本文選題：骨髓間充質干細胞 + 培養(yǎng)　； 參考：《重慶醫(yī)科大學》2011年碩士論文

【摘要】：目的:單純未轉染干細胞移植體內后生存能力低下嚴重限制了其增殖能力。本研究旨在探討存活素(Survivin,svv)基因修飾的骨髓間充質干細胞(MSCs)在離體微環(huán)境中延長生存能力的機制,為新的干細胞抗凋亡移植策略提供理論依據(jù)。 方法:分離提取幼年C57BL/6小鼠骨髓細胞,采用全骨髓法培養(yǎng)擴增骨髓間充質干細胞,取第11代細胞進行試驗。1.未轉染細胞進行連續(xù)培養(yǎng)計數(shù)并繪制生長曲線;同時應用流式細胞技術檢測細胞表面標志物SCA-1、CD29、CD34、CD44和CD117;行成骨、成脂誘導分化測定; rt-PCR測定細胞內survivin表達情況。2.制備DNA-Lipofectamine 2000復合物,同時轉染到293FT細胞中收集含有病毒的培養(yǎng)液,制備Svv重組慢病毒和空白對照病毒,測定病毒滴度;Svv慢病毒和空白病毒分別轉染骨髓間充質干細胞并測出轉染率接近大于90%時的MOI值;分別進行轉染后成骨誘導分化測定;并運用rt-PCR比較轉染前后間充質干細胞內survivin表達變化情況。 結果:培養(yǎng)出的細胞呈長梭成纖維細胞樣,經(jīng)流式細胞儀檢測細胞表面高表達SCA-1、CD29、CD34、CD44,低表達CD117;細胞曲線顯示傳代細胞培養(yǎng)第1-3 d生長緩慢,第4 d生長加快并在第7 d達到高峰;成骨誘導20 d經(jīng)茜素紅染色呈紅色結節(jié),成脂誘導14 d油紅O染色顯示有大量脂質沉淀; rt-PCR結果顯示survivin mRNA陽性表達。成功包裝出Svv慢病毒和空白病毒,實驗組病毒滴度為7.1×10~7 TU/ml,空白病毒組病毒滴度為8.3×10~7 TU/ml;轉染率接近大于90%時的MOI值Svv慢病毒和空白病毒分別為28.4和8.3;轉染后的Svv慢病毒和空白病毒均能向成骨分化;同時rt-PCR結果顯示轉染后明顯過表達survivin。 結論:全骨髓培養(yǎng)法可以培養(yǎng)出大量骨髓間充質干細胞,同時survivin在小鼠骨髓間充質干細胞中正常表達,提示可能參與骨髓間充質干細胞抗凋亡過程;慢病毒轉然后骨髓間充質干細胞仍具有其分化功能,且過表達survivin,為體內研究其存活時間提供了實驗基礎。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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