嵌合性5型腺病毒载体Ad5/F35体外转染大鼠骨髓间充质干细胞的研究

Transduction of rat bone marrow mesenchymal stem cells by a chimeric adenovirus serotype 5(Ad5/F35)in vitro

目的:研究携带35型腺病毒纤毛的嵌合性5型腺病毒载体Ad5/F35能否有效地将增强型绿色荧光蛋白(eGFP)基因体外转染大鼠骨髓间充质干细胞(BMSCs),并观察eGFP基因在大鼠BMSCs内持续表达的时间。方法:贴壁培养法分离、扩增大鼠BMSCs。用带有eGFP基因的Ad5/F35(Ad5/F35-eGFP)分别以1、10、100、1000的感染复数(MOI)转染第2代大鼠BMSCs;荧光倒置显微镜和流式细胞仪(FCM)检测eGFP基因的转染效率与表达水平,观察细胞生长状态以评估病毒对大鼠BMSCs基本生物学特性的影响;对MOI=100的病毒转染的大鼠BMSCs在转染后第2、7、14、21、30天分别用FCM检测大鼠BMSCs内eGFP的表达情况。结果:MOI在1到1000范围内,eGFP基因转染效率和表达水平与Ad5/F35-eGFP的MOI呈正向剂量相关性。MOI=100的病毒可转染90%左右的大鼠BMSCs,且eGFP基因在1个月内均有表达。MOI为1、10、100的病毒不影响大鼠BMSCs的活力和增殖能力。结论:Ad5/F35可以高效地将eGFP基因体外转染大鼠BMSCs,eGFP基因可持续表达1个月。本研究为Ad5/F35作为BMSCs的基因转移和表达载体进行细胞治疗与基因治疗提供了实验依据。

Objective：To investigate the efficiency of transducing rat bone marrow mesenchymal stem cells （BMSCs） with enhanced green fluorescent proteins （eGFP） gene in vitro by a chimeric adenovirus serotype 5 vector Ad5/F35 which carries an adenovirus serotype 35 fiber and observe the longevity of transgene expression in transduced rat BMSCs. Methods：Rat BMSCs was isolated and proliferated by adhesive culture; transduced rat BMSCs of passage 2 with eGFP gene using Ad5/F35 carrying an eGFP gene （Ad5/ F35-eGFP） at a multiplicity of infection （MOI） of 1,10,100 and 1000 respectively; tested the transduction efficiency and expression level of eGFP gene in rat BMSCs by fluorescent inverted microscope and flow cytometer （FCM）; studied the basic biological characteristic of transduced rat BMSCs by observing their growing state; tested the eGFP expression in rat BMSCs at day 2,7,14,21 and 30 respectively after transduction at a MOI of 100 by FCM. Results：A positive dose-response relationship was observed between the transduction efficiency and expression of eGFP gene and the Ad5/F35-eGFP concentrations ranging from 1-1000 MOI. Approximately 90% of the rat BMSCs can be transduced by Ad5/F35-eGFP at a MOI of 100,and the transgene expression can be detected for a month. The virus which infects rat BMSCs at a MOI of 1,10 and 100 can not impair the vitality and proliferative capability of transduced rat BMSCs. Conclusion-Ad5/F35-eGFP can transduce rat BMSCs with eGFP gene efficiently in vitro,the transgene expression in rat BMSCs can last a month. These data suggest that the Ad5/F35 is an effective vector of BMSCs for gene transfer and expression and it will be a powerful tool for gene therapy and cell therapy based on BMSCs .