以Has-miR-100-3 p抑制剂慢病毒表达载体建立稳定感染A549细胞株

目的 用Has-miR-100-3p抑制剂的慢病毒来建立稳定感染人非小细胞肺癌(NSCLC)A549细胞株,为进一步研究miR-100在NSCLC细胞中的机制奠定基础.方法 用has-miR-100-3p抑制剂的重组慢病毒液,以其最适浓度感染A549细胞,获得稳定感染慢病毒的A549细胞株.荧光显微镜观察细胞感染效率,实时定量聚合酶链锁反应(qRT-PCR)和流式细胞术(FCM)分别检测稳定感染慢病毒的A549细胞株中miR-100的表达量和细胞株中绿色荧光蛋白(GFP)表达情况.结果 荧光显微镜下可见绿色荧光在细胞中表达.qRT-PCR检测结果显示,感染抑制剂的A549细胞中miR-100的表达量显著减少;流式细胞术检测感染组细胞中均有GFP的表达.结论 获得了has-miR-100-3p抑制剂的慢病毒稳定感染的A549细胞株,该细胞株中内源性miR-100的表达下调.

Establishment of A549 stable cell lines with lentivirus-based has-miR-100-3 p inhibitor

Objective To establish a stable infection of human non-small cell lung cancer ( A549 ) cell lines with has-miR-100-3p inhibitors lentiviral, provide material for further study of miR-100 in NSCLC cell mechanism. Methods We obtain a stable infected A549 cell lines by infecting A549 cells with optimal concentration of has-miR-100-3p inhibitor recombinant lentivirus. The high cell infection efficiency was observed by fluorescence mi-croscopy. The expression of miR-100 in A549 cells with or without infecting lentivirus was used to detect by quanti-tative real-time polymerase chain reaction ( qRT-PCR ) , and the green fluorescent protein ( GFP ) expression was used to detect by flow cytometry. Results We observed fluorescence expression in all A549 cells by fluorescence microscope. The expression of miR-100 in A549 cells after infecting with has-miR-100-3p inhibitors lentiviral was reduced significantly, measured by qRT-PCR. And the GFP expression was observed in all infected cell lines by flow cytometry. Conclusion The A549 stable cell lines with lentivirus-based has-miR-100-3p inhibitor is estab-lished in which the expression of miR-100 is knockdowned.