人脐带间充质干细胞体外向心肌细胞样分化及HLA-G在人脐带间充质干细胞中的表达

发布时间：2018-05-23 19:59

本文选题：人脐带间充质干细胞 + 心肌细胞　；参考：《遵义医学院》2012年硕士论文

【摘要】：目的：探讨人脐带间充质干细胞的培养及冻存复苏的方法。初步探讨人脐带间充质干细胞向心肌样细胞诱导分化的方法及HLA-G蛋白在脐带间充质干细胞和心肌样细胞中的表达。方法：第4代人脐带间充质干细胞进行传代培养,并观察其形态。取第5代细胞进行冻存3个月后复苏细胞,显微镜下观察细胞形态。取第8代细胞经胰酶(0.125%)消化后接种于T75培养瓶中。待细胞生长达到70%-80%融合时,用10μmol/L5-Aza进行诱导24h,对照组用含10%FBS的α-MEM培养液培养。每2d换1次培养液,并在不同诱导阶段进行细胞形态学观察。诱导第四周行免疫细胞化学,RT-PCR检测。结果：第4代人脐带间充质干细胞在传代后24小时贴壁,一周时观察到细胞已长满瓶底。细胞呈长梭形或不规则形,大小不一,类似成纤维细胞。第5代细胞冻存后细胞的贴壁生长能力及形态与冻存前相同。诱导第2周可见部分细胞体积逐渐增大,呈球状和杆状细胞。诱导4周时经免疫细胞化学可检测到分化后的细胞表达心肌细胞标记cTnT、Cx43和desmin, RT-PCR检测到有心肌细胞标记desmin的表达。诱导前后行免疫细胞化学法可观察到脐带MSCs及心肌样细胞均表达HLA-G。结论：人脐带间充质干细胞在体外具有较强的增殖能力,并能在5-Aza诱导下分化为心肌样细胞,表达心肌细胞的表面特异性标志,人脐带间充质干细胞可做为作心肌细胞移植的来源。HLA-G在脐带MSCs及心肌样细胞表达持续阳性,并不因为其分化而丧失。
[Abstract]:Objective: To investigate the culture of human umbilical cord mesenchymal stem cells and the methods of cryopreservation and resuscitation. The method of inducing differentiation of human umbilical cord mesenchymal stem cells to cardiac myocytes and the expression of HLA-G protein in umbilical cord mesenchymal stem cells and cardiac myocytes were discussed.
Methods: fourth generations of human umbilical cord mesenchymal stem cells were cultured and observed, and their morphology was observed. Fifth generation cells were recovered after 3 months of cryopreservation. The cell morphology was observed under microscope. Eighth generation cells were inoculated into T75 culture bottle after trypsin (0.125%). When the cell growth reached 70%-80% fusion, 24 cells were induced to induce 24. H, the control group was cultured with 10%FBS containing alpha -MEM culture. 1 cultures were changed per 2D, and the cell morphology was observed at different induction stages. The induction of immunocytochemistry and RT-PCR detection were induced for fourth weeks.
Results: the fourth generations of human umbilical cord mesenchymal stem cells were adhered to the wall for 24 hours after the passage, and the cells were observed at the bottom of the bottle at one week. The cells showed long shuttle or irregular shape, different size, similar to fibroblasts. The cell wall growth ability and shape of the cells after the fifth generation of cells were the same as that before the freezing. The volume of the cells was gradually induced to be visible for second weeks. CTnT, Cx43 and desmin were detected by immunocytochemistry at 4 weeks by immunocytochemistry. The expression of desmin in cardiac myocytes was detected by RT-PCR. The expression of HLA-G. in umbilical cord MSCs and myocardial like cells could be observed before and after induction by immunocytochemical method.
Conclusion: human umbilical cord mesenchymal stem cells have strong proliferation ability in vitro, and can differentiate into myocardial like cells under the induction of 5-Aza, and express the specific markers of the surface of cardiac myocytes. Human umbilical cord mesenchymal stem cells can be used as the source of.HLA-G in the umbilical cord MSCs and cardiac myocyte like cells to be the source of continuous positive expression of the human umbilical cord mesenchymal stem cells. Its differentiation is lost.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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