【摘要】 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。更多还原

【Abstract】 Objective: To construct eukaryotic expression plasmid of small hairpin RNA （shRNA） of PCNA, and to investigate the inhibitory effect of shRNA of PCNA on HeLa cell proliferation. Methods: The cDNA shRNA prime of PCNA was inserted into downstream of the vector pGenesil-1 to obtain the recombinant eukaryotic expression plasmid pGenesil-1-PCNA1-4 which was identified with the methods of enzyme digestion and sequence analysis. And then transfected the recombinant plasmid pGenesil-1-PCNA1-4 into eukaryotic HeLa cells by cationic polymer-mediated transfection. The inhibition of HeLa cell proliferation was estimated by immunohistochemistry and Western blot method, and the cell cycle was analyzed by flow cytometry. Results: The shRNA of PCNA inhibited the growth rate of HeLa cells and arrested proliferation of HeLa cells in stage of G 0/G₁-S. Conclusion: The shRNA of PCNA might inhibit the prdiferation of HeLa cell lines by inbiting expression of PCNA, and the results are helpful for further researching in gene therapy of uterine cervix carcinoma.更多还原