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目的:构建增殖细胞抗原(PCNA)的小发夹结构RNA(shRNA)的真核表达载体并在HeLa细胞表达,同时观察PCNA的shRNA对人HeLa细胞株体外增殖的影响及生物学特性的改变。方法:将PCNA的cDNA的shRNA引物插入真核表达载体pGenesil1,构建真核表达质粒pGenesil1PCNA14,并通过酶切和测序等方法进行鉴定,将真核表达质粒pGenesil1PCNA14转染子宫颈癌细胞,运用Westernblot检测PCNA的蛋白表达,流式细胞仪分析细胞周期变化。结果:PCNA的shRNA能显著抑制人HeLa细胞的生长,阻滞细胞G0/G1S期,PCNA的蛋白表达同时受到抑制。结论:PCNA的shRNA能显著抑制人HeLa细胞的生长,其抑制作用可能通过阻断PCNA蛋白表达实现,实验结果为进一步研究PCNA的shRNA对子宫颈癌的基因治疗奠定了基础。
OBJECTIVE: To construct eukaryotic expression vector of small hairpin RNA (shRNA) of proliferating cell antigen (PCNA) and express in HeLa cells. At the same time, observe the effects of shRNA of PCNA on the proliferation of human HeLa cell line and its biological characteristics. Methods: The shRNA of PCNA cDNA was inserted into the eukaryotic expression vector pGenesil1 to construct the eukaryotic expression plasmid pGenesil1PCNA14, which was identified by restriction enzyme digestion and sequencing. The eukaryotic expression plasmid pGenesil1PCNA14 was transfected into cervical cancer cells and detected by Western blot PCNA protein expression, flow cytometry analysis of cell cycle changes. Results: shRNA of PCNA significantly inhibited the growth of human HeLa cells, and inhibited the expression of PCNA protein in G0 / G1S phase. CONCLUSION: shRNA of PCNA can significantly inhibit the growth of human HeLa cells. The inhibitory effect may be through blocking the expression of PCNA protein. The results of this study lay the foundation for the further study of shRNA of PCNA for gene therapy of cervical cancer.