摘要
目的:研究含增强型绿色荧光蛋白(EGFP)基因的9型重组腺相关病毒(rAAV9)对大鼠心肌H9C2细胞的转染及对细胞生长的影响。方法:rAAV9-EGFP按转染复数(MOI)1×105、1×106、1×107转染H9C2细胞,转染后在倒置荧光显微镜下观察H9C2细胞中EGFP阳性表达情况,采用流式细胞仪检测rAAV9-EGFP对H9C2细胞的转染效率。观察转染后细胞生长形态,并用AlamarBlue法检测rAAV9-EGFP对H9C2细胞增殖影响。结果:转染后第1天,MOI=1×106、1×107组中EGFP开始表达;第2天,MOI=1×105组开始表达,各组EGFP表达强度随MOI值的增高而增强,同时EGFP的表达强度随着时间延长而逐渐增强;转染后第4天达到高峰,此时流式细胞术检测rAAV9-EGFP对H9C2细胞的转染率分别为MOI=1×105组:(14.1±0.2)%、MOI=1×106组:(35.1±4.8)%、MOI=1×107组:(56.8±0.1)%。rAAV9-EGFP转染H9C2细胞后,细胞生长及形态正常,AlamarBlue法进一步检测表明转染组对H9C2细胞增殖无影响。结论:rAAV9-EGFP能够有效地转染H9C2细胞,且rAAV9-EGFP对H9C2细胞增殖无明显抑制。
AIM：To evaluate the transfection efficiency using recombinant adeno-associated virus Serotype 9 mediated enhanced green fluorescent protein（rAAV9-EGFP）to rats H9C2 cells and the impact on growth of H9C2 cells.METHODS：rAAV9-EGFP was transfected into H9C2 cells at different multiplicities of infection（MOI=1×10^5,1×10^6,1×10^7）.EGFP expression in the cells was observed under inverted fluorescence microscope,and the EGFP-positive cell percentage determined by flow cytometry.Alamar Blue assay was used to assess the proliferation of the transfected cells.RESULTS：The cells with rAAV9-EGFP transfection at MOI of 1×10^6 and 1×10^7 began to exhibit EGFP expression 1 days after transfection and the cells transfection at MOI of 1×10^5 began to exhibit EGFP expression 2 days after transfection.the fluorescence intensity increased with the MOI used for transfection.EGFP expression reached the maximum on day 4,at the point of which the transduction efficiency of rAAV9-EGFP in H9C2 cells was（14.1±0.2）%,（35.1±4.8）% and（56.8±0.1）%.Corresponding to MOIs of 1×10^5,1×10^6 and 1×10^7,respectively.Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells after transfection.CONCLUSION：rAAV9-EGFP gene can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition.This study played foundation for further research.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第1期18-20,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30760263)
新疆自治区卫生厅青年科技人才专项基金(2007Y23)
关键词
9型重组腺相关病毒
增强型绿色荧光蛋白
心肌细胞
转染
recombinant adeno-associated virus serotype 9
enhanced green fluorescent protein
H9C2 cells
transfection
