2种腺相关病毒介导的增强型绿色荧光蛋白对人成纤维细胞转染效率的研究  

AAV2-mediated versus AAV2/1-mediated enhanced green fluorescent protein transfection into human skin fibroblast in vitro

作　　者：郑钧丰[1] 孔永[2] 葛良鹏[1] 魏泓[1] 

机构地区：[1]第三军医大学基础医学部实验动物学教研室,重庆400038 [2]西南大学生命科学院,重庆400017

出　　处：《第三军医大学学报》2008年第12期1111-1113,共3页Journal of Third Military Medical University

基　　金：国家“十五”科技攻关计划重点项目(2004BA717B23)~~

摘　　要：目的比较2种不同腺相关病毒(adeno-associated virus,AAV)介导的增强型绿色荧光蛋白对人皮肤成纤维细胞(human fibroblast,HFB)的转染效率。方法原代分离培养人皮肤成纤维细胞并鉴定,按照感染复数(multiple of in-fection,MOI)为104、105、106转染传2代的人成纤维细胞,流式细胞检测AAV2-EGFP和AAV2/1-EGFP对HFB的转染效率,倒置荧光显微镜观察转染后的荧光强度。MTT法检测AAV2-EGFP和AAV2/1-EGFP对HFB的毒性。结果腺相关病毒AAV2-EGFP对HFB的转染效率随着MOI增加而增高,MOI为104时转染效率为(7.68±1.18)%,当MOI达到105后,转染效率稳定在(22.16±5.59)%,随着MOI增加,最高至MOI 106转染效率无显著提高。而病毒AAV2/1对于HFB的转染效率在MOI为106转染效率仅为(3.47±0.93)%。2种病毒在转染过程中,细胞生长正常,MTT显示各转染组与未转染组的吸光度值无统计学差异。结论AAV2载体对体外培养的HFB转染效率高于AAV2/1,但2种病毒载体对人皮肤成纤维细胞转染效率均不高,AAV2/1-EGFP对HFB几乎无感染能力。Objective To compare the transfection rate of AAV2-EGFP and AAV2/1-EGFP to human skin fibroblast （HFB）. Methods Two recombinant adeno-assoeiated viruses （AAV） encoding enhanced green fluorescent protein （EGFP） were constructed and transfeeted to HFB at multiple of infection MOI ranging from 10^4 to 10^6. Twenty-four hours after the infection, the expression rates of EGFP on cultured HFB were assessed by flow eytometry and the transfeeted cells were observed under fluorescence microscope. The killing effect of the virus on the infected cells was assayed by MTT. Results The transfeetion efficiency of AAV2-EGFP was increased as MOI increased. When MOI was 10^4, the transfeetion efficiency of AAV2-EGFP was 7.68%±1.18% ; When MOI was 10^5, that was 52.12% ±1.59% ; When MOI was 10^6, that showed no significant increase. However, the transfeetion efficiency of AAV2/1-EGFP showed no obvious changes at any MOI. Conclusion AAV2 is more efficient than AAV2/1 in transfeeting HFB, but neither AAV has a high transfection efficiency.

关 键 词：腺相关病毒 人皮肤成纤维细胞 增强型绿色荧光蛋白 流式细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学] R373[医药卫生—基础医学]