人骨髓间充质干细胞增殖、分泌、凋亡、迁移和黏附影响因素及机制

发布时间：2018-04-15 13:02

  本文选题：骨髓间充质干细胞 + 分化　； 参考：《复旦大学》2009年博士论文

【摘要】： 骨髓干细胞是一类具有高度自我更新和多向分化潜能的细胞，利用其可分化为多种细胞的能力，可作为受损心肌组织修复的供体细胞。骨髓间充质干细胞(mesenchymal stem cells，MSCs)是骨髓中除造血干细胞以外的另一类多能干细胞，可能成为一种理想的修复损伤心肌的移植用细胞，其治疗缺血性心脏病、扩张性心肌病等原因引起的心功能不全的研究尚处于起步阶段，，正日益受到关注。本研究以人MSCs为研究对象，探讨其增殖、旁分泌、迁移、凋亡过程中不同药物的影响，及相关的细胞信号传导途径。 第一部分人骨髓间充质干细胞的分离培养、鉴定及诱导分化 [目的]分离并培养人MSCs，纯化扩增，为进一步进行药物干预研究打基础，并检测细胞表型。使用5-氮杂胞苷(5-azacytidine，5-aza)将人MSCs向心肌细胞方向诱导分化，进行细胞鉴定，以满足实验的需要。 [方法]抽取志愿者骨髓液，使用密度梯度离心法分离骨髓单个核细胞，并借助MSCs黏附于塑料瓶底这一特性进行纯化。相差显微镜观察形态变化，流式细胞仪检测CD34、CD105、CD106表面抗原表达，鉴定MSCs。将MSCs传代扩增培养，取第6代MSCs经5-aza诱导分化，相差显微镜观察形态变化，免疫化学鉴定细胞诱导前后肌球蛋白重链β和心肌肌钙蛋白T(cardiac troponin T，cTnT)表达。 [结果]将密度梯度离心法与贴壁法相结合，可获得较多的MSCs，通过传代后细胞进一步纯化，每个25cm~2细胞培养瓶接种2×10~5个细胞，完全培养液胎牛血清浓度为10％，80％～90％细胞融合后按1：3或1：2比例传代，MSCs能稳定地传代扩增而无明显分化迹象。原代细胞培养2周左右可首次传代，以后7d左右传代一次，传到第6代未发现属性改变，呈现成纤维细胞样生长。应用5-aza对第6代细胞进行诱导分化2周，发现细胞形态变大，连接紧密，呈条索状，外表似心肌细胞，诱导前MSCs肌球蛋白重链β和cTnT表达均阴性，诱导后则均为阳性表达，阳性细胞率约为21％。 [结论]应用密度梯度离心法密度梯度离心法与贴壁法相结合，可建立人MSCs体外稳定的纯化扩增方法。体外5-aza诱导后2周，MSCs从形态和蛋白表达水平上向心肌细胞方向分化，符合实验要求。 第二部分促红细胞生成素、普伐他汀、红景天苷、抗血小板药物对人骨髓间充质干细胞增殖、旁分泌和分化的干预研究 [目的]观察重组人促红细胞生成素(recombinant human EPO，rhEPO)、普伐他汀、红景天苷、抗血小板药物干预对人MSCs增殖、血管内皮生长因子(vascularendothelial growth factor，VEGF)分泌、分化的影响。 [方法]体外扩增培养人MSCs，绘制生长曲线，使用上述药物干预第6代人MSCs，相差显微镜观察药物作用后细胞形态学变化，四甲基偶氮唑盐[3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide，MTT]比色法检测细胞增殖情况，酶联免疫吸附法(enzyme-linked immunosorbent assay，ELISA)法测定细胞VEGF分泌，流式细胞仪检测细胞抗原CD34、CD105、CD106表达变化，免疫化学观察药物预处理后经5-aza诱导，人MSCs cTnT抗体染色阳性细胞率的变化。 [结果](1)细胞增殖情况：传代细胞潜伏期为48h左右，对数增殖期约为接种后第4～6d，至接种后第7d进入平台期。经药物作用后的人MSCs形态和细胞表面抗原CD34、CD105、CD106表达无明显变化。反映MSCs增殖变化的OD570值在一定浓度rhEPO(0．01U／mL～10U／mL)、普伐他汀(0．2μmol／L～10μmol／L)、红景天苷(0．2mg／L～2mg／L)、氯吡格雷(0．02μmol／L～40μmol／L)和噻氯匹定(0．02μmol／L～40μmol／L)作用后明显高于对照组(P＜0．05)，而部分浓度的阿司匹林组(60μmol／L～2000μmol／L)则显著低于对照组(P＜0．05)。(2)细胞VEGF分泌：人MSCs分泌的VEGF水平在高浓度红景天苷(5mg／L)、高浓度氯吡格雷(40μmol／L)和噻氯匹定(40μmol／L)作用后明显低于对照组(P＜0．01)，而低浓度普伐他汀(0．2μmol／L)、阿司匹林组和低浓度氯吡格雷组(0．02μmol／L)VEGF水平与对照组无明显差异，rhEPO、高浓度普伐他汀(100μmol／L)、低浓度红景天苷(0．2mg／L)和低浓度噻氯匹定(0．02μmol／L)VEGF水平则显著高于对照组。(3)细胞分化情况：细胞表面抗原CD34、CD105、CD106在6种药物作用后与对照组相比无明显差异。促红细胞生成素、普伐他汀、红景天苷、氯吡格雷、噻氯匹定和阿司匹林预处理后经5-aza诱导，人MSCs cTnT单克隆抗体染色阳性细胞百分率无明显变化(P值分别为0．597、0．952、0．325、0．141、0．830和0．072)。 [结论]rhEPO、普伐他汀、红景天苷、氯吡格雷和噻氯匹定对人MSCs有明显的促进增殖作用，而阿司匹林则抑制其增殖。高浓度红景天苷、氯吡格雷和噻氯匹定有抑制人MSCs分泌VEGF的作用，rhEPO、高浓度普伐他汀、低浓度红景天苷和低浓度噻氯匹定可促进VEGF分泌。这些药物对人MSCs的分化影响不明显。 第三部分：促红细胞生成素和普伐他汀对人骨髓间充质干细胞增殖、旁分泌、凋亡、迁移和黏附的影响和作用机制的实验研究 [目的]观察促红细胞生成素和普伐他汀干预对人MSCs增殖、VEGF分泌、凋亡、迁移和黏附的影响及相关细胞信号传导通路。 [方法]体外培养人MSCs，使用上述药物干预第6代人MSCs，Western blot方法检测作用前后磷酸化细胞外信号调节激酶1／2(extracellular signal-regulated kinase1／2，ERK1／2)、p38促分裂原活化蛋白激酶(p38 mitogen activated protein kinase，p38MAPK)及磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase，PI3K)／丝氨酸／苏氨酸激酶(serine／threonine kinase，Akt)蛋白的表达情况，进一步用特异的细胞信号通路抑制剂或激动剂阻断或激活ERK1／2、p38MAPK及PI3K／AKT途径，通过MTT法测定对人MSCs的增殖影响，ELISA法测定细胞VEGF的分泌，用流式细胞仪进行rhEPO和普伐他汀对MSCs的抗凋亡研究，Transwell小室进行细胞迁移实验，并进行细胞黏附性测定。 [结果]rhEPO和普伐他汀可使人MSCs的PI3K／Akt通路磷酸化水平升高，抑制p38MAPK通路磷酸化水平，对人MSCs的ERK通路和总Akt、总p38MAPK水平无明显影响。经p38MAPK通路特异激动剂anisomycin预处理后，rhEPO组促增殖作用较前减弱(P＜0．05)，而普伐他汀组作用消失，PI3K／AKT通路特异抑制剂Ly294002预处理影响不明显。Ly294002或anisomycin预处理均可使rhEPO促进人MSCs分泌VEGF的作用减弱(P＜0．01)，Ly294002预处理可使普伐他汀的这种作用减弱(P＜0．01)，但anisomycin作用不明显。rhEPO和普伐他汀能降低H_2O_2诱导的人MSCs凋亡(P＜0．01)，Ly294002预处理后这种作用消失，anisomycin预处理对这种作用影响不明显。经rhEPO或普伐他汀作用后迁移细胞显著增多(P＜0．01)，Ly294002预处理后这种作用消失，anisomycin预处理对这种作用影响不明显。经rhEPO或普伐他汀作用后贴壁细胞显著增多(P＜0．01)，但Ly294002或anisomycin预处理对这种作用影响均不明显。 [结论]rhEPO及普伐他汀可促进人MSCs的增殖、分泌VEGF、迁移和黏附能力，并具有抗凋亡作用。这两种药物的促进增殖作用与其抑制p38MAPK通路有关，p38MAPK通路受抑制也与rhEPO促进VEGF分泌相关，而PI3K／Akt通路的激活参与了它们的刺激VEGF分泌、增强迁移能力以及抗凋亡作用的调控。这两种药物促进黏附能力与PI3K／Akt和p38MAPK通路无关。
[Abstract]:Bone marrow stem cells are a kind of highly self-renewal and pluripotent cells, which can differentiate into a variety of cells, can be used as donor cells to repair damaged myocardial tissue. Bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) bone marrow mesenchymal stem cells outside of another type of pluripotent stem cells may be an ideal repair of myocardial injury by cell transplantation in the treatment of ischemic heart disease, heart function, study the cause of dilated cardiomyopathy insufficiency is still in its infancy, is getting more and more attention. The research on MSCs as the research object, to explore the proliferation, paracrine, migration. Effect of different drugs in the process of apoptosis, and cell related signal pathways.
Part one, isolation, identification and differentiation of human bone marrow mesenchymal stem cells
[Objective] to isolate and culture human MSCs, purify and amplify it, lay the foundation for further drug intervention research, and detect cell phenotypes. 5- 5-azacytidine (5-aza) is used to induce the differentiation of human MSCs into cardiomyocytes, and cell identification is performed to meet the needs of experiment.
[Methods] from volunteer bone marrow, bone marrow mononuclear cells were isolated by density gradient centrifugation, and with the help of MSCs adhesion to the plastic bottom this characteristic were purified. Morphologic changes were observed by phase contrast microscopy, CD34 assay, flow cytometry and CD105, expression of CD106 surface antigen, MSCs. MSCs identification of cultured passaged sixth generations MSCs by 5-aza induced differentiation, morphologic changes were observed by phase contrast microscope, immunohistochemical identification of cells before and after induction of beta myosin heavy chain and cardiac troponin T (cardiac troponin T, cTnT) expression.
[results] the combination of density gradient centrifugation and adherent method can obtain more MSCs, subcultured cells was further purified by 25cm~2, each cell culture bottle with 2 * 10~5 cells, completely cultured fetal bovine serum concentration was 10%, from 80% to 90% after cell fusion by 1:3 or 1:2 cases than the passage MSCs can be stably passaged and amplified without obvious signs of differentiation. The primary cell culture 2 weeks after the first passage, a passage about 7d, reached the sixth generation did not find the property change, showing fibroblastic growth. Application of 5-aza to the sixth generation cells were differentiated for 2 weeks, found that the cells became larger, connection close a funicular look like myocardial cells before induction of MSCs myosin heavy chain and beta cTnT expression were negative after induction were positive, positive rate is about 21%.
[Conclusion] the combination of density gradient centrifugation and density gradient centrifugation combined with adherence can establish a stable amplification method for human MSCs in vitro. After 2 weeks of 5-aza induction, MSCs will differentiate into cardiomyocytes from morphology and protein expression, which is in line with the experimental requirements.
Second parts of erythropoietin, pravastatin, salidroside, antiplatelet drugs on the proliferation, paracrine and differentiation of human bone marrow mesenchymal stem cells
[Objective] to observe the effects of recombinant human EPO (rhEPO), pravastatin and salidroside on the proliferation of MSCs, the secretion and differentiation of vascularendothelial growth factor (VEGF) in human MSCs.
[Methods] human MSCs cultured in vitro, draw the growth curve, the use of drug intervention and sixth generations of MSCs, phase contrast microscope and morphological changes were observed after treatment with four methyl thiazolyl tetrazolium [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, MTT] colorimetric assay for cell proliferation, enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) method for the determination of cell secretion of VEGF cell antigen detection, CD34, flow cytometry, CD105, expression of CD106, immunohistochemical observation of drug pretreatment induced by 5-aza MSCs cTnT, changes in antibody staining positive cell rate.
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