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目的建立稳定感染人hsa-miR-100-3p抑制剂慢病毒表达载体的人支气管上皮(human bronchial epithelial,BEAS-2B)细胞株,为研究miR-100的功能奠定基础。方法根据hsa-miR-100-3p的成熟序列,设计其反向互补序列,用聚合酶链式反应(polymerase chain reaction,PCR)的方法扩增目的基因,将目的基因与慢病毒载体GV280经双酶切后,进行定向连接,产生GV280-hsa-miR-100-3p-inhibitor慢病毒表达载体。将制备好的重组慢病毒质粒与两种辅助包装原件载体质粒共转染人胚肾上皮细胞293(human embryo kidney epithelial cell,HEK-293)T细胞,包装产生病毒颗粒并以最适滴度感染BEAS-2B细胞以获得稳定感染的细胞株。倒置荧光显微镜观察感染效率,用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)的方法检测重组慢病毒感染后BEAS-2B细胞中miR-100的相对表达量。结果测序结果表明,目的基因成功的连接到慢病毒载体上,重组慢病毒高效的感染了BEAS-2B细胞。RT-PCR检测发现,BEAS-2B细胞中miR-100的相对表达明显的下调了。结论稳定感染hsa-miR-100-3p抑制剂的BEAS-2B细胞株构建成功,敲低了细胞中内源性miR-100的表达。
Objective To establish a human bronchial epithelial (BEAS-2B) cell line stably infected with the hsa-miR-100-3p inhibitor lentiviral vector and lay a foundation for the study of the function of miR-100. Methods According to the mature sequence of hsa-miR-100-3p, the reverse complementary sequence of hsa-miR-100-3p was designed and the target gene was amplified by polymerase chain reaction (PCR). The target gene was ligated with the lentiviral vector GV280 After digestion, directional connection was made to generate lentiviral expression vector of GV280-hsa-miR-100-3p-inhibitor. The prepared recombinant lentiviral plasmid was co-transfected with two kinds of auxiliary packaging original vector plasmids into human embryo kidney epithelial cell (HEK-293) T cells and packaged to produce virus particles and infected with the optimal titer BEAS-2B cells to obtain stable infected cell lines. Infection efficiency was observed by inverted fluorescence microscope. The relative expression of miR-100 in BEAS-2B cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results The sequencing results showed that the target gene was successfully ligated into the lentiviral vector and the recombinant lentivirus efficiently infected the BEAS-2B cells. The relative expression of miR-100 in BEAS-2B cells was significantly down-regulated by RT-PCR. Conclusion The BEAS-2B cell line stably infected with hsa-miR-100-3p inhibitor was successfully constructed and knocked down the expression of endogenous miR-100 in cells.