小G蛋白基因重组腺病毒转染乳鼠心肌细胞实验研究

Transfection of neonatal rat cardiomyocytes with small G protein gene recombinant adenovirus

目的研究含有小G蛋白基因Rad重组腺病毒在体外转染乳鼠心肌细胞后的转染效率及表达。方法构建Rad腺病毒载体,分别以感染复数5、10、15和20转染体外培养的乳鼠心肌细胞,48h后,采用荧光显微镜观察转染后荧光强度,流式细胞仪检测转染率。以感染复数为20转染后分3组:空白对照组,阴性对照组和Rad过表达组,3d后,采用RT-PCR、Western blot检测Rad基因表达情况。结果感染复数5、10、15和20对应转染率分别为29.84%、69.69%、77.20%和84.81%。腺病毒转染3d后,Rad过表达组心肌细胞Rad基因mRNA和蛋白表达水平较空白对照组和阴性对照组显著增加(P<0.05)。结论外源性Rad基因可在乳鼠心肌细胞中稳定表达。

Objective To study the transfection efficiency of the adenovirus vector coding tor small G protein Rad and the expression of Rad in the cultured neonatal rat cardiomyocytes. Methods A Rad adenovirus vector was constructed and different multiplicity of infection of 5,10,15 and 20 was used to transfect the cultured neonatal rat cardiomyocytes. The fluorescence intensity and transfection efficiency were detected by fluorescence microscopy and flow cytometry respectively after 48 hours. Multiplicity of infection 20 after transfection was divided into control group,negative group and Rad gene overexpression group. Rad gene expression was detected by RT-PCR and Western blot after three days. Results The multiplicity of infection of 5,10,15 and 20 corresponding to the transfection efficiency was 29.84% ,69.69% ,77.20% and 84. 81%. The rnRNA and protein levels of Rad were higher in Rad gene overexpression group than in blank group and negative group（P〈0.05）. Conclusion The exogenous Rad gene can express in rat myocardial cells.