小鼠脂肪源干细胞的体外分离培养及成骨诱导前后造血调控相关因子表达的实验研究

发布时间：2018-12-14 19:02

【摘要】：目的:探寻小鼠ADSCs的体外分离培养方法以及成骨诱导前后造血调控因子分子水平的表达情况。方法:采用酶消化法获得脂肪来源的细胞,传代培养至第三代,分别进行成脂与成骨细胞诱导,用实时荧光定量多聚酶链反应(qRT-PCR)检测成骨诱导前后的造血调控因子β-catenin、SDF-1、N-cadherin、SCF、MIP-1α和Jag 1,GM-CSF的表达。同时采用流式细胞仪测定ADSCs细胞表面标志物CD29、CD34、CD106、CD49d的表达。结果:小鼠脂肪来源的细胞在体外培养过程中,呈梭形贴壁生长,可稳定传代;细胞表面标志的流式细胞仪检测:CD29表达率强阳性,CD34表达率较低,CD106、CD49d低表达;进行成脂细胞诱导后,油红“O”染色呈阳性,成骨细胞诱导后,茜素红染色和von Kossa染色均为阳性,表明其是ADSCs。通过qRT-PCR检测多种造血调控因子相对表达量,发现β-catenin表达量最高,SDF-1和N-cadherin次之,其余各因子SCF、MIP-1α、Jag1、GM-CSF均为低表达。统计分析后,发现各因子在成骨诱导前后表达差异均无统计学意义。结论:本研究证实,小鼠脂肪来源的细胞具有MSC特征就是ADSCs,其成骨诱导前后的造血调控因子均有不同程度的表达,但差异无统计学意义。
[Abstract]:Aim: to investigate the isolation and culture of mouse ADSCs in vitro and the expression of hematopoietic regulatory factors before and after osteogenesis. Methods: the adipose derived cells were obtained by enzyme digestion and cultured to the third generation. The adipogenic cells and osteoblasts were induced respectively. The expression of hematopoietic regulatory factors 尾-catenin,SDF-1,N-cadherin,SCF,MIP-1 伪 and Jag 1 GM-CSF before and after osteogenesis was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). At the same time, flow cytometry was used to detect the expression of CD29,CD34,CD106,CD49d on the surface of ADSCs cells. Results: the murine adipose derived cells were fusiform adherent to the wall in vitro and could be subcultured stably. Flow cytometry showed that the expression rate of CD29 was strongly positive, the expression rate of CD34 was low, and the expression of CD106,CD49d was low. After adipoblast induction, oil red "O" staining was positive, and osteoblast induction, alizarin red staining and von Kossa staining were positive, indicating that it was ADSCs.. The relative expression of various hematopoietic regulatory factors was detected by qRT-PCR. It was found that the expression of 尾-catenin was the highest, followed by SDF-1 and N-cadherin, and the other factors SCF,MIP-1 伪 and Jag1,GM-CSF were all low expression. After statistical analysis, it was found that there was no significant difference in the expression of each factor before and after osteogenesis. Conclusion: this study confirmed that the expression of hematopoietic regulatory factors before and after osteogenesis induced by ADSCs, was the characteristic of MSC in adipose derived cells of mice, but the difference was not statistically significant.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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