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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。
OBJECTIVE: To study the function of perforin (PF) using adenovirus-mediated RNAi technique in vitro in order to lay the foundation for the further study of the mechanism of PF. Methods: To construct adenovirus-mediated perforin shRNA (Ad-shPF). First, the pShuttle-shPF sequence was verified by mRNA level validation and then packaged into Ad-shPF to verify its effect on the expression of perforin mRNA and protein in NK-92 cells. Results: The interference efficiency of transfected pShuttle-shPF (1-3) were (74.2 ± 4.1)%, (43.2 ± 3.2)% and (61.7 ± 2.6)%, respectively. The interference effect is obvious, among which the interference effect of pShuttle-shPF1 is the best. Ad-shPF1 was successfully packaged to infect NK-92 cells. The mRNA expression of Ad-shPF1 was reduced to 32% of control cells. Western blot analysis showed that Ad-shPF1 significantly inhibited the expression of PF at protein level. Conclusion: PF shRNA recombinant adenovirus can inhibit the expression of PF in NK-92 cells at mRNA and protein levels.