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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。
Objective To construct eukaryotic expression vector of small hairpin RNA (shRNA) of PCNA and express it in Hela cells. At the same time, observe the effects of shRNA of PCNA on the proliferation of human Hela cell line in vitro and its biological characteristics. METHODS: The cD-NA shRNA of PCNA was inserted into the eukaryotic expression vector pGenesil-1 to construct the eukaryotic expression plasmid pGenesil-1-PCNA1-4. The recombinant plasmid pGenesil-1-PCNA1-4 was identified by restriction enzyme digestion and sequencing. 1-PCNA1-4 was transfected into cervical cancer cells. The protein expression of PCNA was detected by Western blot. The cell cycle was analyzed by flow cytometry. Results shRNA of PCNA could significantly inhibit the growth of human Hela cells and arrest the expression of PCNA protein in G0 / G1-S phase. Conclusion The 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 a foundation for further study of gene therapy of cervical cancer by PC-NA shRNA.