PCNA的shRNA的真核表达载体的构建及对子宫颈癌细胞的干预性研究

目的构建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cD-NA的shRNA引物插入真核表达载体pGenesil-1,构建真核表达质粒pGenesil-1-PCNA1-4,并通过酶切和测序等方法进行鉴定,将真核表达质粒pGenesil-1-PCNA1-4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/G1—S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PC-NA的shRNA对子宫颈癌的基因治疗奠定了基础。

Constructed Eukaryotic Expression Plasmid of Small Hairpin RNA (shRNA) of PCNA and Investigated the Inhibitory Effect on Hela Cell

Objective Constructed eukaryotic expression plasmid of small hairpin RNA (shRNA) of PCNA, then expressed plasmid in Hela cells and investigated the inhibitory effect of shRNA of PCNA on Hela cell carcinoma cell proliferation. Methods The cDNA shRNA the prime of PCNA was inserted into downstream of the vector pGenesil-1 to obtain the recombinant eukaryotic expression plasmid pGenesil-1-PCNA1-4 which we identified with the methods of enzyme digestion and sequence analysis. The results were consistent with what we expected. And then transfected the recombinant plasmid pGenesil-1-PCNA1-4 into eukaryotic Hela cells by cationic polymer-mediated transfection. The inhibition of the Hela cell carcinoma cell proliferation was estimated by immunohistochemistry and western blot method, the cell cycle was analysed by flow cytometric. Results shRNA of PCNA inhibited the growth rate of Hela cell and arrested proliferation of Hela cell in phage of G_ 0/G_1-S. Conclusion shRNA of PCNA might be effective in the behavior of Hela cell lines and inhibited the proliferating cell nuclear antigen by inhibiting expression of PCNA. This result laid the foundation for further researching in gene therapy of uterine cervix carcinoma.