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目的:观察人工构建的microRNA对鼻咽癌CNE-2细胞VEGF基因表达的调控效应,探讨microR-NA在鼻咽癌基因治疗中的应用前景。方法:构建靶向VEGF基因的microRNA表达质粒,脂质体法转染鼻咽癌CNE-2细胞,RT-PCR检测转染细胞中VEGF基因的mRNA水平,Western Blotting法观察microRNA对VEGF蛋白表达水平的调控效应,CCK-8比色法检测靶向VEGF的人工构建microRNA对鼻咽癌细胞生长的抑制作用,皮下注射稳定转染microRNA质粒的CNE-2细胞,构建裸鼠移植瘤模型,观察肿瘤生长情况。结果:在mi-croRNA表达质粒转染CNE-2细胞后,VEGF的mRNA表达水平下调,蛋白表达水平降低,但是光镜和荧光显微镜下的初步观察显示CNE-2细胞生长未发生变化,以CCK-8比色法检测的细胞增殖抑制率也无明显改变,而在初步的裸鼠移植瘤实验中,发现调控VEGF表达的microRNA重组质粒能有效抑制肿瘤生长。结论:人工构建表达microRNA的质粒,可在鼻咽癌CNE-2细胞中有效下调VEGF基因的表达水平,抑制裸鼠移植瘤的生长,为鼻咽癌基因治疗研究奠定了一定的基础。
Objective: To observe the regulatory effect of artificially constructed microRNA on VEGF gene expression in nasopharyngeal carcinoma CNE-2 cells and to explore the potential of microR-NA in the gene therapy of nasopharyngeal carcinoma. Methods: The microRNA expression plasmid targeting VEGF gene was constructed and transfected into CNE-2 cells by lipofectamine. The mRNA level of VEGF gene in transfected cells was detected by RT-PCR. The expression of VEGF protein was detected by Western Blotting The inhibitory effect of artificially constructed microRNA targeting VEGF on the growth of nasopharyngeal carcinoma cells was detected by CCK-8 colorimetric assay. CNE-2 cells stably transfected with microRNA plasmid were injected subcutaneously to construct the xenograft model in nude mice. growing situation. Results: After transfection of CNE-2 cells with mi-croRNA expression plasmid, the mRNA expression of VEGF was down-regulated and protein expression was decreased. However, preliminary observation under light microscope and fluorescence microscope showed that the growth of CNE-2 cells did not change. -8 colorimetric assay also showed no significant change in cell proliferation inhibition, and in preliminary nude mice xenograft experiment, found that the regulation of VEGF expression of microRNA recombinant plasmid can effectively inhibit tumor growth. CONCLUSION: Plasmids expressing human microRNA can effectively reduce the expression of VEGF gene in CNE-2 cells and inhibit the growth of transplanted tumor in nude mice, and lay a solid foundation for gene therapy of NPC.