慢病毒转导增强型绿色荧光蛋白对大鼠骨髓间充质干细胞生物学特性的影响

发布时间：2018-03-21 00:13

本文选题：骨髓间充质干细胞　切入点：基因转染　出处：《中国矫形外科杂志》2013年21期 　论文类型：期刊论文

【摘要】：[目的]研究慢病毒载体(lentiviral vectors,LVs)介导增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)标记大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的可行性及对细胞生物学特性的影响。[方法]采用密度梯度离心法分离培养大鼠BMSCs,经Lenti-emGFP/hygro慢病毒载体转染和潮霉素药筛,镜下观察细胞形态和eGFP表达情况,MTT法检查转染前后细胞增殖活力,流式细胞仪检测细胞表面标记物(CD34、CD44、CD45、CD90)和eGFP,检测转染后细胞的成骨、成脂分化潜能并分别行茜素红染、油红O染色鉴定。[结果]大鼠BMSCs呈成纤维细胞样形态,漩涡样状排列,转染后细胞形态不变,并高效表达eGFP,两组细胞增殖活力一致,流式细胞仪检测显示,转染后的P3代大鼠BMSCs-eGFP的eGFP阳性率为99.97%,转染前后,两组细胞CD34和CD45阳性率低于2%,而CD44和CD90阳性率高于99%。转染后的P3代大鼠BMSCs-eGFP成骨诱导后,茜素红染色,可见呈桔红色的块状钙结节,成脂诱导后,油红O染色显示有大量红色脂质沉淀,成骨成脂诱导过程中,eGFP持续表达。[结论]Lenti-emGFP/hygro转染大鼠BMSCs并经潮霉素药筛后,eGFP可稳定高效表达,对细胞的生物学特性无明显影响,是一种较为理想的标记方法。
[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.

【作者单位】：第三军医大学附属新桥医院骨科;
【基金】：国家自然基金青年项目(编号:31000438) 国家自然科学基金面上项目(编号:81271362) 军队“十二五”重点项目(编号:BWS11C040)
【分类号】：R329

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