SCI. 干细胞国际 1687 - 9678 1687 - 966 x Hindawi 10.1155 / 2021/8819884 8819884. 研究文章 人脱落乳牙干细胞减轻大鼠三叉神经痛 小峰 1 2 Xuedi 2 3. 2 3. 2 4 Xuemei 2 4 扇子 2 5 https://orcid.org/0000-0002-7996-4843 2 3. 巴利尼 andrea. 1 口腔颌面外科 医院口腔科 中国医科大学 117 N 南京街 沉阳 中国 cmu.edu.cn 2 辽宁省口腔疾病重点实验室 117 N 南京街 沉阳 中国 3. 美国麻醉学 医院口腔科 中国医科大学 117 N 南京街 沉阳 中国 cmu.edu.cn 4 儿科牙科学系 医院口腔科 中国医科大学 117 N 南京街 沉阳 中国 cmu.edu.cn 5 病理学系 医院口腔科 中国医科大学 117 N 南京街 沉阳 中国 cmu.edu.cn 2021 18 1 2021 2021 5 5 2020 1 1 2021 7 1 2021 18 1 2021 2021 版权所有©2021白晓峰等。 这是在Creative Commons归因许可下分发的开放式访问文章,其允许在任何介质中不受限制地使用,分发和再现,只要正确引用了原始工作。

三叉神经痛是一种可治区的渐进神经系统疾病,可持续数月或数年。来自人剥落的落叶牙齿(SHED)的干细胞是用于细胞疗法的候选来源。由于它们的神经保护和免疫调节作用,这些神经嵴细胞在介导慢性疼痛方面具有潜在的作用。在这项研究中,我们建立了对眶下神经(CCI-离子)的慢性收缩损伤的大鼠模型,评价血管性疼痛中血液的镇痛作用。研究了局部血液移植对三叉神经中炎症细胞浸润的影响是基于苏木精和曙红染色。定量了在三叉神经和神经节中受伤神经和短暂受体潜在的香草型1(TRPV1)表达的促炎因子的水平。该数据显示,系统性或局部注射SHED在神经损伤后测量大鼠对机械刺激的敏感性,并且这种效果持续到8周的观察期。PKH26标记的血液分布到局部注射后的同侧三叉神经神经枝24和72小时。病变部位的血液移植导致受伤神经中的炎症细胞浸润和促炎细胞因子水平降低,抑制早期三阶段的三叉神经和神经节中的CCI离子诱导的TRPV1表达的上调。因此,这些结果提供了突出的证据,这些证据支持使用Shed治疗三叉神经痛和可能其他慢性疼痛病症。

辽宁省自然科学基金 201602811. 20180530097 国家自然科学基金项目 81400530
1.介绍</gydF4y2Batitle> <p>三叉神经痛是由三叉神经的病变或伤害引起的,并且可以持续数月或数年。典型的三叉神经痛的特点是单侧,驯化,自发或可引发的,通常是休克的面部疼痛,无痛的间隔。三叉神经痛是一种进步神经系统疾病[<gydF4y2Baxref ref-type="bibr" rid="B1"> 1</gydF4y2Baxref>].三叉神经痛的治疗是一项临床挑战,因为其发病机制非常复杂,涉及神经系统的结构和神经生理变化。</p><p>gydF4y2Ba人类脱落乳牙干细胞(SHED)是通过非侵入性程序从自然脱落的乳牙中收集[<gydF4y2Baxref ref-type="bibr" rid="B2"> 2</gydF4y2Baxref>].SHED具有分化为各种类型细胞的能力,包括神经细胞、成骨细胞和成脂细胞[<gydF4y2Baxref ref-type="bibr" rid="B3"> 3.</gydF4y2Baxref>].例如,在患者和动物移植到受伤的牙齿后,SHED已经被证明可以用血管和神经再生整个牙髓[<gydF4y2Baxref ref-type="bibr" rid="B4"> 4</gydF4y2Baxref>].作为神经嵴衍生的干细胞,Shed表达各种神经细胞标志物,例如谷氨酸脱羧酶,Neun,Nestin,胶质纤维酸性酸性蛋白,<gydF4y2Baitalic> β</gydF4y2Baitalic>iii -微管蛋白和神经丝M [<gydF4y2Baxref ref-type="bibr" rid="B2"> 2</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B5"> 5</gydF4y2Baxref>].Shed可以分化为多巴胺能神经元样细胞<gydF4y2Baitalic> 在体外</gydF4y2Baitalic>,血液移植部分地改善了帕金森病的大鼠模型中的仲素诱导的旋转行为[<gydF4y2Baxref ref-type="bibr" rid="B6"> 6</gydF4y2Baxref>].在脊髓损伤的大鼠模型中,显示出脱落释放关键的神经营养因子,抑制星形胶质细胞和神经元的凋亡,促进机车恢复[<gydF4y2Baxref ref-type="bibr" rid="B7"> 7</gydF4y2Baxref>].这些证据表明,SHED可能是一种有吸引力的神经再生治疗的细胞来源。此外,SHED具有优越的免疫调节特性,可降低免疫疾病(如系统性红斑狼疮(SLE))中促炎细胞因子的水平[<gydF4y2Baxref ref-type="bibr" rid="B8"> 8</gydF4y2Baxref>].免疫系统与神经系统相互作用以调节疼痛敏感性。作为神经嵴细胞,除了具有神经保护和免疫调节的特性外,SHED还可能在介导慢性疼痛中发挥作用。然而,关于SHED潜在的镇痛作用的信息很少。</p><p>gydF4y2Ba瞬态受体电位香草醛1型(TRPV1)是一种瞬态受体电位家族的阳离子通道成员,可被热、酸和配体(如辣椒素)激活。在动物和人类中,TRPV1定位于外周和中枢神经系统,并已被证明在介导神经病理性疼痛、内脏疼痛、炎症性疼痛和其他形式的异位性疼痛中发挥关键作用[<gydF4y2Baxref ref-type="bibr" rid="B9"> 9</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B11"> 11</gydF4y2Baxref>].例如,在牙齿运动疼痛后的三叉甘氨酸中的TRPV1表达升高,并且报告的TRPV1拮抗剂缓解了大鼠的正畸疼痛[<gydF4y2Baxref ref-type="bibr" rid="B12"> 12</gydF4y2Baxref>].</p><p>gydF4y2Ba本实验通过诱导眶下神经慢性缩窄损伤(CCI-ION)建立大鼠三叉神经性疼痛模型,评价SHED对神经性疼痛的影响。我们的目的是提供证据,表明SHED提高三叉神经痛大鼠的机械阈值。此外,我们还研究了SHED的镇痛作用是否参与炎症细胞浸润和调节促炎细胞因子(如肿瘤坏死因子- (TNF-))的水平。<gydF4y2Baitalic> α</gydF4y2Baitalic>白细胞介素- (IL-) 1<gydF4y2Baitalic> β</gydF4y2Baitalic>.进一步,我们证实了SHED的长期抗痛觉作用与三叉神经和三叉神经节(TG)中TRPV1的表达有关。</p></gydF4y2Basec> <sec id="sec2"> <title>2.材料和方法</gydF4y2Batitle> <sec id="sec2.1"> <title>2.1.SHED隔离与培养</gydF4y2Batitle> <p>根据机构审查委员会的指导方针,在中国医科大学口腔医院从6- 8岁儿童身上获得了脱落的人类乳牙作为丢弃的生物样本。获得了孩子父母的知情同意。将牙髓从脱落的牙齿中分离出来,用4mg /mL (Boehringer Ingelheim, Mannheim, Germany)和3mg /mL胶原酶类型的溶液消化<gydF4y2Baitalic> Ι</gydF4y2Baitalic>(沃辛顿生物化学有限公司,Lakewood,Co,USA)在37°C持续30分钟。通过通过70通过摘要来获得单细胞悬浮液 <italic> μ</gydF4y2Baitalic>m过滤器(Merck Millipore Ltd.,科克,爱尔兰)。细胞在鹰的培养基(HyClone, Logan, UT, USA)、15%胎牛血清(ExCell Bio,上海,中国)、100 mmol/L L-抗坏血酸(Sigma-Aldrich, St. Louis, MO, USA)和100 U/mL青霉素链霉素(HyClone)的α修饰中培养,37℃,5% CO存在<gydF4y2Basub>2</gydF4y2Basub>.实验使用了第3-5代的SHED。采用无血清培养基作为细胞移植悬浮培养基。</p></gydF4y2Basec> <sec id="sec2.2"> <title>2.2.表面标记表达分析</gydF4y2Batitle> <p>采用流式细胞仪(BD FACSAria II, Becton-Dickinson, Islandia, NY, USA)检测细胞表面标记物。使用的一抗(抗cd73、抗cd105、抗cd34和抗cd45)购自BD生物科学公司(Franklin Lakes, NJ, USA)。干细胞被调整到<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 6</米米l:mn> </mml:mrow> </mml:msup> <mml:mo> /</米米l:mo> <mml:mtext> 毫克</米米l:mtext> </mml:math> </inline-formula>并与各自的一抗在冰上孵育30分钟。</p></gydF4y2Basec> <sec id="sec2.3"> <title>2.3.<italic>In Vitro</italic> Neurogenic and Osteogenic Differentiation assay</gydF4y2Batitle> <p>根据以前的一份报告,可诱导神经源性和成骨性分化[<gydF4y2Baxref ref-type="bibr" rid="B2"> 2</gydF4y2Baxref>].对于神经肝分化,细胞在含有B27补充剂的培养基中培养,20ng / ml碱性成纤维细胞生长因子,20ng / ml表皮生长因子和1%青霉素。培养4周后,将梭在4%甲醛中固定,并分析了神经细胞标志物的表达<gydF4y2Baitalic> β</gydF4y2Baitalic>iii -微管蛋白免疫细胞荧光染色(Sigma-Aldrich)。细胞核染色为4<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> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>,6-二氨基-2-苯基吲哚(DAPI),大连美伦生物技术有限公司,中国大连成骨分化时,将SHED培养至80%汇合,然后在添加10 nM地塞米松和1.8 mM磷酸二氢钾的培养基中培养。诱导4周后,在立体显微镜下用1%茜素红S染色评价矿化结节的形成。</p></gydF4y2Basec> <sec id="sec2.4"> <title>2.4。动物,疼痛模型建立和棚移植</gydF4y2Batitle> <p>雄性Sprague-Dawley大鼠(8周龄,200-250克)被安置在温度控制的房间,可免费获得食物和水。通过使用内部方法通过CCI离子建立三叉神经病疼痛模型[<gydF4y2Baxref ref-type="bibr" rid="B13"> 13</gydF4y2Baxref>].在手术期间,将这些动物用戊巴比妥钠(50mg / kg,腹膜内)麻醉。头部固定在仰卧位的桌子上。粘膜切口沿着左上颌牙龈沟缘。用两个铬肠(4.0)连续树脂松散地连接离子。在恶意大鼠中,单侧眶下神经暴露而不结扎。所有实验均经中国医科大学动物研究伦理委员会(G2019008)批准。</p><p>神经连接后三天,用低剂量注射大鼠一次(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M3"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>细胞,0.2毫升,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M4"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>大鼠,CCI-离子+低剂量injshed组)或高剂量(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M5"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 6</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>细胞,0.2毫升,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M6"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>大鼠,CCI-离子+高剂量injshed组)脱落位点。CCI-ION + INFSHED组的大鼠接受一次输注<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M7"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>棚(0.2毫升,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M8"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>)。对照组大鼠局部注射无血清培养液(CCI-ION+injMedium)或静脉注射无血清培养液(CCI-ION+infMedium)。戊巴比妥钠(70 mg/kg,腹腔注射)过量杀死所有动物。动物经升主动脉灌注生理盐水,然后再灌注4%甲醛。</p></gydF4y2Basec> <sec id="sec2.5"> <title>2.5.行为测试</gydF4y2Batitle> <p>机械阈值分别在神经结扎或给药后3、7、14、28和56天测定,并由同一研究人员在盲条件下进行评估。一系列校准的von Frey毛发,其屈曲重量在2g到15g之间,被应用到离子上方的皮肤上,并施加足够的压力来弯曲灯丝。头部主动从von Frey头发中撤退被定义为积极响应。每根von Frey纤维以几秒为间隔涂敷5次。当大鼠对同一纤维显示5个阳性反应时,这个过程就完成了。计算von Frey灯丝力范围内的响应频率([正响应次数/总刺激次数]× 100%),得到刺激-响应频率曲线(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M9"> <mml:mi> 年代</米米l:mi> </mml:math> </inline-formula>-<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M10"> <mml:mi> R</米米l:mi> </mml:math> </inline-formula>绘制曲线)。我们使用EF50值,定义为von Frey灯丝力(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M11"> <mml:mi> g</米米l:mi> </mml:math> </inline-formula>)从刺激响应频率曲线产生50%的响应频率,作为机械阈值的量度。ef50的减少表明存在机械异常脑[<gydF4y2Baxref ref-type="bibr" rid="B14"> 14</gydF4y2Baxref>].</p></gydF4y2Basec> <sec id="sec2.6"> <title>2.6。流分布</gydF4y2Batitle> <p>根据制造商的说明,Shed标有PKH26红色荧光细胞连接迷你套件(Sigma-Aldrich,ST.Louis,Mo,Mo)。简而言之,1ml稀释剂c和4 <italic> μ</gydF4y2Baitalic>在细胞微球中加入L PKH26染料,室温孵育5min。用5ml血清停止反应。染色细胞以1200转/分离心5分钟后注射(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M12"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 6</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>细胞,0.2毫升,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M13"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>在左离子连接后三天内的大鼠)。截面移植后,收集左TG,三叉脊髓亚核脊髓灰杆菌(vc / C2),rostral envermedial medulla(rvm),心脏,肺,肝,肾,脾和肠道.将组织固定在4%甲醛中2天,然后用15%和30%葡萄糖脱水,切成10 <italic> μ</gydF4y2Baitalic>片。DAPI染色(1<gydF4y2Baitalic> μ</gydF4y2Baitalic>l / ml)室温下15分钟。用LSM 510共聚焦激光扫描显微镜(德国Carl Zeiss)分析PKH26(红色)和DAPI(蓝色)荧光信号。</p></gydF4y2Basec> <sec id="sec2.7"> <title>2.7。苏木精伊红(HE)染色</gydF4y2Batitle> <p>采用HE染色法观察SHED对损伤神经炎性细胞浸润的影响。动物在SHED移植后7天处死。sham大鼠和CCI-ION大鼠有无SHED移植的三叉神经石蜡包埋切至10微米厚度,100%乙醇脱水2次10分钟,用水冲洗。然后用Harris苏木精溶液(Baso, China Zhuhai)染色3分钟,用水冲洗。10%的乙酸和85%的乙醇用于组织分化。然后用伊红Y乙醇溶液(Baso)染色2 min。用95%乙醇和100%乙醇分别脱水两次。提取二甲苯两种变化的乙醇后,用覆膜覆盖切片,在光学显微镜下观察成像(Olympus SZX12;奥林匹斯山,日本东京)。</p></sec> <sec id="sec2.8"> <title>2.8。定量逆转录(RT-q) PCR</gydF4y2Batitle> <p>动物在移植后7天和56天处死。mRNA水平<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>采用Stratagene/SYBR green Supermix系统进行RT-qPCR定量。我们使用PrimeScript RT试剂盒与gDNA橡皮防止基因组DNA污染。用TRIzol试剂(Invitrogen, Carlsbad, CA, USA)从损伤神经中提取总RNA,用RNeasy试剂盒(Qiagen, MD, USA)纯化。从1<gydF4y2Baitalic> μ</gydF4y2Baitalic>g RNA,使用SuperScript II (Invitrogen, Carlsbad, CA, USA)和2.5 ng随机引物。PCR程序如下:94℃5 min, 95℃3 min, 95℃30 s, 58℃45 s, 68℃20 s共30个循环。<gydF4y2Baitalic> β</gydF4y2Baitalic>-肌动蛋白被用作常规的管家基因。使用的引物如下:<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>5、前进<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> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-CCC CGA CTA TGT GCT CCT CAC-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> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>和反向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> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-ggg CTC TTG atg GCG ga-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> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>;<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>5、前进<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M18"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-gga agg cag TGT cac tca TTG tg-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M19"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>和反向5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M20"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-ggt CCT cat CCT gga agc tcc-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M21"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>;和<gydF4y2Baitalic> β肌动蛋白</gydF4y2Baitalic>(家务正常化基因),向前5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M22"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-ggt cca CCG cca cca g-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M23"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>和反向5<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M24"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>-cag GTC cag acg cag gat gg-3<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M25"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> ”</米米l:mo> </mml:mrow> </mml:msup> </mml:math> </inline-formula>.所有PCR均以三份运行。使用比较CT方法计算相对mRNA水平(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M26"> <mml:mi> Δ</米米l:mi> <mml:mi> Δ</米米l:mi> <mml:mtext> CT</米米l:mtext> </mml:math> </inline-formula>方法)。</p></gydF4y2Basec> <sec id="sec2.9"> <title>2.9。免疫组织化学染色</gydF4y2Batitle> <p>动物在SHED移植后7天处死。灌注后,收集左侧TG和三叉神经,用4%多聚甲醛固定过夜。将组织于10<gydF4y2Baitalic> μ</gydF4y2Baitalic>m.采用常规免疫组化方法,首先使用抗TRPV1的一抗(1:10 0,Neuromics, Edina, MN, USA),然后使用cy -3标记的山羊抗兔抗血清(1:25 0,Jackson ImmunoResearch, West Grove, PA, USA)。从6个TGs的每个神经节的6个代表性切片中计数trpv1阳性细胞。只评估细胞核清晰的标记神经元。使用ImageJ软件(美国国立卫生研究院,MA, USA)分析三叉神经TRPV1染色强度。</p></gydF4y2Basec> <sec id="sec2.10"> <title>2.10。免疫印迹</gydF4y2Batitle> <p>动物在SHED移植后7天处死。从左Tg和Sham大鼠的三叉神经中提取总蛋白质,没有血液移植的CCI离子大鼠,以及具有血液移植的CCI离子大鼠。用组织蛋白质提取试剂(Weiao Biotech,Shanghai,China)提取蛋白质和50 <italic> μ</gydF4y2Baitalic>在聚丙烯酰胺凝胶上运行每种样品的G转移到硝酸纤维素膜中,在室温下封闭(含有5%脂肪干乳的TWEEN(TBST)的TRIS缓冲盐水),并在4℃下孵育过夜用针对TRPV1的抗体(1:400; SANTA CRUZ)。洗涤膜并用辣根过氧化物酶 - 缀合的二抗(Santa Cruz生物技术)在37℃下孵育2小时。使用ECL(Western Lightning,USA)检测免疫反应性。TRPV1的蛋白质水平被标准化为<gydF4y2Baitalic> β</gydF4y2Baitalic>-肌动蛋白。</p></gydF4y2Basec> <sec id="sec2.11"> <title>2.11。统计分析</gydF4y2Batitle> <p>所有数据都表示为<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M27"> <mml:mtext> 的意思是</米米l:mtext> <mml:mo> ±</米米l:mo> <mml:mtext> 标准</米米l:mtext> <mml:mtext> </mml:mtext> <mml:mtext> 错误</米米l:mtext> <mml:mtext> </mml:mtext> <mml:mtext> 的</米米l:mtext> <mml:mtext> </mml:mtext> <mml:mtext> 的</米米l:mtext> <mml:mtext> </mml:mtext> <mml:mtext> 的意思是</米米l:mtext> </mml:math> </inline-formula>(SEM)。机械阈值数据采用双向重复测量方差分析,然后采用Bonferroni事后检验。采用单因素方差分析比较三组数据。显著性水平在<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M28"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>.</p></gydF4y2Basec> </sec> <sec id="sec3"> <title>3.结果</gydF4y2Batitle> <sec id="sec3.1"> <title>3.1。移植研究中使用的分离棚的特征</gydF4y2Batitle> <p>流式细胞分析显示,培养的SHED中有99.27%和80.23%的CD73阳性(图)<gydF4y2Baxref rid="fig1a" ref-type="fig"> 1(一)</gydF4y2Baxref>和CD105(图<gydF4y2Baxref rid="fig1c" ref-type="fig"> 1 (c)</gydF4y2Baxref>),分别。只有2.35%和0.56%的细胞CD34阳性(图<gydF4y2Baxref rid="fig1d" ref-type="fig"> 1 (d)</gydF4y2Baxref>)和CD45(图<gydF4y2Baxref rid="fig1e" ref-type="fig"> 1 (e)</gydF4y2Baxref>),分别。这些结果表明,SHED表达一套间充质干细胞标记物,而不表达造血干细胞标记物。培养的SHED大多为梭形(图)<gydF4y2Baxref rid="fig1f" ref-type="fig"> 1 (f)</gydF4y2Baxref>).</p><gydF4y2Bafig-group id="fig1"> <label>图1</gydF4y2Balabel> <p>从人剥离落叶齿中分离的干细胞的表征(SHED)。显示了流血清胞流分析的结果。(A)同种型匹配的IgG1抗体作为对照。通过流式细胞术分析在SHED中表达的细胞标志物CD73(B),CD105(C),CD34(e)的百分比。(f)形态学上,培养的脱落是主轴形的。(g)神经标记的表达<gydF4y2Baitalic> β</gydF4y2Baitalic>神经诱导培养4周后,在SHED中培养iii -微管蛋白。(h) SHED诱导成骨分化4周后形成茜素红s阳性矿化结节。规模<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M29"> <mml:mtext> 酒吧</米米l:mtext> <mml:mo> =</米米l:mo> <mml:mn> 20.</米米l:mn> <mml:mtext> </mml:mtext> <mml:mi> μ</米米l:mi> <mml:mtext> 米</米米l:mtext> </mml:math> </inline-formula>(g)、规模<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M30"> <mml:mtext> 酒吧</米米l:mtext> <mml:mo> =</米米l:mo> <mml:mn> One hundred.</米米l:mn> <mml:mtext> </mml:mtext> <mml:mi> μ</米米l:mi> <mml:mtext> 米</米米l:mtext> </mml:math> </inline-formula>(f、h)。</p><gydF4y2Bafig id="fig1a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001a"></graphic> </fig> <fig id="fig1b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001b"></graphic> </fig> <fig id="fig1c"> <label>(C)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001c"></graphic> </fig> <fig id="fig1d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001d"></graphic> </fig> <fig id="fig1e"> <label>(e)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001e"></graphic> </fig> <fig id="fig1f"> <label>(F)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001f"></graphic> </fig> <fig id="fig1g"> <label>(G)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001g"></graphic> </fig> <fig id="fig1h"> <label>(H)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.001h"></graphic> </fig> </fig-group> <p>神经诱导培养4周后,SHED表达了神经标记物<gydF4y2Baitalic> β</gydF4y2Baitalic>iii -微管蛋白,表示其神经分化潜能(图<gydF4y2Baxref rid="fig1g" ref-type="fig"> 1 (g)</gydF4y2Baxref>).在诱导成骨分化4周后,在SHED培养中形成茜素红s阳性结节(图)<gydF4y2Baxref rid="fig1h" ref-type="fig"> 1 (h)</gydF4y2Baxref>),表明SHED有向矿化组织分化的潜力<gydF4y2Baitalic> 在体外</gydF4y2Baitalic>.</p></gydF4y2Basec> <sec id="sec3.2"> <title>3.2.局部注射或输注SHED可逆转CCI-ION术后疼痛过敏反应</gydF4y2Batitle> <p>三叉神经是支配面部皮肤的主要感觉神经。TG的三个大分支是眼神经(V1)、上颌神经(V2)和下颌神经(V3)。我们通过V2的主要分支眶下神经的CCI诱发神经性疼痛。双向重复测量方差分析显示,治疗的主效应显著(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M31"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.009</米米l:mn> </mml:math> </inline-formula>)和时间(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M32"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)和显著交互(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M33"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>).在CCI-ION组中,EF50显著降低(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M34"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>),第3天第一次试验。在该模型中,机械性异常痛持续超过8周,这表明在神经结扎后3,7,14,28和56天,机械性阈值显著且持续下降(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M35"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M36"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.006</米米l:mn> </mml:math> </inline-formula>,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M37"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.002</米米l:mn> </mml:math> </inline-formula>,<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M38"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.008</米米l:mn> </mml:math> </inline-formula>, 和<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M39"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.003</米米l:mn> </mml:math> </inline-formula>分别(图)<gydF4y2Baxref rid="fig2a" ref-type="fig"> 2(一个)</gydF4y2Baxref>).</p><gydF4y2Bafig-group id="fig2"> <label>图2.</gydF4y2Balabel> <p>SHED移植对神经性疼痛的影响。(a)显示SHED移植时机和行为测试的方案。(b) CCI-ION大鼠机械阈值明显降低。(c)直接注射低剂量后机械阈值的变化(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M40"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>CCI-ION+低剂量注射组)或高剂量注射组(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M41"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 6</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>在CCI离子大鼠中测量掉损伤部位的细胞,0.2mL,CCI-离子+高剂量injshed组56天。使用局部注射培养基(CCI-ION + RECMENDIUM组)作为对照。(d)输注棚后的机械阈值的变化(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M42"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>CCI-ION+infSHED组大鼠尾静脉滴注细胞(0.2 mL) 56 d。以输注培养基(CCI-ION+infMedium组)作为对照组。<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M43"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> *</米米l:mo> </mml:mrow> </mml:msup> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>与虚假的(b),<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M44"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> *</米米l:mo> </mml:mrow> </mml:msup> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>Vs.中等(c, d),和<gydF4y2Basup>#</gydF4y2Basup> <inline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M45"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>与天0。所有数据都表示为<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M46"> <mml:mtext> 的意思是</米米l:mtext> <mml:mo> ±</米米l:mo> <mml:mtext> 扫描电镜</米米l:mtext> </mml:math> </inline-formula>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M47"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>每组大鼠)。</p><gydF4y2Bafig id="fig2a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.002a"></graphic> </fig> <fig id="fig2b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.002b"></graphic> </fig> <fig id="fig2c"> <label>(C)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.002c"></graphic> </fig> <fig id="fig2d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.002d"></graphic> </fig> </fig-group> <p>在初步实验中,我们发现第3-5代的SHED有效地产生了长期的疼痛缓解,而在高通道(>20)的SHED没有效果(数据未显示)。</p><p>gydF4y2Ba首先,我们测试了损伤三叉神经直接注射SHED对CCI-ION大鼠行为体征的影响。如图所示<gydF4y2Baxref rid="fig2b" ref-type="fig"> 2 (b)</gydF4y2Baxref>,两种低剂量(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M48"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)及高剂量SHED (<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M49"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>局部注射后3、7、14、28和56 d,与CCI-ION+injMedium相比,机械超敏反应和异常疼痛均得到缓解。培养基对损伤力学阈值无显著影响。</p><p>gydF4y2Ba接下来,我们测试了单一输注是否有效。在接受血液输注的大鼠中,与中等对照相比,在输注后第3天的EF50显着增加(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M50"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)及基线水平(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M51"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>).在8周的观察期间,镇痛作用持续存在,表明与CCI-ION+infMedium相比,SHED输注后3,7,14,28和56天的机械阈值显著增加(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M52"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>在所有时间点)(图<gydF4y2Baxref rid="fig2c" ref-type="fig"> 2 (c)</gydF4y2Baxref>).</p></gydF4y2Basec> <sec id="sec3.3"> <title>3.3.局部移植棚早期的定位</gydF4y2Batitle> <p>24小时后,PKH26和DAPI染色的SHED分布到同侧TGs(图)<gydF4y2Baxref rid="fig3a" ref-type="fig"> 3(一个)</gydF4y2Baxref>- - - - - -<gydF4y2Baxref rid="fig3c" ref-type="fig"> 3 (c)</gydF4y2Baxref>)和72h(图<gydF4y2Baxref rid="fig3d" ref-type="fig"> 3 (d)</gydF4y2Baxref>- - - - - -<gydF4y2Baxref rid="fig3f" ref-type="fig"> 3 (f)</gydF4y2Baxref>)局部注射后。三叉神经脊髓尾侧亚核、C1-C2脊髓颈背角(VC/C2)、延髓前部腹内侧(RVM)、心、肺、肝、肾、脾、肠未见pkh26阳性细胞。</p><gydF4y2Bafig-group id="fig3"> <label>图3.</gydF4y2Balabel> <p>pkh26标记的SHED在同侧TG中的分布:(a)局部注射24 h后pkh26阳性细胞(红色);(b)局部注射后24小时dapi染色细胞(蓝色);(c) (a)和(b)的合并图像;(d)局部注射72 h后pkh26阳性细胞(红色);(e)局部注射72 h后dapi染色细胞(蓝色);(f) (d)与(e)的合并图像。</p><gydF4y2Bafig id="fig3a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.003a"></graphic> </fig> <fig id="fig3b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.003b"></graphic> </fig> <fig id="fig3c"> <label>(C)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.003c"></graphic> </fig> <fig id="fig3d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.003d"></graphic> </fig> <fig id="fig3e"> <label>(e)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.003e"></graphic> </fig> <fig id="fig3f"> <label>(F)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.003f"></graphic> </fig> </fig-group> </sec> <sec id="sec3.4"> <title>3.4.局部SHED移植可抑制损伤神经的炎症细胞浸润</gydF4y2Batitle> <p>损伤部位注射SHED后7天,用HE染色对损伤的三叉神经进行组织学检查。如图所示<gydF4y2Baxref rid="fig4" ref-type="fig"> 4</gydF4y2Baxref>, CCI-ION组三叉神经招募的白细胞数量显著增加(图)<gydF4y2Baxref rid="fig4c" ref-type="fig"> 4(c)</gydF4y2Baxref>),与假手术组(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M53"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)(图<gydF4y2Baxref rid="fig4b" ref-type="fig"> 4(b)</gydF4y2Baxref>).但与CCI-ION+injMedium组相比,CCI-ION+低剂量injSHED组三叉神经炎症细胞浸润减少(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M54"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)(图<gydF4y2Baxref rid="fig4d" ref-type="fig"> 4(d)</gydF4y2Baxref>).</p><gydF4y2Bafig-group id="fig4"> <label>图4.</gydF4y2Balabel> <p>局部注射SHED可抑制损伤神经内炎症细胞浸润。(a)显示SHED移植和组织收集时机的方案。苏木精-伊红染色显示,与假手术组(b)相比,CCI-ION组(c)三叉神经招募的白细胞数量显著增加。局部注射SHED后三叉神经炎症细胞浸润减少(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M55"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>(d).天平<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M56"> <mml:mtext> 酒吧</米米l:mtext> <mml:mo> =</米米l:mo> <mml:mn> 200</米米l:mn> <mml:mtext> </mml:mtext> <mml:mi> μ</米米l:mi> <mml:mtext> 米</米米l:mtext> </mml:math> </inline-formula>.</p><gydF4y2Bafig id="fig4a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.004a"></graphic> </fig> <fig id="fig4b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.004b"></graphic> </fig> <fig id="fig4c"> <label>(C)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.004c"></graphic> </fig> <fig id="fig4d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.004d"></graphic> </fig> </fig-group> </sec> <sec id="sec3.5"> <title>3.5.局部SHED移植对三叉神经TNF-<italic>α</italic>和IL-1<italic>β</italic>水平的影响</gydF4y2Batitle> <p>为了评估SHED移植对促炎细胞因子表达的影响,我们检测了<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>RT-qPCR检测三叉神经损伤后7天和56天的mRNA水平。CCI-ION组的mRNA水平明显高于对照组<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M57"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.002</米米l:mn> </mml:math> </inline-formula>,图<gydF4y2Baxref rid="fig5a" ref-type="fig"> 5(一个)</gydF4y2Baxref>),<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M58"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.011</米米l:mn> </mml:math> </inline-formula>,图<gydF4y2Baxref rid="fig5c" ref-type="fig"> 5 (c)</gydF4y2Baxref>),比假手术组大。而局部SHED移植降低了mRNA水平<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M59"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.049</米米l:mn> </mml:math> </inline-formula>,图<gydF4y2Baxref rid="fig5b" ref-type="fig"> 5 (b)</gydF4y2Baxref>),<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M60"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.025</米米l:mn> </mml:math> </inline-formula>,图<gydF4y2Baxref rid="fig5d" ref-type="fig"> 5(d)</gydF4y2Baxref>局部移植7 d后,与中对照相比,损伤神经的存活率明显提高。上述结果提示,在神经症大鼠损伤神经早期,SHED明显抑制cci - ion诱导的促炎细胞因子上调。</p><gydF4y2Bafig-group id="fig5"> <label>图5.</gydF4y2Balabel> <p>局部SHED移植减少<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>发生发生后三叉神经的mRNA水平。信使rna水平的<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(一)和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>(c)在CCI-ION后7天测量损伤的三叉神经。信使rna水平的<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(b)和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>(d)局部SHED移植后7天测量损伤的三叉神经(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M61"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>细胞,0.2毫升)。<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M62"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> *</米米l:mo> </mml:mrow> </mml:msup> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>Vs. sham (a, c),<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M63"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> *</米米l:mo> </mml:mrow> </mml:msup> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>vs. medium (b, d)<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M64"> <mml:mtext> 的意思是</米米l:mtext> <mml:mo> ±</米米l:mo> <mml:mtext> 扫描电镜</米米l:mtext> </mml:math> </inline-formula>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M65"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>每组大鼠)。</p><gydF4y2Bafig id="fig5a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.005a"></graphic> </fig> <fig id="fig5b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.005b"></graphic> </fig> <fig id="fig5c"> <label>(C)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.005c"></graphic> </fig> <fig id="fig5d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.005d"></graphic> </fig> </fig-group> <p>两组间mRNA水平无显著差异<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(图<gydF4y2Baxref rid="fig6a" ref-type="fig"> 6(一)</gydF4y2Baxref>),<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>(图<gydF4y2Baxref rid="fig6c" ref-type="fig"> 6 (c)</gydF4y2Baxref>)神经结扎56天后,假手术组和CCI-ION组损伤三叉神经(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M66"> <mml:mi> p</米米l:mi> <mml:mo> ></米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>).局部SHED移植无明显改变<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(图<gydF4y2Baxref rid="fig6b" ref-type="fig"> 6 (b)</gydF4y2Baxref>),<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>信使rna水平(图<gydF4y2Baxref rid="fig6d" ref-type="fig"> 6 (d)</gydF4y2Baxref>)损伤三叉神经56天后(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M67"> <mml:mi> p</米米l:mi> <mml:mo> ></米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>).</p><gydF4y2Bafig-group id="fig6"> <label>图6.</gydF4y2Balabel> <p>局部SHED移植减少<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>三叉神经移植后56天的mRNA水平。信使rna水平的<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(一)和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>(c)在CCI-ION后56天测量损伤的三叉神经。信使rna水平的<gydF4y2Baitalic> TNF-α.</gydF4y2Baitalic>(b)和<gydF4y2Baitalic> il - 1β</gydF4y2Baitalic>(d)在局部血液移植后56天测量的受损三叉神经(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M68"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>细胞,0.2毫升)。<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M69"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> *</米米l:mo> </mml:mrow> </mml:msup> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>Vs. sham (a, c),<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M70"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> *</米米l:mo> </mml:mrow> </mml:msup> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>vs. medium (b, d)<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M71"> <mml:mtext> 的意思是</米米l:mtext> <mml:mo> ±</米米l:mo> <mml:mtext> 扫描电镜</米米l:mtext> </mml:math> </inline-formula>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M72"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>每组大鼠)。</p><gydF4y2Bafig id="fig6a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.006a"></graphic> </fig> <fig id="fig6b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.006b"></graphic> </fig> <fig id="fig6c"> <label>(C)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.006c"></graphic> </fig> <fig id="fig6d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.006d"></graphic> </fig> </fig-group> </sec> <sec id="sec3.6"> <title>3.6。局部SHED移植抑制CCI-ION大鼠TRPV1表达上调</gydF4y2Batitle> <p>局部注射SHED后7天拔除三叉神经和TGs。免疫组化染色检测TRPV1表达(图<gydF4y2Baxref rid="fig7" ref-type="fig"> 7</gydF4y2Baxref>)和western blot(图<gydF4y2Baxref rid="fig8" ref-type="fig"> 8</gydF4y2Baxref>).trpv1阳性细胞在TG中显著增加(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M73"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)和三叉神经(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M74"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>),与假手术大鼠比较(图<gydF4y2Baxref rid="fig7" ref-type="fig"> 7</gydF4y2Baxref>).CCI离子诱导TG中TRPV1表达的上调(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M75"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)及主要传入神经(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M76"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)被局部SHED管理显著抑制(图<gydF4y2Baxref rid="fig7" ref-type="fig"> 7</gydF4y2Baxref>).</p><gydF4y2Bafig-group id="fig7"> <label>图7.</gydF4y2Balabel> <p>局部SHED移植抑制CCI-ION大鼠trpv1阳性细胞的上调。假手术大鼠TG (a)和三叉神经(e)中的trpv1阳性细胞。未注射SHED的CCI-ION大鼠TG (b)和三叉神经(f)中trpv1阳性细胞。局部给药的CCI-ION大鼠TG (c)和三叉神经(g)中trpv1阳性细胞(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M77"> <mml:mn> 1</米米l:mn> <mml:mo> ×</米米l:mo> <mml:msup> <mml:mrow> <mml:mn> 10</米米l:mn> </mml:mrow> <mml:mrow> <mml:mn> 5</米米l:mn> </mml:mrow> </mml:msup> </mml:math> </inline-formula>细胞,0.2毫升)。(d) TG中trpv1阳性细胞的定量分析。(h)三叉神经TRPV1染色定量。规模<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M78"> <mml:mtext> 酒吧</米米l:mtext> <mml:mo> =</米米l:mo> <mml:mn> 50</米米l:mn> <mml:mtext> </mml:mtext> <mml:mi> μ</米米l:mi> <mml:mtext> 米</米米l:mtext> </mml:math> </inline-formula>.<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M79"> <mml:msup> <mml:mrow></mml:mrow> <mml:mrow> <mml:mo> *</米米l:mo> </mml:mrow> </mml:msup> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>与骗局,<gydF4y2Basup>#</gydF4y2Basup> <inline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M80"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.05</米米l:mn> </mml:math> </inline-formula>vs.无SHED移植的CCI-ION大鼠。所有数据都表示为<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M81"> <mml:mtext> 的意思是</米米l:mtext> <mml:mo> ±</米米l:mo> <mml:mtext> 扫描电镜</米米l:mtext> </mml:math> </inline-formula>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M82"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>每组大鼠)。</p><gydF4y2Bafig id="fig7a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007a"></graphic> </fig> <fig id="fig7b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007b"></graphic> </fig> <fig id="fig7c"> <label>(C)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007c"></graphic> </fig> <fig id="fig7d"> <label>(d)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007d"></graphic> </fig> <fig id="fig7e"> <label>(e)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007e"></graphic> </fig> <fig id="fig7f"> <label>(F)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007f"></graphic> </fig> <fig id="fig7g"> <label>(G)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007g"></graphic> </fig> <fig id="fig7h"> <label>(H)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.007h"></graphic> </fig> </fig-group> <fig-group id="fig8"> <label>图8.</gydF4y2Balabel> <p>局部SHED移植可抑制CCI-ION大鼠TRPV1蛋白水平的上调。TRPV1蛋白在假手术大鼠TG (a)和三叉神经(b)中的表达。所有数据都表示为<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M83"> <mml:mtext> 的意思是</米米l:mtext> <mml:mo> ±</米米l:mo> <mml:mtext> 扫描电镜</米米l:mtext> </mml:math> </inline-formula>(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M84"> <mml:mi> n</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 6</米米l:mn> </mml:math> </inline-formula>每组大鼠)。</p><gydF4y2Bafig id="fig8a"> <label>(一种)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.008a"></graphic> </fig> <fig id="fig8b"> <label>(b)</gydF4y2Balabel> <graphic xlink:href="//www.newsama.com/downloads/journals/sci/2021/8819884.fig.008b"></graphic> </fig> </fig-group> <p>TG中TRPV1蛋白水平(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M85"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)三叉神经(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M86"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.003</米米l:mn> </mml:math> </inline-formula>)与假大鼠的那些相比,上调神经连接后(图<gydF4y2Baxref rid="fig8" ref-type="fig"> 8</gydF4y2Baxref>).局部SHED移植抑制cci - ion诱导的TG中TRPV1表达上调(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M87"> <mml:mi> p</米米l:mi> <mml:mo> <</米米l:mo> <mml:mn> 0.001</米米l:mn> </mml:math> </inline-formula>)及主要传入神经(<gydF4y2Bainline-formula> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M88"> <mml:mi> p</米米l:mi> <mml:mo> =</米米l:mo> <mml:mn> 0.011</米米l:mn> </mml:math> </inline-formula>)(图<gydF4y2Baxref rid="fig8" ref-type="fig"> 8</gydF4y2Baxref>).</p></gydF4y2Basec> </sec> <sec id="sec4"> <title>4。讨论</gydF4y2Batitle> <p>本研究表明,全身或局部注射SHED均可降低三叉神经损伤后大鼠对机械刺激的敏感性,并持续观察8周。局部注射24、72 h后,pkh26标记的SHED分布到同侧TG。病变部位的SHED移植可降低早期损伤神经的炎症细胞浸润和促炎细胞因子水平。此外,局部SHED移植抑制了cci - ion诱导的三叉神经和神经节TRPV1表达上调。</p><p>gydF4y2Ba牙源性间充质干细胞在组织修复或重建中的应用被认为是转化再生医学的一个有趣的里程碑。牙源性间充质干细胞具有很强的分化为神经系、成骨系和脂肪系的能力[<gydF4y2Baxref ref-type="bibr" rid="B15"> 15</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B17"> 17</gydF4y2Baxref>].例如,血液移植通过增加神经生长因子蛋白水平和糖尿病大鼠毛细血管密度来提供神经保护[<gydF4y2Baxref ref-type="bibr" rid="B18"> 18</gydF4y2Baxref>].近年来的研究表明,骨髓基质细胞(BMSCs)可减少神经性疼痛的进展。全身和局部使用骨髓间充质干细胞可减轻各种神经性疼痛模型的机械和热痛觉过敏。静脉注射骨髓间充质干细胞可逆转CCI-ION模型的伤害性超敏反应[<gydF4y2Baxref ref-type="bibr" rid="B19"> 19</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B20"> 20.</gydF4y2Baxref>]和坐骨神经CCI模型[<gydF4y2Baxref ref-type="bibr" rid="B21"> 21</gydF4y2Baxref>].骨髓间充质干细胞已被证明能在鞘内产生疼痛缓解[<gydF4y2Baxref ref-type="bibr" rid="B22"> 22</gydF4y2Baxref>], intrabrain [<gydF4y2Baxref ref-type="bibr" rid="B23"> 23</gydF4y2Baxref>和椎管内[<gydF4y2Baxref ref-type="bibr" rid="B24"> 24</gydF4y2Baxref>)注入。当骨髓间充质干细胞直接注入病变部位时,CCI- ion大鼠、CCI坐骨神经模型和脊髓损伤模型的机械阈值和热阈值均升高[<gydF4y2Baxref ref-type="bibr" rid="B20"> 20.</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B25"> 25</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B26"> 26</gydF4y2Baxref>].</p><p>gydF4y2Ba据报道,与成体骨髓间充质干细胞和牙髓干细胞相比,SHED显示出更高的增殖率和碱性成纤维细胞生长因子基因表达[<gydF4y2Baxref ref-type="bibr" rid="B2"> 2</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B27"> 27</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B28"> 28</gydF4y2Baxref>].SHED可以很容易获得,不需要侵入性手术,因此代表了潜在临床应用的干细胞的一大来源,没有伦理问题。更重要的是,作为神经嵴细胞相关的产后干细胞,SHED表达多种神经细胞标志物[<gydF4y2Baxref ref-type="bibr" rid="B2"> 2</gydF4y2Baxref>],释放关键神经营养因子,抑制星形胶质细胞和神经元的凋亡[<gydF4y2Baxref ref-type="bibr" rid="B7"> 7</gydF4y2Baxref>].重要的是,血液移植缓解了糖尿病外周神经病变,并增加了糖尿病大鼠的毛细血管密度[<gydF4y2Baxref ref-type="bibr" rid="B18"> 18</gydF4y2Baxref>].因此,SHED在控制疼痛方面可能比骨髓间充质干细胞有优势,并有可能成为治疗神经性疼痛的一种替代和有效的治疗方法。</p><p>gydF4y2Ba目前尚不清楚干细胞是如何抑制疼痛的。已经有人提出,干细胞可以通过细胞间接触激活机制或分泌各种分子如细胞因子来减轻疼痛,在许多病理条件下调节局部环境[<gydF4y2Baxref ref-type="bibr" rid="B21"> 21</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B29"> 29</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B30"> 30.</gydF4y2Baxref>].越来越多的证据表明,大多数全身骨髓间充质干细胞迅速滞留在肺部,其持久的抗痛觉过敏作用远远长于它们在宿主中的生存时间[<gydF4y2Baxref ref-type="bibr" rid="B31"> 31</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B32"> 32</gydF4y2Baxref>].因此,BMSCs治疗慢性疼痛的局部机制是分泌各种分子来抑制神经炎症和调节细胞因子水平。例如,在坐骨神经CCI模型小鼠[<gydF4y2Baxref ref-type="bibr" rid="B22"> 22</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B33"> 33</gydF4y2Baxref>],鞘内骨髓间充质干细胞通过释放抗炎生长因子转化生长因子β发挥持久的镇痛作用,抑制cci诱导的脊髓神经炎症,下调TNF-<gydF4y2Baitalic> α</gydF4y2Baitalic>脊髓和背根神经节神经元兴奋性突触的表达和调节[<gydF4y2Baxref ref-type="bibr" rid="B22"> 22</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B33"> 33</gydF4y2Baxref>].核因子kappa B (NF-<gydF4y2Baitalic> κ</gydF4y2Baitalic>B)信号传导在bmsc诱导的持续性疼痛和脊髓损伤大鼠模型的抗痛觉过敏中起关键作用[<gydF4y2Baxref ref-type="bibr" rid="B34"> 34</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B35"> 35</gydF4y2Baxref>].在脊髓损伤大鼠中,骨髓间充质干细胞下调NF-磷酸化<gydF4y2Baitalic> κ</gydF4y2Baitalic>B P65亚基和下降其他促炎因子,如TNF-<gydF4y2Baitalic> α</gydF4y2Baitalic>和IL-1<gydF4y2Baitalic> β</gydF4y2Baitalic>[<gydF4y2Baxref ref-type="bibr" rid="B35"> 35</gydF4y2Baxref>].人骨髓间充质干细胞通过降低促炎细胞因子IL-1的水平来减轻机械异位痛和热痛觉过敏<gydF4y2Baitalic> β</gydF4y2Baitalic>和IL-17,并释放抗炎细胞因子IL-10在幸免神经损伤的神经性疼痛模型小鼠中[<gydF4y2Baxref ref-type="bibr" rid="B21"> 21</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B23"> 23</gydF4y2Baxref>].</p><p>gydF4y2Ba为了支持骨髓间充质干细胞控制疼痛的机制,我们的研究提供了证据,局部SHED移植通过减少早期损伤神经中的炎症细胞浸润和促炎细胞因子水平来减轻神经性疼痛。有充分的证据表明,细胞因子在神经性疼痛的炎症反应中发挥重要作用。神经损伤引起的促炎细胞因子水平升高有助于缓解疼痛[<gydF4y2Baxref ref-type="bibr" rid="B36"> 36</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B37"> 37</gydF4y2Baxref>].</p><p>gydF4y2Ba值得一提的是,SHED具有免疫调节特性,并在其他条件下调节细胞因子水平。SHED抑制淋巴细胞刺激,伴有促炎因子(TNF-<gydF4y2Baitalic> α</gydF4y2Baitalic>和IL-2)以及抗炎因子(IL-6和IL-10)的增加[<gydF4y2Baxref ref-type="bibr" rid="B38"> 38</gydF4y2Baxref>].此外,SHED可调节免疫系统,缓解免疫紊乱,如SLE [<gydF4y2Baxref ref-type="bibr" rid="B8"> 8</gydF4y2Baxref>].SHED移植降低了促炎细胞因子IL-17的水平,上调了调节性T细胞和辅助性T细胞的比例<gydF4y2Baitalic> 在体外</gydF4y2Baitalic>并逆转小鼠SLE相关疾病和改善SLE表型[<gydF4y2Baxref ref-type="bibr" rid="B8"> 8</gydF4y2Baxref>].进一步研究SHED对神经损伤免疫反应的影响,将是了解SHED镇痛作用机制的一个有希望的方向。</p><p>gydF4y2Ba本研究的另一个重要发现是,SHED移植抑制了cci - ion诱导的原发性传入神经TRPV1表达上调及TG的上调,这可能提示TRPV1参与了SHED的强镇痛作用。TRPV1是疼痛检测和调节的关键分子组成部分。在神经元中,TRPV1的激活增加了钙内流,并有助于对疼痛的更高感知[<gydF4y2Baxref ref-type="bibr" rid="B9"> 9</gydF4y2Baxref>- - - - - -<gydF4y2Baxref ref-type="bibr" rid="B11"> 11</gydF4y2Baxref>].在这项工作中,在大鼠神经损伤后,在损伤部位和IpsilaTalal Ganglia的三叉神经中的TRPV1表达增加,这表明外周Trpv1表达介导三叉神经痛。在此介绍,先前的研究报告说,在脊柱和颅神经损伤后神经纤维和神经节上调TRPV1表达。在坐骨神经连接大鼠中,背根神经节和脊髓背角的TRPV1 mRNA和蛋白质水平损伤后1-4周增加到1-4周[<gydF4y2Baxref ref-type="bibr" rid="B39"> 39</gydF4y2Baxref>,<gydF4y2Baxref ref-type="bibr" rid="B40"> 40</gydF4y2Baxref>].成年雪貂舌神经交易后3周损伤神经和TG神经元TRPV1表达上调[<gydF4y2Baxref ref-type="bibr" rid="B41"> 41</gydF4y2Baxref>].在小鼠三叉神经损伤后,损伤V2中大量大神经元的体细胞可被TRPV1配体辣椒素激活[<gydF4y2Baxref ref-type="bibr" rid="B9"> 9</gydF4y2Baxref>].CCI-ION治疗后,小鼠所有背角板上的TG神经元中枢纤维和末端均发现TRPV1活性增高。我们的数据显示,SHED移植通过下调外周TRPV1的表达和调节神经元可塑性来逆转神经性疼痛。然而,SHED调控TRPV1表达的分子机制尚不清楚,有待进一步研究。本研究的一个局限性是,我们没有研究细胞因子和TRPV1表达在shed介导的镇痛中的关系。未来,我们将重点研究SHED在缓解神经性疼痛中的具体机制。</p></gydF4y2Basec> <sec id="sec5"> <title>5.结论</gydF4y2Batitle> <p>本研究揭示了SHED在扭转大鼠三叉神经损伤后的超敏和异常痛觉方面的新作用。旁分泌功能和外周神经系统TRPV1表达下调可能是shedbased镇痛的机制。这些结果提供了临床前证据,支持使用牙科干细胞治疗三叉神经痛和潜在的其他慢性疼痛。</p></gydF4y2Basec> <back> <sec sec-type="data-availability"> <title>数据可用性</gydF4y2Batitle> <p>用于支持本研究发现的数据可由通讯作者要求提供。</p></gydF4y2Basec> <sec sec-type="COI-statement"> <title>利益冲突</gydF4y2Batitle> <p>作者声明本文的发表不存在利益冲突。</p></gydF4y2Basec> <ack> <title>致谢</gydF4y2Batitle> 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