不同重组病毒载体转染大鼠骨髓间充质干细胞的效率及其基因表达(英文)  

Efficiency of different recombinant viral vectors in the transduction of bone marrow mesenchymal stem cells and exogenous gene expression

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,上海市200080 [2]上海交通大学生命科学技术学院,上海市200240

出　　处：《中国组织工程研究与临床康复》2008年第34期6790-6794,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：Special Funds for Major State Basic Research of China(973program),No.2004CB518804;the National Natural Science Foundation for Excellent Youth,No.30325043~~

摘　　要：背景:利用基因工程细胞进行替代治疗和基因治疗中合适的供体细胞、靶细胞以及相应载体是细胞和基因治疗最为困难的部分。目的:检测重组腺病毒Ad5和Ad5F35、腺相关病毒rAAV1/2和rAAV2、慢病毒LV对大鼠骨髓间充质干细胞的感染效率和外源基因表达水平,拟筛选能够高效转导骨髓间充质干细胞的载体。设计、时间及地点:细胞基因工程对照观察,于2006-10/2007-03在上海市第一人民医院中心实验室完成。材料:清洁级SD大鼠10只用于制备骨髓间充质干细胞。所有重组病毒均携带增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)报告基因,Ad5由本实验室制备,Ad5F35由解放军第二军医大学钱其军教授惠赠,rAAV2及rAAV1/2为本元正阳基因技术有限公司产品,LV由中科院上海生化与细胞生物学研究所郭礼和教授惠赠。方法:采用淋巴细胞分离液密度梯度离心法体外分离培养大鼠骨髓间充质干细胞,传至第4代用于实验。按1×105/孔接种于24孔细胞培养板中,1d后细胞贴壁,设立5组:第1组向细胞培养液中加入10,100,1000MOIAd5-EGFP,第2组加入10,100,1000MOIAd5F35-EGFP,第3组加入1×104,1×105vgrAAV1/2-EGFP,第4组加入1×104,1×105vgrAAV2-EGFP,第5组加入30TULV-EGFP。Ad病毒感染2d,rAAV及LV病毒感染6d。主要观察指标:EGFP阳性表达及荧光强度。结果:Ad5-EGFP感染24h后镜下可见EGFP阳性细胞,随着病毒用量的增加EGFP阳性细胞数目增多,荧光亮度增强,12d后阳性细胞开始逐渐减少,荧光亮度减弱。Ad5F35-EGFP感染情况与Ad5-EGFP基本相似,但EGFP阳性细胞数和荧光亮度均增加。rAAV1/2-EGFP或rAAV2-EGFP感染6d后,EGFP阳性细胞数和荧光亮度均较弱。LV-EGFP感染第2天即可见少量EGFP阳性细胞,随着时间延长EGFP阳性细胞数目逐渐增多,至第6天表达EGFP荧光的细胞数量及亮度不再有明显变化。结论:腺病毒Ad5、Ad5F35与慢病毒LV能够有效感染体外培养的骨髓BACKGROUND： Genetically engineered cells have been used in the replacement therapy and gene therapy. However, how to select proper donor cells, target cells, and corresponding viral vectors is the most difficult in the therapy. OBJECTIVE： To compare the transduction efficiency of recombinant adenovirus Ad5 and Ad5F35, adeno-associated virus rAAV1/2 and rAAV2, and lentivirus LV in bone marrow mesenchymal stem cells （BMSCs） and exogenous gene expression level, so as to select the vectors, which can efficiently transduce BMSCs. DESIGN, TIME AND SETTING： Gene engineering controlled observation, performed in the Central Laboratory, Shanghai First People＇s Hospital between October 2006 and March 2007. MATERIALS： Ten Sprague Dawley rats of clean grade were used to prepare BMSCs. All recombinant viral vectors carded enhanced green fluorescent protein （EGFP） report gene. AdS was prepared by the Central Laboratory, Shanghai First People＇s Hospital. AdSF35 was gifted by professor Qian Qi-jun from the Second Military Medical University of Chinese PLA. rAAV2 and rAAV1/2 were the products of Benyuan Zhengyang Gene Technique Co.,Ltd. LV was gifted by professor Guo Li-he from Shanghai Institute of Biochemistry and Cytobiology, Chinese Academy of Sciences. METHODS： Rat BMSCs were in vitro isolated and cultured by density gradient centrifugation. BMSCs of passage 4 were inoculated into 24-well plate at 1×10^5/well. One day later, cells adhered to the wall and allocated to 5 groups. Ad5-EGFP [10, 100, 1 000 multiplicity of infection （MOI）], AdSF35-EGFP （10,10, 1 000 MOI）, rAAV1/2-EGFP （1×10^4,1×10^5vg）, rAAV2-EGFP （1×10^4, 1×10^5vg）, and LV-EGFP （30 TU） were respectively added into the 5 groups. BMSCs were transduced for 2 days with Ad virus and separately for 6 days with rAAV and LV virus. MAIN OUTCOME MEASURES： EGFP-positive expression and fluorescence intensity. RESULTS： After twenty-four hours of AdS-EGFP transduction, EGFP-positive cells were visible under the microscope. With

关 键 词：骨髓间充质干细胞 细胞/基因治疗 重组病毒载体 转导效率 

分 类 号：R394.2[医药卫生—医学遗传学]