重组腺相关病毒介导增强型绿色荧光蛋白在骨髓间充质干细胞中的表达

Expression of enhanced green fluorescent protein mediated by recombinant adeno-associated virus in mesenchymal stem cells

目的:重组腺相关病毒是携带治疗目的基因的有效载体.骨髓间充质干细胞则可作为外源基因的载体和表达场所实验选择携带绿包荧光蛋白的腺相关病毒体外转染骨髓间充质干细胞,观察转染效果。方法:实验于2006-01/12在咸宁学院心血管疾病研究所和武汉大学心内科实验室完成清洁级成年SD大鼠6只.由武汉大学医学院试验动物中心提供,实验过程中对动物的处置符合动物伦理学标准。携带增强型绿色荧光蛋白的重组腺相关病毒载体由北京本元正阳基因技术公司提供,基因启动子为CMV,有效滴度4×10^(11)v.g/mL。全骨髓法分离大鼠骨髓间充质干细胞,于体积分数为0.1的胎牛血清中培养.扩增纯化至第3代。按感染复数分别为10~2.10~3.10~4,10~5 v.g/cell加入携带增强型绿色荧光蛋白的重组腺相关病毒。于4,8,24、48 h倒置相差荧光显微镜下观察腺相关病毒感染骨髓间充质干细胞的状态,随机记录200个细胞中增强型绿色荧光蛋白阳性表达率.计算感染效率。结果:①骨髓间充质干细胞转染效率:感染复数为10~3.10~4,10~5 v.g/cell时.转染8 h后骨髓间充质干细胞内可见绿色荧光蛋白的表达.其中105 v.g/cell感染效果最佳.于转染48 h时荧光强度达最高值.感染效率约28%②骨髓间充质干细胞经G418筛选后检测增强型绿色荧光蛋白阳性细胞率达98%。结论:介导绿色荧光蛋白的腺相关病毒转染骨髓间充质干细胞简便易行,感染复数为10~5v.g/cell时效果较佳,目的基因表达稳定,用于后续基因治疗具备一定的可行性。

AIM： Recombinant adeno-associated virus is an effective carrier for target gene therapy. Mesenchymal stem cells are carrier and expression locus of exogenous gene. The study observed the transfectior, efficiency of recombinant adeno-associated virus with enhanced green fluorescent protein transduction to rat bone marrow mesenchymal stem cells in vitro. METHODS： Experiments were conducted at the Institute of Cardiovascular Disease, Xianning University and Research Laboratory in the Department of Cardiology, Wuhan University from January to December 2006. Six clean SD rats were supported by the Experimental Animal Center of Medical School of Wuhan University. Animal intervention met the animal ethical standard. Recombinant adeno-associated virus was supported by Benyuanzhenyang Biology Company. Gene promotor was CMV with the effective titre of 4×10^11 v.g/mL. Mesenchymal stem cells were isolated from the femur and tibia of rats, cultured in fetal bovine serum of 0.1 volume fraction, amplified and purified to the 3^rd passage. Mesenchymal stem cells were transfected with recombinant adeno-associated virus combined with enhanced green fluorescent protein at different multiplicity of infection （102, 103, 104, 105 v.g/cell）. The dynamic transfection state was observed by inverted phased fluorescence microscope in 4, 8, 24 and 48 hours after transfection. Positive expression of enhanced green fluorescent protein of 200 cells was randomly recorded to measure infection efficiency. RESULTS： ①Transfection efficiency of mesenchymal stem cells： The expression of enhanced green fluorescent protein was observed after 8 hours of recombinant adeno-associated virus transfection （multiplicity of infection 103, 104,10s v.g/cell）. The best multiplicity of infection is 10s v.g/cell. Transfection efficiency was the highest （28%） after 48 hours. ② After treatment of G418, the efficiency of enhanced green fluorescent protein expression in mesenchymal stem cells was 98%. CONCLUSION： The method of recombinant adeno-associated virus transduction to mesenchymal stem cells is easy and gene expression in mesenchymal stem cells is stable. Transfection efficiency was the highest when the best multiplicity of infection is 105 v.g/cell. Thus, it is feasible for subsequent gene therapy.