介导穿孔素短发夹RNA重组腺病毒的构建及其生物学作用

Recombinant Adenovirus mediate RNA silencing of perforin in NK-92 cells and its function

目的:采用腺病毒介导的RNAi技术对穿孔素(PF)的功能进行体外研究,以期为深入研究PF的作用机制奠定基础。方法:构建腺病毒介导的穿孔素shRNA(Ad-shPF)。首先通过mRNA水平验证所设计的pShuttle-shPF序列的有效性,有效后包装Ad-shPF,验证其对NK-92细胞穿孔素mRNA和蛋白表达水平的影响。结果:转染pShuttle-shPF(1-3)的干扰效率分别为(74.2±4.1)%、(43.2±3.2)%、(61.7±2.6)%。干扰效果明显,其中又以pShuttle-shPF1的干扰效果最佳。成功包装Ad-shPF1,感染NK-92细胞,mRNA表达水平降至对照组的32%;Western blot分析表明Ad-shPF1处理后可在蛋白水平上明显抑制PF的表达。结论:PF shRNA重组腺病毒可以在mRNA和蛋白水平抑制NK-92细胞中PF的表达。

AIM：To adenovirus-mediated RNAi technology be used to down-regulate perforin expression in NK-92 cells.This study will provide convenience for further investigation of the mechanism of perforin.METHODS：First,we designed three perforin shRNAs（shPF） and identified their knockdown efficiency at mRNA levels.Then we constructed recombinant adenovirus with shPF.After that,we transfected NK-92 cells with Ad-shPF and detected the expression of perforin at mRNA and protein levels.RESULTS：We confirm that the design of shPF sequences are effective.Moreover,the mRNA level of NK-92 cells treated by Ad-shPFP1 drop to 32% compare to Ad-scramble treated control.Furthermore,using Western blot,we find Ad-shPF1 treatment can obviously down-regulate the expression of perforin protein.CONCLUSION：Recombinant adenovirus with perforin shRNA can knockdown the expression of Perforin in NK-92 cells.