。这些细胞都是骨修复和再生的关键。此外,伴着强大的旁分泌功能,包括生长因子和神经营养因子<gydF4y2Baxref ref-type="bibr" rid="B59"> 59</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B61"> 61年</gydF4y2Baxref>]。和维护具备干细胞保持这种能力是很重要的。此外,本研究提出了一个新的视角对于理解神经调节的骨头,但它仍然需要进一步研究有关的因素,除了P物质,在DRG行动bmsc bmsc以及它们如何工作。</gydF4y2Bap> </sec> <sec id="sec5"> <title>5。结论</tgydF4y2Baitle> <p>DRG中有助于维护bmsc的具备干细胞transwell coculture系统,和这个函数是通过增强的自噬水平BSMCs通过AMPK / mTOR信号,这也许可以解释感觉神经促进骨生成的机制,和P物质在这个过程中起着重要的作用。</gydF4y2Bap> </sec> <back> <sec sec-type="data-availability"> <title>数据可用性</tgydF4y2Baitle> <p>使用的数据来支持本研究的发现可以从相应的作者。</gydF4y2Bap> </sec> <sec> <title>信息披露</tgydF4y2Baitle> <p>江Shuaishuai张、李黄俊钦Huijie共同第一作者。</gydF4y2Bap> </sec> <sec> <title>的利益冲突</tgydF4y2Baitle> <p>作者宣称没有利益冲突有关的出版。</gydF4y2Bap> </sec> <sec> <title>作者的贡献</tgydF4y2Baitle> <p>刘洋,Guoxian裴,王纯美少女,Shuaishuai张设计实验。Shuaishuai张黄俊钦,易高,和江Huijie CCK8和分化诱导实验。专业Shuaishuai张Pengzhen Cheng曹,冀萌当初王执行rt - pcr和免疫印迹实验。刘曰歌,本,吴皓执行主BMSC文化实验。张Shuaishuai收集数据。刘洋,Shuaishuai张,黄俊钦李生成的数据和数据分析。Shuaishuai张,刘洋和Guoxian裴写道,修订后的手稿。</gydF4y2Bap> </sec> <ack> <title>确认</tgydF4y2Baitle> <p>本研究在经济上支持的中国国家自然科学基金重点项目(81430049和81430049)。</gydF4y2Bap> </ack> <sec sec-type="supplementary-material" id="supplementary-material-1"> <title>补充材料</tgydF4y2Baitle> <supplementary-material id="supp-1" xlink:href="//www.newsama.com/downloads/journals/sci/2018/8478953.f1.docx" mimetype="application/msword"> <label>补充材料</gydF4y2Balabel> <p>图S1:复合C单独并没有改变bmsc的自噬水平或具备干细胞基因。(一)蛋白表达LC3I LC3II。(b)分析LC3II / LC3I两组之间。(c)干细胞相关基因表达在两组之间。</gydF4y2Bap> </supplementary-material> </sec> <ref-list> <ref id="B1" content-type="article"> <label>1</gydF4y2Balabel> <element-citation publication-type="journal"> <person-group person-group-type="author"> <name> <surname> Dumic-Cule</gydF4y2Basurname> <given-names> 我。</ggydF4y2Baiven-names> </name> <name> <surname> Pecina</gydF4y2Basurname> <given-names> M。</ggydF4y2Baiven-names> </name> <name> 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