人骨髓间充质干细胞体外诱导分化为角膜缘干细胞的研究

发布时间：2018-03-29 19:33

本文选题：骨髓间充质干细胞　切入点：角膜缘干细胞　出处：《南昌大学》2009年硕士论文

【摘要】： 目的:探讨体外诱导人骨髓间充质干细胞(HBMSCs,human bone mesenchymal stem cells)分化为角膜缘干细胞(LSCs,limbal stem cells)的可行性。方法:用密度梯度离心法体外分离HBMSCs,在含10%胎牛血清(FBS,fetal bovine serum)的LG-DMEM/F12培养基中扩增培养,流式细胞仪检测细胞的表面抗原;用消化后组织块培养法分离培养LSCs,在含20%FBS的HG-DMEM/F12培养基中扩增培养,流式细胞仪检测细胞的核心抗原P63。将第四次传代的HBMSCs按5 X 104/ml的细胞密度接种于六孔Transwell培养板底层,分成两组:实验组将第二次传代的LSCs按5 X 104/ml接种于Transwell培养板上层,用一种特殊的培养基(含90% LG-DMEM/F12,10%FBS,10μg/ml EGF, 10ng/ml bFGF,1μg/mLPS)共同培养10天。对照组不加任何诱导剂,以原LG-DMEM/F12培养基培养。在相差显微镜下进行形态学观察。结果:密度梯度离心法能分离出纯度较高的HBMSCs。典型的HBMSCs贴壁生长,呈长梭形,漩涡状盘旋排列。流式细胞分析结果显示第四代HBMSCs表达相关的抗原标记CD29(96.78%), CD44(96.23%), CD105(91.45%), CD166(81.70%),不表达造血细胞系的表面标志CD34(2.34%), CD45(3.92%)及主要组织相容性抗原HLA-DR(1.11%)。在体外成功培养出LSCs,细胞呈圆形、椭圆形、多角形,胞体透亮,约2周后细胞融合呈镶嵌状排列。第二代LSCs表达P63(83.35%)。实验组共同培养72小时后,贴壁HBMSCs部分呈椭圆形、圆形改变,在体外微环境中诱导10天后大部分细胞呈多边形、圆形、椭圆形,少量细胞呈梭形。对照组细胞呈典型HBMSCs贴壁生长,以长梭形为主。试验组P63弱阳性(7.16±0.56%),对照组P63阴性(0.74±0.49%)。结论:与受炎性刺激的LSCs共培养后,HBMSCs有横向分化为类LSCs细胞的能力。
[Abstract]:Aim: to investigate the feasibility of inducing the differentiation of human bone mesenchymal stem cells from human bone marrow mesenchymal stem cells into limbal stem cells in vitro. Methods: HBMSCs were isolated by density gradient centrifugation in vitro and cultured in LG-DMEM/F12 medium containing 10% fetal bovine serum (FBS). Flow cytometry was used to detect the surface antigen of the cells. LSCs were isolated and cultured by digested tissue mass culture method, and amplified in HG-DMEM/F12 medium containing 20s. The core antigen P63of the cells was detected by flow cytometry. The fourth passage of HBMSCs was inoculated at the bottom of the six-well Transwell culture plate according to the cell density of 5 X 104/ml. The experimental group was divided into two groups: the second passage of LSCs was inoculated on the top of the Transwell culture plate according to 5X 104/ml, and co-cultured on a special medium (90% LG-DMEM / F12, 10s 10 渭 g/ml EGF, 10ng/ml bFGF-1 渭 g / mLPS-1 渭 g / mLPS1 渭 g / mL PSS) for 10 days, the control group was not treated with any inducer. The morphology was observed under phase contrast microscope in the culture medium of LG-DMEM/F12. Results: high purity HBMSCs could be isolated by density gradient centrifugation. Typical HBMSCs adherent growth was long fusiform. The results of flow cytometry showed that the antigen markers associated with the expression of HBMSCs in the fourth generation were CD2996.78, CD44C96.23K, CD105, 91.4545, CD166, 81.70T, the surface markers of hematopoietic cell line CD34C ~ 2.34, CD453.922), and the main histocompatibility antigen HLA-DR1.11T. The cells were round, and the cells were round and round. Oval, polygonal and bright cell bodies. After about 2 weeks, the cells were arranged in mosaics. The second generation of LSCs expressed P63O83.35. After 72 hours of co-culture, the HBMSCs parts of the experimental group were oval and round. After 10 days of induction in microenvironment in vitro, most of the cells were polygonal, round, oval, and a few cells were fusiform. The cells of the control group were typical HBMSCs adherent growth, mainly long fusiform. In the test group, the P63 weak positive cells were 7.16 卤0.56 and those in the control group were 0.74 卤0.49m. Conclusion: hBMSCs co-cultured with inflammatory LSCs have the ability to differentiate into LSCs like cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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