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目的:建立稳定表达RFX6基因的肝癌细胞株。方法:构建人源RFX6基因的重组质粒(pLNCX2-RFX6),pVSV-G质粒分别与pLNCX2-RFX6和空载体pLNCX2共转染至GP2-293细胞中进行包装,并用包装的逆转录病毒感染人肝癌QGY-7703细胞株,G418筛选出阳性克隆。RT-PCR和蛋白质印迹法验证稳定细胞株RFX6基因表达情况。采用Image J软件进行灰度值分析比较两株细胞系的差异。结果:在QGY-7703-pLNCX2-RFX6和QGY-7703-pLNCX2细胞中,RFX6/18SmRNA电泳条带灰度比值分别为5.044 0±1.384 0和0.357 2±0.407 9,QGY-7703-pLNCX2-RFX6细胞RFX6/18S是QGY-7703-pLNCX2细胞的14.1倍,差异有统计学意义,t=4.143,P<0.05;而在两细胞株中,RFX6/GAPDH的蛋白质印迹条带灰度值分别为1.391 4±0.141 3和0.127 8±0.037 3,QGY-7703-pLNCX2-RFX6细胞RFX6/GAPDH是QGY-7703-pLNCX2细胞的10.9倍,差异有统计学意义,t=14.966,P<0.05。结论:成功构建稳定表达RFX6基因的肝癌细胞株,为研究RFX6基因在肝癌细胞中的功能奠定了基础。
Objective: To establish a hepatocellular carcinoma cell line stably expressing RFX6 gene. Methods: Recombinant plasmids (pLNCX2-RFX6) and pVSV-G were constructed by co-transfecting pLNCX2-RFX6 and empty vector pLNCX2 into GP2-293 cells for packaging. The packaged retroviruses were used to infect human hepatocellular carcinoma QGY-7703 cell line, G418 positive clones were screened. The expression of RFX6 gene in stable cell lines was verified by RT-PCR and Western blotting. Gray value analysis was performed using Image J software to compare the differences between the two cell lines. Results: The gray scale ratios of RFX6 / 18S mRNA bands in QGY-7703-pLNCX2-RFX6 and QGY-7703-pLNCX2 cells were 5.044 ± 1.3840 and 0.357 2 ± 0.407 9, respectively. QGY-7703-pLNCX2-RFX6 cells RFX6 / 18S was 14.1-fold higher than QGY-7703-pLNCX2 cells, t = 4.143, P <0.05; however, the gray value of RFX6 / GAPDH in two cell lines was 1.391 4 ± 0.141 3 and 0.127 8 ± 0.037 3 respectively. The RFX6 / GAPDH of QGY-7703-pLNCX2-RFX6 cells was 10.9 times of QGY-7703-pLNCX2 cells, the difference was statistically significant, t = 14.966, P <0.05. Conclusion: The successful construction of a hepatoma cell line stably expressing the RFX6 gene lays the foundation for the study of the function of the RFX6 gene in hepatocellular carcinoma cells.