人脐带胶质间充质干细胞的分离培养与特性研究

发布时间：2018-06-17 15:27

本文选题：脐带胶质 + 人脐带间充质细胞　；参考：《内蒙古农业大学》2011年硕士论文

【摘要】：细胞治疗已经走过十多年的历程,起初科学家对免疫细胞进行大量研究,如细胞因子诱导的杀伤细胞(CIK)治疗癌症具有可观的前景;而目前科学界对人脐带间充质干细胞(Human umbilical cord mesenchymal stem cell, hUC-MSC)的研究较为关注,由于hUC-MSC比胚胎干细胞(ESC)、骨髓间充质干细胞(BM-MSC)等具有更好的临床应用潜能,从而成为人类再生医学和组织工程的研究热点。本研究目的是探索更有效的hUC-MSC分离培养方法和进一步研究hUC-MSC生物学特性,通过建立优化的实验方法,能为再生医学科研和临床应用提供操作规范标准和参考依据。方法:实验随机选取足月顺产健康胎儿脐带,采取手工机械分离法和3种酶组合消化法从脐带胶质中释放人脐带间充质细胞;并用优化完全培养基纯化培养;MTT法检测各代脐带间充质细胞生长增殖活性并绘制生长曲线;流式细胞术检测脐其生长周期和表面标志物;RT-PCR扩增基因P53、C-myc和OCT-4;结果:培养脐带间充质细胞形态为梭形、呈纤维样,并出现流水状或漩涡样生长;生长增殖速度较快,第7代脐带间充质细胞指数倍增时间小于30h;80%以上细胞处于G0/G1期;培养脐带间充质细胞均一稳定高表达CD29、CD70、CD105、CD90和CD44,而几乎不表达CD45、CD34和HLA-DR;P53基因在各代脐带间充质细胞均有表达,C-myc基因在第4代和第18代出现明显表达,OCT-4基因在不同代数均出现表达;结论:1)1h可分离30cm脐带胶质,获得的大量脐带间充质细胞(9×10~4/cm),经培养检测证明具有hUC-MSC的形态特征、增殖活性,表面抗原一致,是原始的hUC-MSC;2)通过RT-PCR检测各代脐带间充质细胞的P53、C-myc和OCT-4基因,发现培养10代以内hUC-MSC状态较正常。本研究意义:提示在临床应用hUC-MSC时,对其癌基因和抑癌基因全面检测以及衰老凋亡系统检测很有必要;本实验可为短期培养扩增出临床治疗所需要安全可靠的MSC提供研究基础,可为避免hUC-MSC体内移植治疗产生致瘤性提供参考依据。
[Abstract]:Cell therapy has gone through more than ten years. At first, scientists carried out a lot of research on immune cells, such as cytokine induced killer cells (CIK) treatment of cancer has considerable prospects; Human umbilical cord mesenchymal stem cell, hUC-MSC (human umbilical cord mesenchymal stem cell) has been paid more attention to by the scientific community at present. Because hUC-MSC has better clinical application potential than embryonic stem cell (ESC), bone marrow mesenchymal stem cell (BM-MSC), and so on, human umbilical cord mesenchymal stem cell (hUC-MSC) has better clinical application potential. Therefore, it has become the research hotspot of human regenerative medicine and tissue engineering. The purpose of this study was to explore a more effective method for the isolation and culture of hUC-MSC and to further study the biological characteristics of hUC-MSC. By establishing an optimized experimental method, it can provide the standard and reference for the scientific research and clinical application of regenerative medicine. Methods: human umbilical cord mesenchymal cells were released from cord glia by manual mechanical separation and three enzyme digestion methods. The growth and proliferation activity of umbilical cord mesenchymal cells was determined by MTT method and the growth curve was drawn. Flow cytometry was used to detect the growth cycle and surface marker of umbilical cord by RT-PCR. Results: the morphology of cultured umbilical cord mesenchymal cells was fusiform, fibrous, and the growth of umbilical cord mesenchymal cells was like water or whirlpool. The doubling time of the seventh passage mesenchymal cell index was less than 30 h and more than 80% of the cells were in G _ 0 / G _ 1 phase. In cultured umbilical cord mesenchymal cells, the expression of CD29, CD70, CD105, CD90 and CD44, and almost no expression of CD45, CD34 and HLA-DRN p53 genes in all generations of umbilical cord mesenchymal cells, were significantly expressed in the 4th and 18th generation of umbilical cord mesenchymal cells, and OCT-4 genes were expressed in different generations. Conclusion 30cm cord glia can be isolated from 30cm for 1 hour. A large number of umbilical cord mesenchymal cells (9 脳 10 ~ (4) / cm ~ (-1) 路cm ~ (-1) were obtained. The morphological characteristics, proliferative activity and surface antigen of HUC-MSC were confirmed by culture. The P53C-myc and OCT-4 genes of umbilical cord mesenchymal cells were detected by RT-PCR. It was found that the status of hUC-MSC was normal within 10 generations. The results indicate that it is necessary to detect the oncogenes and tumor suppressor genes and the aging apoptosis system in the clinical application of hUC-MSC, and this study can provide a research basis for the amplification of safe and reliable MSC for clinical treatment in short term culture. It can provide reference for avoiding tumorigenicity of hUC-MSC transplantation in vivo.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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