论文部分内容阅读
目的:构建稳定表达LBH基因的人前列腺癌细胞株PC-3M-LBH,探讨LBH基因对PC-3M细胞增殖能力的影响。方法:构建表达LBH基因的重组慢病毒载体并制备出相应的慢病毒,感染低表达LBH基因的人前列腺癌PC-3M细胞后,经嘌呤霉素筛选获得细胞克隆;实时荧光定量PCR和蛋白印迹法(Western-Blot)分别检测细胞株中LBH的mRNA、蛋白表达水平;采用CCK-8法检测表达LBH基因后细胞增殖能力的改变。结果:成功构建了重组慢病毒表达质粒p Lenti-LBH并包装出了慢病毒,感染前列腺癌细胞后经嘌呤霉素筛选得到PC-3M-LBH细胞株;PC-3M-LBH细胞株中LBH基因的mRNA和蛋白表达显著上调;相对母细胞和NC对照组,PC-3M-LBH细胞在接种后第4天即出现明显的生长抑制,到第6天其生长抑制率达到19.7%。结论:构建的细胞株能稳定表达LBH基因,该基因的表达能显著抑制前列腺癌PC-3M细胞的体外增殖。
OBJECTIVE: To construct a PC-3M-LBH cell line stably expressing LBH gene and investigate the effect of LBH gene on the proliferation of PC-3M cells. Methods: The recombinant lentiviral vector expressing LBH gene was constructed and the corresponding lentivirus was constructed. The human prostate cancer PC-3M cells with low expression of LBH gene were infected and cloned by puromycin. Real-time fluorescence quantitative PCR and Western blot (Western-Blot) were used to detect the mRNA and protein expression of LBH in the cell lines respectively. The changes of cell proliferation were detected by CCK-8 assay. Results: The recombinant lentiviral plasmid p Lenti-LBH was successfully constructed and packaged into lentivirus. The PC-3M-LBH cell line was obtained by puromycin selection after infection of prostate cancer cells. The expression of LBH gene in PC-3M-LBH cell line The mRNA and protein expression of PC-3M-LBH cells were significantly up-regulated on the 4th day after inoculation compared with NC and NC control groups. The growth inhibition rate reached 19.7% on the 6th day. CONCLUSION: The constructed cell line stably expresses the LBH gene. The expression of this gene can significantly inhibit the proliferation of prostate cancer PC-3M cells in vitro.