反转录病毒载体体外转染人间充质干细胞的可行性

Feasibility of human mesenchymal stem cells transfected with retroviral vector in vitro

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?

AIM:To investigate the feasibility of human mesenchymal stem cells(hMSCs) tran sfected with retroviral vector in vitro so as to label hMSCs. METHODS:The experiment was conducted in the Key Research Laboratory for Wound Repair of Chinese PLA from October 2003 to July 2004.The posterior superior ilia c spine of health adults was punctured under sterility condition for drawing bon e marrow.hMSCs were isolated and purified by means of direct adherent plastic,an d amplified in vitro.Then they were identified using immunocytochemical methods and flow cytometry.Package cell PT67 was transfected by using retroviral vector pLEGFP C1 encoding incompetent retrovirus,genes of enhanced green fluorescent protein(EGFP) and anti G418 as the carrier mediated by cationic liposome.The t ransfected monoclonal PT67 was screened using G418 and limited dilution,then the fresh supernatant of transfected PT67 carrying recombinant retroviral vector wa s collected by adding Polybrene(6 mg/L) to infect hMSCs.The results were observe d under fluorescence microscope. RESULTS:In primary cultured in vitro hMSCs,a few fibroid cells adhered to plas tic surface within 24 hours and fused at 7 days.Immunocytochemistry showed that hMSCs were positive for CD44 and CD105,and negative for CD34; BrdU of partial hM SC nuclei was stained positive.The positive rate of CD44 was 89.64%and CD34 was 0.11%shown by flow cytometry.There were small quantity of package PT67 cells t ransfected with EGFP mediated by cationic liposome.The transfected monoclonal PT 67 cells were screened by using G418 and limited dilution.The hMSCs exhibited gr een fluorescence a little after they were transfected with the supernatant of PT 67 carrying granules of incompetent recombinant retroviral vector.The transfecte d hMSCs proliferated at a decreasing velocity. CONCLUSION:Though hMSCs have been used in many clinical and pre clinical rese arches,the transfection rate is low using retroviral vector and the proliferatio n velocity of the transfected hMSCs is slowered,so it provides a theoretical bas is for the study of skin tissue engineering from another aspect.