靶向Survivin的siRNA对鼻咽癌细胞生长抑制作用的研究

发布时间：2018-04-15 10:41

本文选题：Survivin + 真核表达载体　；参考：《南华大学》2012年硕士论文

【摘要】：第一部分目的:通过构建靶向Survivin的shRNA真核表达载体的方法来探讨RNAi技术对鼻咽癌细胞生长抑制作用，为鼻咽癌的基因治疗提供新的实验依据。方法:以凋亡抑制基因Survivin为靶位，构建表达小干扰RNA(siRNA)的真核表达载体，经脂质体转染鼻咽癌细胞系CNE-2，应用RT-PCR检测鼻咽癌细胞中SurvivinmRNA表达抑制情况，初步鉴定干扰效果。结果:重组载体pGPU6/GFP/Neo-shRNA1,2,3,4,N经双酶切和基因测序检测,证实碱基序列与模板序列相符，无基因突变，载体构建成功。重组载体经脂质体Lipofectamine2000介导转染鼻咽癌细胞，细胞发生不同程度凋亡，转染效率约为55%。RT-PCR方法初步鉴定其干扰效果，以Survivin/GAPDH的相对表达来表示SurvivinmRNA的变化,各组的mRNA表达相差较大，24h测得的mRNA相对于转染前有一定下降，大约在50%左右；但是随着时间延长，重组质粒的干扰作用逐渐减弱。结论：本实验通过构建靶向Survivin的shRNA真核表达载体的方法，经脂质体介导，，转染鼻咽癌细胞系CNE-2，在一定程度上抑制细胞中Survivin的基因表达水平。证实了应用RNAi技术对鼻咽癌细胞生长有一定程度的抑制作用，并为鼻咽癌的基因治疗提供了新的实验数据。第二部分目的：通过化学合成的小干扰RNA(smallinterferingRNA，siRNA)的方法来探讨RNAi技术对鼻咽癌细胞生长抑制作用，为鼻咽癌的基因治疗提供新的实验依据。方法：根据siRNA的设计原则设计针对survivin基因序列特异性的siRNA，在脂质体介导下转染CNE-2细胞。采用RT-PCR和Westernblotting检测Survivin在干扰后mRNA和蛋白的表达情况，并筛选出干扰效果最好的siRNA序列进行细胞生长抑制试验（CCK-8实验）检测对细胞生长抑制作用。结果：转染siRNA24h、48h和72h后，用RT-PCR和Westernblotting检测，CNE-2Z细胞survivin表达明显下降，并筛选出抑制效果最好1号序列进行CCK-8实验，结果显示能明显抑制细胞生长增殖。结论：靶向Survivin的siRNA转染人鼻咽癌CNE-2细胞有效抑制细胞中Survivin基因的表达水平，并对细胞生长增殖等生物学行为产生抑制作用。证实了应用通过化学合成的siRNA的RNAi技术对鼻咽癌细胞生长有显著的抑制作用为鼻咽癌的基因治疗提供了新的实验数据。
[Abstract]:Part oneAim: to investigate the inhibitory effect of RNAi on the growth of nasopharyngeal carcinoma cells by constructing shRNA eukaryotic expression vector targeting Survivin, and to provide a new experimental basis for gene therapy of nasopharyngeal carcinoma.Methods: the eukaryotic expression vector expressing small interfering RNAs siRNAs was constructed with Survivin as the target. The nasopharyngeal carcinoma cell line CNE-2 was transfected with liposome. The inhibition of SurvivinmRNA expression in nasopharyngeal carcinoma cells was detected by RT-PCR, and the interference effect was preliminarily evaluated.Results: the recombinant vector pGPU6 / GFPU / Neo-shRNA1, 2, 2, 3, 4N was detected by double enzyme digestion and gene sequencing. It was confirmed that the base sequence was consistent with the template sequence, and there was no gene mutation. The vector was successfully constructed.The recombinant vector was transfected into nasopharyngeal carcinoma cells mediated by liposome Lipofectamine2000. The transfection efficiency was about 55%.RT-PCR method to identify its interfering effect. The relative expression of Survivin/GAPDH was used to express the changes of SurvivinmRNA.The expression of mRNA in each group was significantly lower than that before transfection, but the interference effect of the recombinant plasmid gradually weakened with the prolongation of time, compared with that before transfection, the expression of mRNA decreased to a certain extent (about 50%).Conclusion: in this experiment, shRNA eukaryotic expression vector targeting Survivin was constructed and transfected into nasopharyngeal carcinoma cell line CNE-2 by liposome, and the expression level of Survivin gene was inhibited to some extent.It is confirmed that RNAi can inhibit the growth of nasopharyngeal carcinoma cells to a certain extent and provide new experimental data for gene therapy of nasopharyngeal carcinoma.Part twoAim: to investigate the inhibitory effect of RNAi on the growth of nasopharyngeal carcinoma cells by chemically synthesized small interfering RNAs, and to provide a new experimental basis for gene therapy of nasopharyngeal carcinoma.Methods: according to the design principle of siRNA, survivin gene sequence specific siRNAs were designed and transfected into CNE-2 cells by liposome.RT-PCR and Westernblotting were used to detect the expression of mRNA and protein in Survivin after interference, and the best siRNA sequence was screened for cell growth inhibition test (CCK-8).Results: after transfection of siRNA24 h for 48 h and 72 h, the expression of survivin in CNE-2Z cells was detected by RT-PCR and Westernblotting, and the best inhibitory sequence was selected for CCK-8 assay. The results showed that the expression of survivin was significantly inhibited by RT-PCR and Westernblotting.Conclusion: siRNA targeting Survivin can effectively inhibit the expression of Survivin gene in human nasopharyngeal carcinoma CNE-2 cells and inhibit cell growth and proliferation.It is confirmed that the application of chemically synthesized siRNA RNAi technique can significantly inhibit the growth of nasopharyngeal carcinoma cells and provide new experimental data for gene therapy of nasopharyngeal carcinoma.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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