IFI16基因在人喉鳞癌细胞中的作用研究

发布时间：2018-03-25 15:19

本文选题：IFI16　切入点：Hep-2　出处：《杭州师范大学》2012年硕士论文

【摘要】：喉鳞癌是头颈部常见恶性肿瘤,其发病率呈逐年上升趋势。目前,对喉癌等恶性肿瘤的传统治疗方法是手术、放疗和化疗。这些传统的方法对于治疗晚期的喉癌病人没有很大意义,所以近年来兴起综合治疗备受关注,干扰素就是其中一种。研究发现,作为干扰素可诱导家族HIN-200蛋白家族成员,人中的IFI16蛋白和小鼠中的p202蛋白都与系统性红斑狼疮(SLE)发病有关。多个研究证明HIN-200家族蛋白具有抑制细胞增殖的作用。由于这个原因,研究人员开始着手于研究IFI16基因的抗肿瘤作用。目前已知IFI16基因具有抗乳腺癌,前列腺癌的作用,并且在头颈部鳞状细胞癌HNO136细胞中表现出抑制细胞生长的作用。本研究将RT-PCR扩增得到的IFI16基因构建到真核表达质粒pVR1012,得到PVR1012-IFI16重组质粒,用脂质体将其转染导入Hep-2细胞,半定量RT-PCR分析IFI16基因在Hep-2细胞中的外来表达情况,细胞生长曲线绘制法及MTT法测定IFI16基因外来表达对Hep-2细胞增殖的影响,流式细胞术检测IFI16基因外来表达对Hep-2细胞周期及凋亡的影响；合成针对IFI16基因的shRNA,构建慢病毒表达载体pGreen Puro-IFI16-shRNA,用磷酸钙将其转染Hep-2细胞构建稳定表达这个载体的Hep-2细胞系.半定量RT-PCR分析结果发现,pVR1012-IFI16重组质粒转染的Hep-2细胞IFI16基因mRNA水平显著升高；细胞生长曲线测定结果发现,第2天开始pVR1012-IFI16重组质粒转染的Hep-2细胞生长变慢,至第3天时pVR1012-IFI16重组质粒转染Hep-2细胞其生长速度明显变慢,与mock转染及空载体转染对照细胞相比差异显著(P0.05)；MTT测定结果发现,pVR1012-IFI16重组质粒转染Hep-2细胞48h后,其相对活细胞数目显著降低,与mock转染及空载体转染对照细胞相比差异十分显著(P0.05及P0.01)；流式细胞术检测结果发现,与mock转染及空载体转染对照细胞相比,pVR1012-IFI16重组质粒转染48h后Hep-2细胞亚GO期细胞比例以及凋亡细胞比例都显著升高。上述研究结果说明,构建得到的pVR1012-IFI16重组质粒能在Hep-2细胞中外来表达IFI16基因,IFI16基因外来表达抑制Hep-2细胞增殖、阻滞Hep-2细胞周期在亚GO期并诱导Hep-2细胞发生凋亡；成功构建得到pGreen Puro-IFI16-shRNA,并且构建稳定表达pGreen Puro-control-shRNA的Hep-2细胞系,稳定表达pGreen Puro-IFI16-shRNA也已经在构建当中。本实验希望采用外来过表达技术来研究IFI16基因对于Hep-2细胞生长的影响,并且希望通过外来沉默技术作为辅助,验证IFI16基因在喉癌Hep-2细胞中的作用。从上述结果中看出IFI16基因抑制喉癌Hep-2细胞生长增殖方面的作用,并且使用脂质体转染,流式细胞仪等检测实验有效地验证这个猜测。这将为进一步阐明IFI16基因抑制喉癌Hep-2细胞生长增殖的分子机制奠定基础。
[Abstract]:Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor in the head and neck, and its incidence is increasing year by year. Radiotherapy and chemotherapy. These traditional methods do not have much significance in the treatment of advanced laryngeal cancer patients, so the rise of comprehensive therapy in recent years has attracted much attention. Interferon is one of them. As a member of the family of HIN-200 proteins induced by interferon, both IFI16 protein in human and p202 protein in mice are involved in the pathogenesis of SLEs. Several studies have shown that HIN-200 family proteins can inhibit cell proliferation. The researchers began to study the antitumor effects of IFI16 gene, which is known to have anti-breast and prostate cancer effects, and to inhibit cell growth in HNO136 cells of head and neck squamous cell carcinoma. In this study, the IFI16 gene amplified by RT-PCR was constructed into eukaryotic expression plasmid pVR1012, and the recombinant PVR1012-IFI16 plasmid was obtained. The recombinant plasmid was transfected into Hep-2 cells by liposome, and the extraneous expression of IFI16 gene in Hep-2 cells was analyzed by semi-quantitative RT-PCR. Cell growth curve drawing and MTT assay were used to detect the effect of foreign expression of IFI16 gene on the proliferation of Hep-2 cells. Flow cytometry was used to detect the effect of foreign expression of IFI16 gene on the cell cycle and apoptosis of Hep-2 cells. IFI16 gene was synthesized and lentivirus expression vector pGreen Puro-IFI16-shRNAwas constructed. The Hep-2 cell line expressing the vector stably was constructed by calcium phosphate transfection into Hep-2 cells. Semi-quantitative RT-PCR analysis showed that the IFI16 gene of Hep-2 cells transfected with recombinant plasmid pVR1012-IFI16 was constructed by semi-quantitative RT-PCR analysis. The level of mRNA increased significantly. The results of cell growth curve showed that the growth of Hep-2 cells transfected with pVR1012-IFI16 recombinant plasmid became slower on the second day, and at the third day, the growth rate of Hep-2 cells transfected with pVR1012-IFI16 recombinant plasmid was obviously slower. Compared with the control cells transfected with mock and empty vector, the results showed that the number of relative living cells decreased significantly 48 hours after transfection of pVR1012-IFI16 recombinant plasmid into Hep-2 cells. Compared with the control cells transfected with mock and empty vector, the difference was significant (P0.05) and P0.01G, the results of flow cytometry showed that, Compared with the control cells transfected with mock and empty vector, the proportion of subgo phase cells and the proportion of apoptotic cells in Hep-2 cells were significantly increased after transfection of pVR1012-IFI16 recombinant plasmid for 48 h. The constructed pVR1012-IFI16 recombinant plasmid could express IFI16 gene in Hep-2 cells and inhibit the proliferation of Hep-2 cells, block the cell cycle of Hep-2 cells in subgo phase and induce apoptosis of Hep-2 cells. PGreen Puro-IFI16-shRNAs were successfully constructed, and Hep-2 cell lines stably expressing pGreen Puro-control-shRNA were constructed. The stable expression of pGreen Puro-IFI16-shRNA was also in the process of construction. The purpose of this study is to study the effects of IFI16 gene on the growth of Hep-2 cells by using foreign overexpression techniques, and to use foreign silencing as an aid. To verify the role of IFI16 gene in laryngeal carcinoma Hep-2 cells. From the above results, we can see the role of IFI16 gene in inhibiting the growth and proliferation of laryngeal cancer Hep-2 cells, and transfection with liposome, Flow cytometry and other assays can effectively verify this hypothesis, which will lay a foundation for further elucidation of the molecular mechanism of IFI16 gene inhibiting the growth and proliferation of laryngeal cancer Hep-2 cells.
【学位授予单位】：杭州师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.65

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