体外诱导人羊水来源间充质干细胞向软骨细胞分化的实验研究

发布时间：2018-03-24 16:12

本文选题：人羊水来源间充质干细胞　切入点：软骨细胞　出处：《复旦大学》2009年硕士论文

【摘要】： 研究目的探讨分离、提纯和扩增孕中期羊水来源间充质干细胞(AF-MSCs)的方法,以及体外诱导AF-MSCs向软骨细胞分化的可能性。研究方法机械手段挑取孕中期人羊水细胞培养体系中的MSCs集落,培养扩增后,通过流式细胞术对表面抗原表达情况进行鉴定,并通过RT-PCR检测多能性基因Oct4及Nanog的表达情况。取第3代AF-MSCs,经TGF-β1、IGF-1、地塞米松等生长因子向软骨细胞方向诱导分化。倒置相差显微镜观察诱导后细胞形态的变化。诱导3周后,免疫荧光染色及RT-PCR分别检测软骨特异性基质CollengonⅡ、Aggrecan、Chondromudulin-1、S100的蛋白及其基因的表达情况。研究结果机械手段分离出的AF-MSCs为CD105、CD166阳性,CD29若阳性,CD34、CD45阴性,符合MSCs特点。AF-MSCs表达多能性基因Oct4,但不表达Nanog。诱导2周后,细胞开始呈多角形样改变;诱导后3周时,大部分细胞由长梭形转变为多角形。免疫荧光及RT-PCR检测显示诱导后的AF-MSCs软骨特异性基质CollengonⅡ、Aggrecan、Chondromudulin-1和S100的蛋白和基因呈阳性表达,而未经诱导培养的AF-MSCs呈阴性表达。研究结论机械分离法可有效获得孕中期羊水来源间充质干细胞,经TGF-β1,IGF-1,地塞米松联合诱导3周后,AF-MSCs可以分化为软骨细胞样细胞。
[Abstract]:Research purpose. To explore the methods of isolation, purification and amplification of AF-MSCs derived from amniotic fluid in the second trimester of pregnancy, and the possibility of inducing AF-MSCs to differentiate into chondrocytes in vitro. Research method. In the second trimester of human amniotic fluid cell culture system, the MSCs colony was selected by mechanical means, and the expression of surface antigen was identified by flow cytometry. The expression of Oct4 and Nanog was detected by RT-PCR. The third generation AF-MSCs were induced to differentiate into chondrocytes by growth factors such as TGF- 尾 1 IGF-1 and dexamethasone. The morphologic changes of the cells were observed by inverted phase contrast microscopy. The expression of Chondromudulin-1 S100 protein and its gene were detected by immunofluorescence staining and RT-PCR, respectively. Research results. The AF-MSCs isolated by mechanical means was CD105, CD166 positive and CD29 positive. If CD34 + CD45 was negative, it was consistent with the characteristics of MSCs. AF-MSCs expressed multipotent gene Oct4, but did not express Nanog.After 2 weeks of induction, the cells began to show polygonal changes, and at 3 weeks after induction, the cells began to show polygonal changes. Immunofluorescence and RT-PCR analysis showed that the AF-MSCs chondromudin-1 and S100 chondromudin-1 and S100 chondromudin-1 were expressed positively, but the uninduced AF-MSCs showed negative expression. Research conclusion. Mesenchymal stem cells derived from amniotic fluid in the second trimester of pregnancy could be obtained by mechanical isolation. AF-MSCs could differentiate into chondrocyte-like cells after induction of TGF- 尾 _ 1 IGF-1 and dexamethasone for 3 weeks.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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