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本研究旨在对骨髓间充质干细胞(mesenchymal stem cells,MSC)进行移植前增殖分化能力测定,病毒学检查,核型分析,PCR-STR和HLA的检测,以便为临床应用提供安全、可靠的移植细胞。采用贴壁法分离骨髓MSC,在相同条件下分别考察各代细胞形态、生长、表面标记、成骨、成脂分化能力的变化情况,并进行不同生长时期的核型分析,利用HLA-SBT技术检测4个不同供者来源的MSC的HLA-I和HLA-II位点的高分辨分型;应用PCR-STR技术检测4个不同供者来源的MSC基因遗传标记。并且利用ELISA方法检测HIV,HBV,HCV和TP,使用PCR方法检测支原体污染。结果表明:随着传代次数增加,MSC的增殖能力、成骨能力均有所下降。在扩增过程中,MSC始终保持较高的纯度,CD29、CD44、CD105、CD166、CD73均表达阳性,CD14、CD34、CD45、CD80和CD86均表达阴性。在8代以前未发现核型变异。4个不同供者来源的MSC的HLA高分辨分型和STR基因分型结果为MSC3的TP表达阳性,MSC2在5代出现支原体污染。结论:在体外培养过程中MSC干细胞特性逐渐丢失,其中向成骨方向的分化潜能降低。MSC在8代以前可作为实验研究及临床应用的良好对象。
The aim of this study was to determine the ability of mesenchymal stem cells (MSCs) to proliferate and differentiate before transplantation, to detect virulence, to karyotype analysis, to PCR-STR and HLA to provide a safe and reliable Transplant the cells. The bone marrow MSCs were isolated by adherence method. The morphology, growth, surface marker, osteogenic and adipogenic differentiation of the cells were examined under the same conditions. The karyotype analysis of different growth stages was performed. HLA-SBT The HLA-I and HLA-II loci of four MSCs from different donors were detected by high-resolution genotyping. The genetic markers of MSCs from four different donors were detected by PCR-STR. HIV, HBV, HCV and TP were detected by ELISA. Mycoplasma contamination was detected by PCR. The results showed that with the increase of passages, the proliferation and osteogenic capacity of MSC all decreased. In the process of amplification, MSC maintained high purity. CD29, CD44, CD105, CD166 and CD73 all expressed positive, while CD14, CD34, CD45, CD80 and CD86 all expressed negative. No karyotype variation was found before the 8th generation. High-resolution HLA typing and STR genotyping of MSCs from 4 different donors resulted in a positive TP expression of MSC3, and MSC2 showed Mycoplasma contamination in 5th generation. CONCLUSION: The characteristics of MSC stem cells gradually lost during in vitro culture, and the differentiation potential of osteoblasts decreased. MSC can be used as a good target for experimental research and clinical application before 8 generations.