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  本文選題：骨髓間充質干細胞　切入點：基因轉染　出處：《中國矯形外科雜志》2013年21期 　論文類型：期刊論文

【摘要】：[目的]研究慢病毒載體(lentiviral vectors,LVs)介導增強型綠色熒光蛋白(enhanced green fluorescent protein,eGFP)標記大鼠骨髓間充質干細胞(bone marrow mesenchymal stem cells,BMSCs)的可行性及對細胞生物學特性的影響。[方法]采用密度梯度離心法分離培養(yǎng)大鼠BMSCs,經(jīng)Lenti-emGFP/hygro慢病毒載體轉染和潮霉素藥篩,鏡下觀察細胞形態(tài)和eGFP表達情況,MTT法檢查轉染前后細胞增殖活力,流式細胞儀檢測細胞表面標記物(CD34、CD44、CD45、CD90)和eGFP,檢測轉染后細胞的成骨、成脂分化潛能并分別行茜素紅染、油紅O染色鑒定。[結果]大鼠BMSCs呈成纖維細胞樣形態(tài),漩渦樣狀排列,轉染后細胞形態(tài)不變,并高效表達eGFP,兩組細胞增殖活力一致,流式細胞儀檢測顯示,轉染后的P3代大鼠BMSCs-eGFP的eGFP陽性率為99.97%,轉染前后,兩組細胞CD34和CD45陽性率低于2%,而CD44和CD90陽性率高于99%。轉染后的P3代大鼠BMSCs-eGFP成骨誘導后,茜素紅染色,可見呈桔紅色的塊狀鈣結節(jié),成脂誘導后,油紅O染色顯示有大量紅色脂質沉淀,成骨成脂誘導過程中,eGFP持續(xù)表達。[結論]Lenti-emGFP/hygro轉染大鼠BMSCs并經(jīng)潮霉素藥篩后,eGFP可穩(wěn)定高效表達,對細胞的生物學特性無明顯影響,是一種較為理想的標記方法。
[Abstract]:[Objective] to study the lentiviral vector (lentiviral, vectors, LVs) - mediated enhanced green fluorescent protein (enhanced green fluorescent protein, eGFP) labeled rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs)]. The feasibility of the method and effect of the biological characteristics of the cells isolated from rat BMSCs by density gradient centrifugation with Lenti-emGFP/hygro lentiviral vector transfection and hygromycin drug screening, microscopic observation of the cell morphology and the expression of eGFP, MTT before and after transfection method were used to examine the cell proliferation, cell surface markers were detected by flow cytometry (CD34, CD44, CD45, CD90 and eGFP), detection of osteoblasts after transfection the cells of the adipogenic differentiation potential and were stained by alizarin red staining, oil red O staining. The BMSCs rats showed fibroblast like morphology, the vortex like like arrangement, cell morphology did not change after transfection, and high expression of eGFP, two groups of fine Cell proliferation, flow cytometry showed that the positive rate of eGFP P3 in rat BMSCs-eGFP after transfection was 99.97%, two groups of cells before and after transfection, CD34 and CD45 positive rate of less than 2%, while the positive rate of CD44 was higher than that of 99%. and CD90 after transfection of P3 rats BMSCs-eGFP after osteogenic induction, alizarin red staining, there were small orange red calcium nodules, after adipogenic induction, oil red O staining showed a large number of red lipid deposition, osteogenic and adipogenic induction process, eGFP continued to express. Conclusion]Lenti-emGFP/hygro transfected rat BMSCs and hygromycin drug screening, eGFP stable high expression, no obvious effect the biological characteristics of the cells, is an ideal marker method.

【作者單位】： 第三軍醫(yī)大學附屬新橋醫(yī)院骨科;
【基金】：國家自然基金青年項目(編號:31000438) 國家自然科學基金面上項目(編號:81271362) 軍隊“十二五”重點項目(編號:BWS11C040)
【分類號】：R329

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本文編號：1641415