低氧培养对人脐带间充质干细胞成软骨分化特性的影响

发布时间：2018-01-25 01:30

  本文关键词： 脐带间充质干细胞 低氧诱导因子 培养环境 成软骨分化　出处：《中南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的体外分离培养人脐带间充质干细胞并对其进行鉴定,探讨体外低氧培养环境对其成软骨分化能力的影响。 方法采用酶消化法分离培养人脐带沃顿胶组织来源间充质干细胞,加入含10%FBS的DMEM/F12培养基中进行原代培养；待细胞生长至80-90%融合时,1：3传代培养。通过倒置显微镜观察细胞形态变化,采用MTT法检测细胞增殖能力,应用流式细胞仪检测细胞表面抗原。取传3代细胞采用条件培养基诱导其向成软骨方向分化,根据培养氧浓度分为实验组(2%氧浓度)及对照组(20%氧浓度)。诱导2周后细胞爬片进行甲苯胺蓝染色及II型胶原免疫组化染色,应用RT-PCR技术检测HIF-1α、Sox-9及CoL2al mRNA表达,研究低氧环境对人脐带干细胞成软骨分化的影响。 结果原代培养细胞贴壁生长,初始细胞形态呈短梭形、梭形或多角形；传代后细胞多呈均一的成纤维样细胞形态,细胞贴壁和增殖速度明显加快,呈平行排列生长或旋涡状生长；MTT法检测第3、5、7代脐带干细胞均具有较强的增殖能力；流式细胞仪检测传3代脐带干细胞95%表达CD29,96%表达CD44,仅有0.8%表达CD45、1.8%表达CD31,与间充质干细胞表面抗原表达一致；诱导2周后低氧培养组细胞甲苯胺兰染色及Ⅱ型胶原免疫组化染色均显示强阳性反应,胞浆及胞膜异染明显,而对照组染色显示弱阳性反应；RT-PCR检测结果显示实验组HIF-1α、Sox-9及CoL2al mRNA表达均显著强于对照组,差异有统计学意义(P0.05)。 结论采用酶消化法可以成功分离培养人脐带间充质干细胞；低氧培养环境可促进人脐带间充质干细胞向成软骨方向分化,其机制可能与HIF-1α基因表达上调有关。
[Abstract]:Objective to isolate and identify human umbilical cord mesenchymal stem cells (HMSCs) in vitro and to investigate the effect of hypoxia culture environment on the ability of chondrogenic differentiation of human umbilical cord mesenchymal stem cells. Methods mesenchymal stem cells derived from human umbilical cord Wharton gum tissue were isolated and cultured by enzyme digestion method. The mesenchymal stem cells were cultured in DMEM/F12 medium containing 10s. When the cells grew to 80-90% fusion, they were subcultured with 1: 3. The morphologic changes of cells were observed by inverted microscope, and the proliferative ability of cells was detected by MTT method. Flow cytometry was used to detect cell surface antigen. The third passage of cells was used to induce chondrogenic differentiation in conditioned medium. According to the culture oxygen concentration, the cells were divided into experimental group (2% oxygen concentration) and control group (20% oxygen concentration). After 2 weeks of induction, toluidine blue staining and type II collagen immunohistochemical staining were used. The expression of HIF-1 伪 -Sox-9 and CoL2al mRNA was detected by RT-PCR technique, and the effect of hypoxia on chondrogenic differentiation of human umbilical cord stem cells was studied. Results the primary cultured cells were adherent to the wall, and the initial cells were fusiform, fusiform or polygonal. After passage, most of the cells showed uniform fibroblast morphology, and the cell adhesion and proliferation were accelerated obviously, and the cells grew in parallel arrangement or swirl shape. All the umbilical cord stem cells of the 3rd and 5th generation were detected by MTT method, and all of the umbilical cord stem cells had strong proliferative ability. Flow cytometry was used to detect the expression of CD29996% in umbilical cord stem cells (95%) and CD31 in 0.8% (1.8%). The expression of surface antigen was consistent with that of mesenchymal stem cells. After 2 weeks of induction, toluidine blue staining and type 鈪，

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