SDF-1α-GFP基因重组腺病毒载体的构建及其转染大鼠心肌细胞的条件优化研究  

Construction of recombinant adenoviral vector of SDF-1α-GFP gene and the investigation of optimal transfection condition for rat myocardium

作　　者：郑栋[1] 郭军[1] 李海瑞[1] 李自成[1] 

机构地区：[1]暨南大学附属第一医院心内科,广州510630

出　　处：《临床心血管病杂志》2014年第12期1086-1090,共5页Journal of Clinical Cardiology

基　　金：国家自然科学基金青年项目资助(No:81100078);教育部科学研究重点项目资助(NO:211207);广州珠江科技新星资助计划项目(No:2012J2200063);广东省科技厅博士启动项目资助(No:S2011040001392)

摘　　要：目的：构建基质细胞衍生因子1α-绿色荧光蛋白（SDF-1α-GFP）重组腺病毒载体并摸索其转染大鼠心肌细胞系的最佳条件。方法：人工合成SDF-1α基因并插入线性化表达载体,转化感受态E.coliDH5a细胞。筛选阳性菌落,采用DNA测序技术测定目的基因序列。用获得的重组腺病毒（adenovirus,Ad）载体转染293T细胞,采用qPCR技术测定SDF-1α的mRNA表达水平。用不同滴度感染复数Ad-SDF-1α-GFP转染H9C2心肌细胞系,采用倒置相差显微镜及荧光显微镜分别在24和48h检测细胞形态及GFP蛋白阳性的细胞比率。结果：DNA测序证实SDF-1α基因构建成功。qPCR结果显示,与阴性对照组相比,MOI 2000组的SDF-1α的表达水平增加了约10 000倍,MOI 8000组的SDF-1α表达水平增加了约210 000倍。转染H9C2心肌细胞系的结果显示,MOI值为2 000、3 000、4 000、5 000、6 000、7 000及8 000的SDF-1α-GFP转染H9C2细胞48h后,其转染率分别为（45±5.04）%、（75±5.70）%、（80±5.67）%、（85±6.45）%、（90%±6.90）%、（90±5.22）%和（95±5.36）%,但是当MOI值区间为5 000～8 000时,细胞形态上出现明显损伤。结论：MOI值为4 000的Ad-SDF-1α-GFP转染H9C2细胞系48h能达到最佳的转染效率,并最小程度损伤细胞。Objective：To construct a recombinant adenoviral vector （Ad） carrying the SDF-1 gene and to inves- tigate the optimal condition of transfection. Method：The SDF-1 gene sequence was artificially synthesized and then was inserted into shuttle plasmid. The recombinant shuttle plasmid was constructed and transformed into compe- tent E. coliDH5a cells. Screening the positive colony, DNA sequencing proved Ad-SDF-1α-GFP was constructed successfully. Using the obtained recombinant adenoviral vector to transfect 293T cells, the expression level of SDF-1 a was measured by qPCR. H9C2 myocardial cells were transfected by the recombinant adenoviral solution with different multiplicity of infection （MOI） , the morphology and percentage of GFP-positive cells was observed and calculated post 24 and 48 hours of transfection respectively. Result：Compared to the negative control group, the expression level of SDF-1α increased about 10 000 times in MOI 2000 group and 210 000 times in MOI 8000 group according to the result of qPCR. The result of transfecting H9C2 cells showed that when the MOI was 2 000, 3 000, 4 000, 5 000, 6 000, 7 000, 8 000, the corresponding transfection efficiency was （45±5.04）%,（75 ±5.70）%,（80±5.67）%,（85±4.6.45）%,（90±6.90）%,（90±5.22）%,（95±5.36）% at 48 h post transfection, but when MOI was 5 000, 6 000, 7 000, 8 000, the cell morphology showed significant changes, which indicated cellular injury under such conditions. Conclusion：It might be the optimal transfection efficiency and the less slight cell injury that H9C2 cells are transfected with Ad-SDF-1α-GFP in MOI 4 000 for 48 hours.

关 键 词：基质细胞衍生因子1α基因 重组腺病毒 载体构建 H9C2细胞 

分 类 号：R373.1[医药卫生—病原生物学]