重组腺病毒介导的绿色荧光蛋白基因转染体外培养的人气道平滑肌的实验研究

Recombinant adenovirus vector mediated transfection of green fluorescent protein gene into human airway smooth muscles in vitro

目的:研究重组腺病毒载体介导的绿色荧光蛋白基因转染体外培养的人气道平滑肌(HASMCs)的方法。方法:用组织贴块原代培养传代至3～8代的HASMCs,重组绿色荧光蛋白基因的腺病毒载体(Ad-GFP)以不同的感染复数(multiplicity of infection,MOI)转染,荧光显微镜和流式细胞术检测其转染效率,CCK-8法检测其对细胞的毒性。结果:随着MOI增高,转染效率增高。MOI=100时Ad-GFP对HASMCs的感染效率可达(96±0.32)%,以感染后48h达高峰,持续7d仍有绿色荧光表达;转染后的细胞与未转染细胞相比,细胞毒性无统计学差异(P>0.05)。结论:重组腺病毒载体介导的绿色荧光蛋白基因Ad-GFP能高效转染HASMCs,转染后对细胞无毒性,是一种安全的基因载体。

Objective To evaluate the efficiency of recombinant adenovirus vector mediated transfection of green fluorescent protein gene (GFP) into human airway smooth muscle (HASMCs) in vitro. Methods Three to eight passages of HASMCs cultured by tissue adherence method from the trachea were infected with different multiplicity of infection(MOI). The transfection efficiency was assessed by fluorescence microscope and flow cytometry,and the cell cytotoxicity was measured by CCK-8. Results The transfection efficiency was promoted with increasing MOI. The infection efficiency of Ad-GFP to HASMCs was about (96 ± 0.32)% at 100 MOI after 48 hours of infection,and the expression of GFP gene maintained 7 days after infection. There were no statistically differences in cell cytotoxicity between the un-infected cells and the Ad-GFP infected cells (P > 0.05). Conclusion Ad-GFP can infect HASMCs efficiently, and is a safe gene vector with no cell cytotoxicity in vitro.