靶向沉默HIF-1α和Survivin基因对鼻咽癌裸鼠移植瘤放疗敏感性的影响

发布时间：2018-04-04 01:55

本文选题：缺氧诱导因子-1a　切入点：生存素(Survivin)　出处：《贵州医科大学》2017年硕士论文

【摘要】：目的:通过siRNA技术靶向沉默人鼻咽癌细胞株CNE-2缺氧诱导因子-1a(Hypoxia inducible factor 1-alpha,HIF-1α)与生存素(Survivin),观察HIF-1α与Survivin低表达的CNE-2裸鼠移植瘤对放疗敏感性的影响,为探讨鼻咽癌基因治疗奠定一定的基础;方法:设计合成靶向沉默HIF-1α与Survivin的microRNA干扰质粒,采用脂质体转染法将含HIF-1α、Survivin的microRNA干扰质粒转染CNE-2细胞,荧光显微镜观察其转染率,RT-qPCR检测其基因干扰效率。取35只4~6周龄的雌性裸鼠,随机分为5组,每组7只,分为HIF-1α干扰组,Survivin干扰组,HIF-1αSurvivin干扰组(联合干扰组)、空载组、阴性对照组。将转染48h后的各组CNE-2细胞分别注射至BALA/c裸鼠体内成瘤,待各组裸鼠成瘤后,每次每只接受5Gy X射线照射,每隔三天放疗一次,共15Gy X,观察各组裸鼠放射治疗后不同时间肿瘤体积,3天后处死动物,迅速剥离瘤体测量体积,观察放射治疗对肿瘤的抑制作用,分析HIF-1α和Survivin基因沉默对肿瘤放射敏感性的影响。RT-qPCR检测瘤体组织中的HIF-1α和Survivin表达水平,蛋白免疫印记(Western Blot)检测HIF-1α和Survivin蛋白表达情况,TUNEL试剂盒检测鼻咽癌裸鼠移植瘤中细胞凋亡。结果:HIF-1α干扰组、Survivin干扰组与HIF-1αSurvivin干扰组经过放射治疗后肿瘤体积增加率明显小于空载体组与阴性对照组(P0.05);HIF-1α和Survivin的mRNA、蛋白质在基因干扰组中较空载组与阴性对照组均明显下降;凋亡率较空载组与阴性对照组升高(P0.05),HIF-1α干扰组、Survivin干扰组与联合干扰组三者之间无统计学意义(P0.05)。结论:单独干扰HIF-1α、Survivin基因与联合干扰HIF-1α和Survivin基因均能够增加鼻咽癌裸鼠移植瘤对放疗敏感性,但联合干扰HIF-1α和Survivin基因并没有进一步提高鼻咽癌裸鼠移植瘤放疗敏感性。
[Abstract]:Objective: to investigate the effects of siRNA targeting CNE-2 hypoxia inducible factor 1-alpha-HIF-1 伪 and survivin survivin on the radiosensitivity of CNE-2 xenografts with low expression of HIF-1 伪 and Survivin in nude mice.Methods: microRNA interference plasmids targeting silencing HIF-1 伪 and Survivin were designed and synthesized, and microRNA interference plasmids containing HIF-1 伪 survivin were transfected into CNE-2 cells by liposome transfection.The transfection rate was observed by fluorescence microscope and RT-qPCR was used to detect its gene interference efficiency.Thirty-five female nude mice aged 4 and 6 weeks were randomly divided into 5 groups, 7 in each group, and divided into HIF-1 伪 interference group, survivin interference group and HIF-1 伪 Survivin interference group (combined interference group, no-load group, negative control group).After 48 hours of transfection, the CNE-2 cells of each group were injected into BALA/c nude mice for tumorigenesis. After tumorigenesis, each nude mouse was irradiated by 5Gy X-ray every time, and every three days was treated with radiotherapy.15Gy X was used to observe the tumor volume of the nude mice at different time after radiotherapy for 3 days. The tumor volume was quickly stripped off and the tumor inhibitory effect was observed.The effect of HIF-1 伪 and Survivin gene silencing on radiosensitivity of nasopharyngeal carcinoma was analyzed. RT-qPCR was used to detect the expression of HIF-1 伪 and Survivin in tumor tissues. Western blot was used to detect the expression of HIF-1 伪 and Survivin protein. Tunel kit was used to detect apoptosis in transplanted nasopharyngeal carcinoma (NPC) nude mice.Results the tumor volume increase rate of survivin interference group and HIF-1 伪 Survivin interference group was significantly lower than that of empty vector group and negative control group after radiotherapy. The protein in gene interference group was significantly lower than that in no-load group and negative control group.The apoptosis rate was higher than that in the no-load group and the negative control group. There was no significant difference between the survivin interference group and the combined interference group.Conclusion: interference of HIF-1 伪 survivin gene and HIF-1 伪 and Survivin genes alone can increase the radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice, but the combined interference of HIF-1 伪 and Survivin genes does not further improve the radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.63

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