文章摘要
杨 鎏,毛 曦,刘 英,李建勇.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[J].南京医科大学学报,2007,(10):1075~1079
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
投稿时间:2007-03-10  
DOI:10.7655
中文关键词: 间充质干细胞  Ad5/F35腺病毒载体  转染  绿色荧光蛋白  大鼠
英文关键词: mesenchymal stem cells  Ad5/F35 adenovirus vector  transfection  green fluorescent proteins  rats
基金项目:江苏省基础研究重大招标项目资助(BK2004004)
作者单位
杨 鎏 东南大学基础医学院 人体解剖与组织胚胎学系,江苏 南京 210009 
毛 曦 东南大学基础医学院 人体解剖与组织胚胎学系,江苏 南京 210009 
刘 英 东南大学基础医学院 人体解剖与组织胚胎学系,江苏 南京 210009 
李建勇 南京医科大学第一附属医院血液科,江苏 南京 210029 
摘要点击次数: 1425
全文下载次数: 97
中文摘要:
      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?
英文摘要:
      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.
查看全文   查看/发表评论  下载PDF阅读器
关闭