人IL-24基因?qū)D133陽(yáng)性喉癌細(xì)胞作用的實(shí)驗(yàn)研究
[Abstract]:Objective: to clone human IL-24 gene and construct its eukaryotic expression vector. CD133 positive cells (CD133 Hep-2) were isolated from Hep-2 laryngeal carcinoma cell lines. The effect of IL-24 gene on CD133 Hep-2 cells was studied by studying the expression of human IL-24 gene in CD133 Hep-2 cells. Methods: normal human peripheral blood mononuclear cells (PBMC) were isolated. Total RNA, of mononuclear cells was extracted by Trizol method. Total RNA was reverse transcribed into cDNA, designed primer P1CP2to amplify human IL-24 gene. The TA was cloned into pMD19-T simple vector after correct agarose gel electrophoresis and gene sequencing. The eukaryotic expression vector pIRES2-ZsGreen1-hIL-24 of human IL-24 gene was constructed by NheI,XhoI double enzyme digestion of human IL-24 gene and eukaryotic expression vector pIRES2-ZsGreen1, and verified by enzyme digestion and gene sequencing. Flow cytometry (FCM) (FCM) was used to separate CD133 Hep-2 cells from Hep-2 cells. The correct pIRES2-ZsGreen1-hIL-24 was transferred into CD133 Hep-2 cells mediated by liposome 2000 and its expression was confirmed. The effect of human IL-24 gene on the growth of CD133 Hep-2 cells was detected by MTT,FCM, and the tumorigenic ability of CD133 Hep-2 cells transfected with human IL-24 gene was detected by tumor transplantation assay in nude mice. Results: the human IL-24 gene of 621bp was cloned successfully in human peripheral blood mononuclear cells, and the results of gene sequencing were consistent with those reported in GenBank. The recombinant plasmid pIRES2-ZsGreen1-hIL-24 was successfully transfected into CD133 Hep-2 cells by liposome-mediated transfection, and the human IL-24 gene was integrated into the transfected cells and expressed stably. The results of MTT assay showed that the growth of the cells transferred into the IL-24 gene group was significantly inhibited compared with the untransfected and empty vector control group (P0.05). The results of FCM showed that the apoptotic rate of the experimental group was significantly higher than that of the untransfected and empty vector control group (P0.05). Tumor transplantation in nude mice confirmed that the volume and weight of transplanted tumor of CD133 Hep-2 cells transferred to hIL-24 gene in nude mice were significantly lower than those in untransfected and empty vector control group (P0.05). Conclusion: the expression of human IL-24 gene can inhibit the proliferation of CD133 Hep-2 cells, promote their apoptosis and inhibit the growth of xenografts of CD133 Hep-2 cells in nude mice.
【學(xué)位授予單位】:吉林大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2014
【分類號(hào)】:R739.65
【共引文獻(xiàn)】
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