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目的:研究热休克蛋白60(HSP60)敲低对结肠癌SW480细胞增殖的影响,并进一步探究其作用机制。方法:通过含HSP60sh RNA载体的慢病毒感染加上流式细胞仪无菌分选的方法构建结肠SW480 HSP60基因稳定RNA干扰(RNAi)单克隆细胞系,利用Western blot和q-PCR验证结肠癌细胞中HSP60的敲低效率;使用CCK-8试剂检测结肠癌细胞增殖能力,并用流式细胞仪检测其HSP60敲低对细胞周期的影响。结果:Western blot和q-PCR结果验证了HSP60在结肠癌细胞中的敲低效率,与对照组细胞相比,实验组细胞HSP60的m RNA水平和蛋白水平均降低了60%以上。CCK-8实验结果表明,敲低HSP60后SW480细胞的增殖能力下降了约70%;流式细胞周期实验显示敲低HSP60后SW480细胞中G0/G1期、S期、G2/M期的分布比例变化不大。结论:敲低HSP60能够显著抑制SW480细胞的增殖能力,而SW480细胞周期并没有发生明显变化,推测HSP60的敲低引起的线粒体损伤导致细胞生长速度变慢。
Objective: To investigate the effect of heat shock protein 60 (HSP60) knockdown on the proliferation of colon cancer SW480 cells and to explore its mechanism. Methods: The colon cancer SW480 HSP60 gene stable RNA interference (RNAi) monoclonal cell line was constructed by lentivirus infection combined with HSP60 shRNA vector and sterile selection by flow cytometry. Western blot and q-PCR were used to confirm the colon cancer cells HSP60 knockdown efficiency; using CCK-8 reagent to detect the proliferation of colon cancer cells, and using flow cytometry to detect HSP60 knockdown on cell cycle. Results: The efficiency of knockdown of HSP60 in colon cancer cells was verified by Western blot and q-PCR. Compared with the control cells, the m RNA and protein levels of HSP60 decreased by more than 60%. The results of CCK-8 assay showed that the proliferation of SW480 cells was reduced by about 70% after knockdown of HSP60. Flow cytometry showed that the distribution of G0 / G1 phase, S phase and G2 / M phase in SW480 cells after knockdown of HSP60 Has not changed much. CONCLUSION: Knockdown of HSP60 can significantly inhibit the proliferation of SW480 cells, but the cell cycle of SW480 cells did not change significantly. It is speculated that the mitochondrial damage caused by HSP60 knockdown leads to slow cell growth.