9型重组腺相关病毒转染大鼠心脏成纤维细胞的体外研究  

Recombinant adeno-associated virus serotype 9 transfection of rat cardiac fibroblasts in vitro

作　　者：杜雷[1] 马依彤[1] 杨毅宁[1] 向阳[1] 陈邦党[1] 刘芬[1] 高霞[1] 

机构地区：[1]新疆医科大学第一附属医院心脏中心,新疆乌鲁木齐市830054

出　　处：《心血管康复医学杂志》2010年第2期121-125,共5页Chinese Journal of Cardiovascular Rehabilitation Medicine

基　　金：国家自然科学基金项目(30760263);新疆自治区卫生厅青年科技人才专项基金(2007Y23);新疆维吾尔自治区重点实验室(XJDX0903)

摘　　要：目的:研究9型重组腺相关病毒(rAAV9)对大鼠心脏成纤维细胞的转染效率及其对细胞生长的影响。方法:分离、获取并培养大鼠心脏成纤维细胞,以携带增强型绿色荧光蛋白(EGFP)报告基因的rAAV9(rAAV9-EGFP)转染大鼠心脏成纤维细胞,以转染复数(MOI)将细胞分为未转染病毒A组、B组MOI=1×104、C组MOI=1×105、D组MOI=1×106,倒置荧光显微镜下观察大鼠心脏成纤维细胞中EGFP的表达情况,观察转染后细胞生长形态,应用流式细胞仪检测rAAV9-EGFP对大鼠心脏成纤维细胞的转染效率,应用AlamarBlue法检测rAAV9-EGFP对大鼠心脏成纤维细胞增殖的影响。结果:D组的细胞于转染后24h即可观察到EGFP表达,B组与C组细胞则于48h观察到EGFP的表达。倒置荧光显微镜下观察转染后大鼠心脏成纤维细胞生长及形态正常,镜下观察显示各组细胞表达EGFP的强度随MOI值的增高而增强,同时亦随着时间延长而增强,EGFP表达在转染120h达到高峰,此时检测rAAV9-EGFP对心脏成纤维细胞的转染效率,分别为B组:(2.6±0.2)%,C组:(7.3±1.4)%,D组:(45.1±2.7)%。AlamarBlue法检测表明转染组细胞各时点与未转染组的还原率比值接近于1。结论:rAAV9-EGFP能够有效转染大鼠心脏成纤维细胞,转染后EGFP基因能够有效得以表达,rAAV9对细胞增殖无明显抑制。9型重组腺相关病毒可作为基因治疗心脏疾病研究的理想载体。Objective： To assess the transfection efficiency of recombinant adeno--associated virus serotype 9 mediated enhanced green fluorescent protein （rAAV9-EGFP） to rat cardiac fibroblasts and the impact on growth of rat cardiac fibroblasts. Methods： The rat cardiac fibroblasts were cultured and dissociated. The rAAV9-EGFP was transfected into rat cardiac fibrohlasts at different multiplicities of infection （MOI= 1 × 10^4 , 1 × 10^5 , 1 × 10^6 ） . EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP--positive cell percentage was determined by flow cytometry. AlamarBlue assay was used to assess the proliferation of the transfected cells. Results： The cells with rAAV9-EGFP transfection at MOI of 1 ×10^6 began to exhibit EGFP expression 24 h after transfection and the cells transfection at MOI of 1 ×10^5 and 1 × 10^4 began to exhibit EGFP expression 48 h after transfection. The cells growth was normal in all three groups under inverted fluorescence microscope. EGFP expression reached the maximum at 120 h, at the point of＇which the transduetion efficiency of rAAVg--EGFP in rat cardiac fibroblasts ceils was （2. 6±0. 2）%, （7.3±1.4）% and （45.1±2.7）% corresponding toMOIsof 1×10^4, 1×10^5and 1×10^6, respectively. The fluorescence intensity increased with the MOI and time increasing. AlamarBlue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells. Conclusion; rAAV9--EGFP gene can be stably and efficiently expressed in rat cardiac fibroblasts without inhibiting cell growth , suggesting the potential of rAAV9 as a safe and efficient vector for gene therapy of heart disease research.

关 键 词：病毒 绿色荧光蛋白类 成纤维细胞 转染 

分 类 号：R542.2[医药卫生—心血管疾病]