慢病毒介导survivin体外转染MSCs实验研究

发布时间：2018-06-27 10:36

本文选题：骨髓间充质干细胞 + 培养　；参考：《重庆医科大学》2011年硕士论文

【摘要】：目的:单纯未转染干细胞移植体内后生存能力低下严重限制了其增殖能力。本研究旨在探讨存活素(Survivin,svv)基因修饰的骨髓间充质干细胞(MSCs)在离体微环境中延长生存能力的机制,为新的干细胞抗凋亡移植策略提供理论依据。方法:分离提取幼年C57BL/6小鼠骨髓细胞,采用全骨髓法培养扩增骨髓间充质干细胞,取第11代细胞进行试验。1.未转染细胞进行连续培养计数并绘制生长曲线;同时应用流式细胞技术检测细胞表面标志物SCA-1、CD29、CD34、CD44和CD117;行成骨、成脂诱导分化测定; rt-PCR测定细胞内survivin表达情况。2.制备DNA-Lipofectamine 2000复合物,同时转染到293FT细胞中收集含有病毒的培养液,制备Svv重组慢病毒和空白对照病毒,测定病毒滴度;Svv慢病毒和空白病毒分别转染骨髓间充质干细胞并测出转染率接近大于90%时的MOI值;分别进行转染后成骨诱导分化测定;并运用rt-PCR比较转染前后间充质干细胞内survivin表达变化情况。结果:培养出的细胞呈长梭成纤维细胞样,经流式细胞仪检测细胞表面高表达SCA-1、CD29、CD34、CD44,低表达CD117;细胞曲线显示传代细胞培养第1-3 d生长缓慢,第4 d生长加快并在第7 d达到高峰;成骨诱导20 d经茜素红染色呈红色结节,成脂诱导14 d油红O染色显示有大量脂质沉淀; rt-PCR结果显示survivin mRNA阳性表达。成功包装出Svv慢病毒和空白病毒,实验组病毒滴度为7.1×10~7 TU/ml,空白病毒组病毒滴度为8.3×10~7 TU/ml;转染率接近大于90%时的MOI值Svv慢病毒和空白病毒分别为28.4和8.3;转染后的Svv慢病毒和空白病毒均能向成骨分化;同时rt-PCR结果显示转染后明显过表达survivin。结论:全骨髓培养法可以培养出大量骨髓间充质干细胞,同时survivin在小鼠骨髓间充质干细胞中正常表达,提示可能参与骨髓间充质干细胞抗凋亡过程;慢病毒转然后骨髓间充质干细胞仍具有其分化功能,且过表达survivin,为体内研究其存活时间提供了实验基础。
[Abstract]:Objective: the low viability of untransfected stem cells after transplantation severely limits their proliferative ability. The purpose of this study was to investigate the mechanism of survivin (survivin) gene modified bone marrow mesenchymal stem cells (MSCs) to prolong their viability in vitro, and to provide a theoretical basis for new anti-apoptotic transplantation strategies of stem cells. Methods: bone marrow mesenchymal stem cells (BMSCs) were isolated from young C57BL / 6 mouse bone marrow cells and cultured with whole bone marrow method. The cell surface markers SCA-1, CD29, CD34, CD44 and CD117 were detected by flow cytometry. Osteogenesis, adipogenic differentiation and rt-PCR were used to detect the expression of survivin in the cells. DNA-Lipofectamine 2000 complex was prepared and transfected into 293FT cells to collect virus-containing culture medium to prepare SVV recombinant lentivirus and blank control virus. Bone marrow mesenchymal stem cells (BMSCs) were transfected with SvV lentivirus and blank virus respectively, and the moi values of bone marrow mesenchymal stem cells were measured when the transfection rate was higher than 90, and the osteogenic differentiation was determined after transfection. The changes of survivin expression in mesenchymal stem cells before and after transfection were compared by rt-PCR. Results: the cultured cells showed long fusiform fibroblasts. The high expression of SCA-1T CD29 CD34 CD44 and the low expression of CD117 were detected by flow cytometry, and the cell curve showed that the cells grew slowly on the 1-3 day, accelerated the growth on the 4th day and reached the peak on the 7th day. After 20 days of osteogenesis induction, alizarin red staining showed red nodules, oil red O staining showed a large number of lipid precipitates at 14 days of fat-forming induction, and rt-PCR showed positive expression of survivin mRNA. Successful packaging of SVV lentivirus and blank virus, The titer of virus in the experimental group was 7.1 脳 10 ~ (7) TU / ml, the titer of the blank virus group was 8.3 脳 10 ~ (7) TU / ml, the moi value of Svv lentivirus and blank virus were 28.4 and 8.3 when the transfection rate was higher than 90, respectively. After transfection, both SVV lentivirus and blank virus could differentiate into osteogenesis. At the same time, the results of rt-PCR showed that survivin was significantly overexpressed after transfection. Conclusion: bone marrow mesenchymal stem cells can be cultured by whole bone marrow culture method, and survivin is expressed normally in mouse bone marrow mesenchymal stem cells, suggesting that it may be involved in the anti-apoptosis process of bone marrow mesenchymal stem cells. Lentivirus transformed bone marrow mesenchymal stem cells still have its differentiation function and overexpression of survivin, which provides an experimental basis for the study of its survival time in vivo.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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