逆转录病毒介导EGFP基因转染骨髓间充质干细胞及类Schwann细胞诱导分化

发布时间：2018-04-05 13:03

本文选题：骨髓间充质干细胞　切入点：增强型绿色荧光蛋白　出处：《山东大学》2006年硕士论文

【摘要】：目的体外扩增培养兔骨髓间充质干细胞(MSCs)。定向诱导MSCs为类Schwann细胞。逆转录病毒载体法使MSCs转染增强型绿色荧光蛋白基因(EGFP)并稳定表达，观测转染后荧光表达强度及时间，，转染前后骨髓间充质干细胞的生物学特性变化，及转染对MSCs向类Schwann细胞诱导分化的影响。探讨MSCs作为组织工程人工神经种子细胞及利用EGFP作为体内试验示踪剂的可行性。方法密度梯度离心联合贴壁筛选法分离扩增培养兔骨髓间充质干细胞。采用β-巯基乙醇、全反视黄酸、福斯克林、碱性成纤维细胞生长因子、重组人血小板衍生生长因子等依次诱导MSCs向类Schwann细胞定向分化，S100免疫细胞化学染色鉴定。同时用pLEGFP-N1逆转录病毒载体质粒转染PT67包装细胞，提取病毒上清液后感染骨髓间充质干细胞，G418筛选及单克隆培养得到稳定表达绿色荧光蛋白的MSCs细胞(EGFP-MSCs)。观察测定转染前后骨髓间充质干细胞的生长曲线，细胞活性和贴壁率并比较。并以相同条件诱导EGFP-MSCs为类Schwann细胞，比较转染前后S100阳性表达率。结果 1．培养获取了较高纯度的MSCs，细胞增殖迅速，细胞活性保持较好。 2．类Schwann细胞诱导分化：经β-巯基乙醇诱导24 h后，细胞体
[Abstract]:Objective to expand and culture rabbit bone marrow mesenchymal stem cells (MSCs) in vitro.MSCs was induced to be Schwann-like cells.MSCs was transfected and stably expressed by retrovirus vector method. The intensity and time of fluorescence expression after transfection were observed, and the biological characteristics of bone marrow mesenchymal stem cells before and after transfection were observed.And the effect of transfection on the differentiation of MSCs into Schwann-like cells.To explore the feasibility of MSCs as artificial neural seeding cells for tissue engineering and EGFP as tracer in vivo.Methods Bone marrow mesenchymal stem cells were isolated and cultured by density gradient centrifugation and adherent screening.尾 -mercaptoethanol, total retinoic acid, forskine, basic fibroblast growth factor and recombinant human platelet-derived growth factor were used to induce MSCs to differentiate into Schwann like cells by immunocytochemical staining.At the same time, the PT67 packaging cells were transfected with pLEGFP-N1 retrovirus vector plasmid. The MSCs cells expressing green fluorescent protein (GFP) were obtained by G418 screening of bone marrow mesenchymal stem cells infected with virus supernatant, and the EGFP-MSCs cells were obtained by monoclonal culture.The growth curve, cell activity and adherent rate of bone marrow mesenchymal stem cells before and after transfection were observed and compared.The positive expression rate of S100 was compared before and after transfection of EGFP-MSCs as Schwann cells induced by the same conditions.Result 1.High purity MSCs were obtained by culture, the cells proliferated rapidly and the cell activity remained better.2.Differentiation of Schwann like cells: after induced by 尾 -mercaptoethanol for 24 h, cell body
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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