不同重组病毒载体在大鼠骨髓间充质干细胞中的转染效率及基因表达  

Transfection efficacy and gene expression of different recombinant viral vectors in rat mesenchymal stem cells in vitro

作　　者：潘虹[1] 刘新建[1] 吴继红[1] 田毓华[1] 谢匡成[1] 陈霞芳[1] 张圣海[1] 黄倩[1] 林志新[2] 

机构地区：[1]上海交通大学附属第一人民医院中心实验室,上海200080 [2]上海交通大学生命科学技术学院,上海200240

出　　处：《中国肿瘤生物治疗杂志》2007年第4期306-311,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家重点基础研究发展计划(973)资助项目(No2004CB518804);国家自然科学基金杰出青年资助项目(No30325043)~~

摘　　要：目的:探讨不同的重组病毒载体对体外培养的SD大鼠骨髓间充质干细胞(rMSCs)的转染效率和外源基因表达水平,为利用骨髓间充质干细胞进行细胞和基因治疗提供实验依据。方法:采用淋巴细胞分离液、密度梯度离心及体外培养方法分离rMSCs,流式细胞仪检测细胞表面CD11b、CD45和CD90的表达鉴定细胞类型。进一步用携带绿色荧光蛋白(EGFP)报告基因的重组Ad5-EGFP、Ad5F35-EGFP、rAAV1/2-EGFP、rAAV2-EGFP及Lentivirus-EGFP转染体外培养的rMSCs,荧光显微镜观察、流式细胞仪检测EGFP阳性率和荧光强度。结果:rMSCs细胞表面CD11b、CD45和CD90阳性率分别为(14.1±3.3)%、(1.1±0.4)%和(82.3±5.7)%。Ad5-EGFP按10、100和1000MOI转染rMSCs,2d后流式细胞仪检测,EGFP阳性率分别为(33.6±2.7)%、(88.6±1.0)%及(99.9±0.1)%,荧光强度为4.4±0.3、39.8±1.5及811.4±3.9;Ad5F35-EGFP按10、100和1000MOI转染rMSCs,2d后阳性率分别为(96.9±0.4)%、(99.9±0.1)%及(99.7±0.1)%,荧光平均强度为369.3±14.8、895.4±7.5及703.2±38.4;rAAV1/2-EGFP及rAAV2-EGFP按1×104和1×105vg/细胞转染rMSCs,6d后阳性率分别为(0.94±0.31)%及(1.30±0.36)%,和(2.16±0.38)%及(3.90±0.33)%;LV-EGFP按30(TU/细胞)转染rMSCs,6d后阳性率为(60.5±3.2)%,荧光强度为27.0±3.6。结论:Ad5、Ad5F35及LV能够有效转染体外培养的rMSCs并表达外源基因,转染效率与病毒的用量间存在量效关系。Objective ： To investigate the transfection efficacy and exogenous gene expression of different recombinant viral vectors in the rat mesenchymal stem cells （rMSCs） in vitro, so as to provide evidence for using MSCs in gene therapy. Methods： rMSCs were isolated by Filcoll-hypaque gradient centrifuging method （lymphocytes separation） and the cell types were identified by detecting CD1 lb, CD45 and CD90 by flow cytometry. Ad5-, AdSF35-, rAAV1/2-, rAAV2- and Lentivirus-encoding EGFP were used to transfect cultured mesenchymal stem cells separately, then the GFP positive rate and the mean intensity of GFP fluorescence were examined by flow cytometry and fluorescence microscopy, respectively. Results ： Flow cytometry assay showed that the positive rates of CD11 b, CD45 and CD90 were （ 14.1 ± 3.3 ） % , （ 1.1 ± 0. 4）% and （82.3 ±5.7 ）%, respectively. Two days after rMSCs were transfected with AdS-EGFP at 10,100, 1 000 MOI, the EGFP positive rates were respectively （33.6 ± 2.7） %, （ 88.6 ±1.0） % and （99.9 ±0.1 ） % and the mean intensity of EGFP fluorescence were respectively 4.4 ± 0.3, 39.8 ±1.5 and 811.4±3.9 ; the numbers for transfection with AdSF35-EGFP at 10,100, 1 000 MOI were respectively （96.9 ±0.4）% , （99.9 ±0.1）% , and （99.7 ±0.1）% and 369.3 ± 14.8, 895.4 ±7.5 and 703.2 ± 38.4. The EGFP positive rates in rMSCs transfected with 1 × 10^4 and 1 × 10^5 vgicell of rAAV1/2-EGFP and rAAV2-EGFP were （0.94±0.31）%, （1. 30 ±0. 36）%, and （2. 16±0.38）%, （ 3.90 ± 0.33 ） % , respectively. Six days after rMSCs were transfected with 30 TU/cell of Lentivirus-GFP, the EGFP positive rates and mean intensity of GFP fluorescence were （ 60.5 ±3.2 ） % and 27.0 ± 3.6, respectively. Conclusion： Ad5, Ad5F35 and Lentivirus can effectively transfeet rMSCs in vitro and express exogenous genes; the transfeetion efficacy is associated with the amount of the virus.

关 键 词：骨髓间充质干细胞 基因治疗 重组病毒载体 转染效率 基因表达 

分 类 号：R730.5[医药卫生—肿瘤]