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目的:为标记人间充质干细胞,探讨用反转录病毒转染人间充质干细胞的可行性。方法:实验于2003-10/2004-07在全军创伤修复重点实验室完成,无菌条件下穿刺人髂后上嵴抽取骨髓,采用直接贴壁法分离纯化人间充质干细胞,体外扩增,分别用免疫细胞化学法及流式细胞仪法对培养的人间充质干细胞进行鉴定。以含不完整反转录病毒、增强型绿色荧光蛋白、G418抗性基因的pLEGFP-C1为载体,用阳离子脂质体介导法转染包装细胞PT67,收集新鲜的含重组反转录病毒载体的PT67上清,加入终浓度为6mg/L的Polybrene感染人间充质干细胞,荧光显微镜观察。结果:体外原代培养的人间充质干细胞,24h内有少量成纤维样细胞贴壁,7d达到融合,免疫细胞化学示CD44和CD105表达阳性,CD34表达阴性,部分人间充质干细胞核5-溴脱氧尿嘧啶尿苷染色阳性。流式细胞仪示CD44阳性率为89.64%,CD34为0.11%。用脂质体介导法转染包装细胞PT67,有少量发绿色荧光细胞,通过G418筛选和有限稀释后,筛选出单克隆的转染PT67细胞。用含不完整重组反转录病毒颗粒的PT67上清,转染人间充质干细胞,有少量人间充质干细胞发绿光,转染后的人间充质干细胞增殖速度减慢。结论:虽然人间充质干细胞已用于许多临床前和临床研究,但观察结果表明用反转录病毒载体转染人间充质干细胞,转?
OBJECTIVE: To investigate the feasibility of transfection of human mesenchymal stem cells with retroviruses for the purpose of labeling human mesenchymal stem cells. Methods: The experiment was performed in the key laboratory of trauma repairing in the whole army from October 2003 to July 2004. The bone marrow was obtained by puncture of the posterior crista of ilium under aseptic conditions. Human mesenchymal stem cells were isolated and purified by direct adherent method. The cultured human mesenchymal stem cells were identified by immunocytochemistry and flow cytometry, respectively. Using pLEGFP-C1 with incomplete retrovirus, enhanced green fluorescent protein and G418 resistance gene as the carrier, the packaging cells PT67 were transfected by cationic liposome, and fresh recombinant retroviral vector Of PT67 supernatant was added to a final concentration of 6mg / L Polybrene infected mesenchymal stem cells, fluorescence microscopy. RESULTS: In vitro cultured human mesenchymal stem cells were adherent to a small number of fibroblast-like cells within 24h and reached confluence on day 7. Immunocytochemistry showed that CD44 and CD105 were positive and CD34 was negative. Some human mesenchymal stem cells showed nuclear 5-bromine Deoxyuridine uridine positive staining. Flow cytometry showed CD44 positive rate of 89.64%, CD34 0.11%. Transfection of PT67 with liposome-mediated packaging cells, a small amount of green fluorescent cells, through G418 screening and limited dilution, the monoclonal transfected PT67 cells were screened. Transfection of human mesenchymal stem cells with PT67 supernatant containing incompletely recombinant retroviral particles resulted in a small amount of human mesenchymal stem cells emitting green light and the proliferation of transfected human mesenchymal stem cells slowed down. CONCLUSIONS: Although human mesenchymal stem cells have been used in many preclinical and clinical studies, the observations indicate that transfection of human mesenchymal stem cells with retroviral vectors,