人羊水间充质干细胞生物学特征及其体外向内皮细胞诱导分化的研究

发布时间：2018-04-14 01:28

本文选题：间充质干细胞 + 羊水　；参考：《中国人民解放军医学院》2012年硕士论文

【摘要】：目的体外分离培养人羊水间充质干细胞(HAFMSCs, human amniotic fluid mesenchymal stem cells),观察其生物学特性。低温冻存HAFMSCs三个月,观察冻存后细胞的生物学特性,找出最佳的冻存方案。体外诱导HAFMSCs向内皮细胞分化,分别在常氧及低氧环境下观察其分化为内皮细胞的能力。方法采用四种方法分离培养HAFMSCs,用MTT法观察各组细胞的增殖能力。流式细胞技术(FACS)检测各组细胞表面抗原CD29, CD34, CD44, CD45, CD73, CD90, HLA-ABC, HLA-DR等的表达。比较各组细胞成脂、成骨的分化能力,及检测各组细胞中OCT-4, Nanog等mRNA表达水平。用不同成分的冻存液冻存HAFMSCs,冻存三个月后复苏细胞,并分析其存活率、生物学特性及分化能力。体外采用VEGF和bFGF联合诱导HAFMSCs向内皮细胞分化,FACS分析细胞vWF、CD31及CD133mRNA的表达水平,采用细胞免疫荧光的方法鉴定诱导分化后细胞vWF的表达情况,观察诱导后细胞在Matrigel上形成毛细血管样结构的能力。比较低氧环境与常氧环境下上述指标的差异。结果四种不同的培养方法均培养出HAFMSCs。其中Chang培养基两步培养法培养的细胞,其贴壁时间、细胞集落形成时间及细胞传代时间较其余三组更短,培养1.5天即有细胞贴壁,4-5天可见细胞集落形成,细胞增殖快。四组细胞均表达间充质干细胞标记物CD29、CD44、CD73、CD90,而不表达造血干细胞标记物CD34、CD45,符合间充质干细胞的特征,各组之间无明显差异。四组细胞成骨、成脂诱导成功,并分别通过油红O染色及von Kossa染色证实。四组细胞中均检测到OCT-4, Nanog mRNA的表达。冻存三个月后的HAFMSCs,以冻存液方案为DMEM:FBS:DMSO=5:4:1勺细胞存活率最高,细胞增殖能力最强。而复苏后细胞的生长曲线、细胞表型、分化能力及OCT-4, Nanog6勺表达与冻存前细胞相比无统计学差异。HAFMSCs体外诱导分化为内皮细胞,流式细胞技术分析比较各组诱导后细胞中vWF, CD31,CD133等内皮相关标记物抗原的水平,以VEGF和bFGF联合诱导组诱导效率最高,Mtrigel上形成毛细血管结构的能力也最强。低氧环境下经VEGF和bFGF联合诱组HAFMSCs内VEGF, v WF和CD31mRNA水平高于其余组，，毛细血结构能力也明显强于其余组。结论HAFMSCs具有易获、体外增殖快、多向分化能力等优势。Chang培基两步培法能够在较短时间内培大量HAFMSCs。短期冻存HAFMSCs对于其生物学特无明显影，最佳冻存配方为DMEM:FBS:DMSO=5:4:1。VEGF和bFGF联合诱较为理体外诱HAFMSCs分化为内皮细胞诱方案。低氧能够促进这过程，在短期低氧环境下，HAFMSCs诱分化为内皮细胞能力与暴于低氧环境时间关。
[Abstract]:Objective to isolate and culture human amniotic fluid mesenchymal stem cells from human amniotic fluid mesenchymal stem cells in vitro and observe their biological characteristics.After cryopreservation of HAFMSCs for three months, the biological characteristics of cryopreserved cells were observed and the best cryopreservation scheme was found out.HAFMSCs was induced to differentiate into endothelial cells in vitro and its ability to differentiate into endothelial cells was observed under normoxic and hypoxic conditions.Methods Haf MSCs were isolated and cultured by four methods. The proliferative ability of each group was observed by MTT method.The expression of CD29, CD34, CD44, CD45, CD73, CD90, HLA-ABC and HLA-DR were detected by flow cytometry.The ability of adipogenic and osteogenic differentiation and the expression of OCT-4 and Nanog were measured.HAF MSCs were frozen with different components of cryopreservation solution for 3 months, and the survival rate, biological characteristics and differentiation ability of Hafs cells were analyzed.In vitro, VEGF and bFGF were used to induce the differentiation of HAFMSCs into endothelial cells. The expression levels of vWF-1 CD31 and CD133mRNA were analyzed in vitro, and the expression of vWF in differentiated cells was identified by cell immunofluorescence.The ability of induced cells to form capillary-like structure on Matrigel was observed.To compare the difference of above indexes between hypoxic environment and normoxic environment.Results HAF MSCs were obtained from four different culture methods.The adherent time, colony forming time and cell passage time of Chang culture medium were shorter than those of the other three groups.All the cells in the four groups expressed mesenchymal stem cell marker CD29, CD44, CD73, CD90, but not hematopoietic stem cell marker CD34, CD45, which was consistent with the characteristics of mesenchymal stem cells, and there was no significant difference among the four groups.Osteogenesis and adipogenesis were successfully induced in the four groups, and were confirmed by oil red O staining and von Kossa staining, respectively.OCT-4 and Nanog mRNA were detected in all four groups.After three months of cryopreservation, the cryopreservation solution was DMEM:FBS:DMSO=5:4:1 with the highest cell survival rate and the strongest cell proliferation.However, there was no significant difference in growth curve, phenotype, differentiation ability and expression of OCT-4 and Nanog6 between resuscitated cells and pre-cryopreserved cells. HAFMSCs were induced to differentiate into endothelial cells in vitro.The levels of endothelium-related markers such as vWF, CD31 and CD133 were analyzed by flow cytometry, and the ability to form capillary structure on Mtrigel was the highest in the combination of VEGF and bFGF.In hypoxic environment, the levels of VEGF, vWF and CD31mRNA in HAFMSCs induced by VEGF and bFGF were higher than those in other groups, and the capillary blood structure ability was significantly stronger than that in other groups.Conclusion HAFMSCs has the advantages of easy to obtain, rapid proliferation in vitro and multidirectional differentiation ability. Chang Peiji two-step culture method can cultivate a large number of HAF MSCs in a short time.Short-term cryopreservation of HAFMSCs had no obvious effect on its biology. The best formula of cryopreservation was the combination of DMEM:FBS:DMSO=5:4:1.VEGF and bFGF to induce HAFMSCs to differentiate into endothelial cells in vitro.Hypoxia can promote this process. The ability of HAFMSCs to differentiate into endothelial cells under short-term hypoxia is related to the time of exposure to hypoxia.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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