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目的:探讨人端粒酶逆转录酶(hTERT),癌基因蛋白(C-myc),存活素(Survivin),血管内皮生长因子(VEGF)基因对人类鼻咽癌细胞生长的影响,以及同时编码四个基因的短发夹重组质粒对人鼻咽癌细胞生长抑制作用及机制。方法:利用基因重组技术构建一个同时靶向作用四个基因的短发夹双链RNA(shRNA)真核表达载体和靶向单独作用hTERTmRNA的shRNA真核表达载体,脂质体法转染人CNE-2Z细胞;试验分组:空白对照组BC组(不进行干扰),阴性对照组NC组(加入阴性质粒),A组(hTERT单基因质粒组),B组(多基因联合质粒组)。激光共聚焦显微镜观察转染情况;MTT法检测细胞增殖活性;RT-PCR和Western blot法分别检测转染后细胞内各基因mRNA和蛋白表达情况。结果:MTT法检测,与BC相,NC组相比,A组和B组的细胞增殖活性均降低,与A组相比,B组的增殖活性降低更显著;RT-PCR,Western blot法,A组和B组mRNA和蛋白表达水平均降低,B组降低更显著。结论:四个基因共同参与了鼻咽癌细胞的发生和发展。多个基因的联合干扰与单基因干扰相比,能更高效下调各基因蛋白在鼻咽癌细胞的表达水平,更好抑制鼻咽癌细胞的增殖。
Objective: To investigate the effects of human telomerase reverse transcriptase (hTERT), C-myc, Survivin and VEGF gene on the growth of human nasopharyngeal carcinoma cells, Inhibitory effect of four genes of short hairpin recombinant plasmids on human nasopharyngeal carcinoma cells and its mechanism. Methods: A short hairpin double-stranded RNA (shRNA) eukaryotic expression vector targeting shRNA targeting four genes and a shRNA eukaryotic expression vector targeting hTERT mRNA alone were constructed by gene recombination technique. Human CNE The cells were divided into two groups: control group BC (no interference), negative control group NC (negative plasmid added), group A (hTERT single gene plasmid group) and group B (multiple gene combination plasmid group). The transfected cells were observed by laser confocal microscopy. The cell proliferation activity was detected by MTT assay. The mRNA and protein expression of each gene were detected by RT-PCR and Western blot respectively. Results: MTT assay showed that compared with BC and NC groups, the proliferation activity of cells in group A and group B was decreased. Compared with group A, the cell proliferation activity in group B was significantly reduced. The mRNA and protein expression of group A and group B were decreased, while group B was more significantly reduced. Conclusion: The four genes are involved in the occurrence and development of nasopharyngeal carcinoma cells. Compared with single gene interference, the combined interference of multiple genes can lower the expression level of each gene protein in nasopharyngeal carcinoma cells more effectively and inhibit the proliferation of nasopharyngeal carcinoma cells more effectively.