【作者】 郑威楠；

【导师】 潘泽民；

【作者基本信息】 石河子大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 目的:探讨IFITM1基因在新疆维吾尔族妇女子宫颈鳞癌组织中转录和翻译水平的表达及临床意义,进一步研究IFITM1基因对子宫颈癌HeLa细胞存活率、凋亡的影响方法:采用(1)免疫组织化学(S-P法):检测IFITM1在子宫颈癌及慢性宫颈炎蛋白质表达。(2)实时定量PCR:检测5例子宫颈鳞癌及5例相应的宫颈癌旁组织中IFITM1基因mRNA表达水平。(3)构建IFITM1真核表达重组质粒,通过脂质体法将质粒转染入子宫颈癌HeLa细胞。a.通过RT-PCR检测IFITM1基因在HeLa细胞中mRNA的表达水平。b.免疫组化实验检测IFITM1在HeLa细胞蛋白表达水平。c.采用MTT法分析存活细胞数及用AnnexinV/PI双色法流式细胞仪分析IFITM1基因对子宫颈癌HeLa细胞凋亡的影响。结果:(1)IFITM1蛋白质在维吾尔族子宫颈鳞癌组织表达百分比15.0%,在维吾尔族慢性子宫颈炎组织中的IFITM1蛋白质强阳性表达百分比56.5% ,两者比较有统计学有显著性差异(P<0.05)。通过非参数检验,各临床分级Z=3.344,P>0.05,无显著性差异,表明IFITM1蛋白表达与新疆维吾尔族妇女子宫颈鳞癌的病理分级、临床分期不相关。(2)5例子宫颈鳞癌癌患者组织中IFITM1基因的mRNA表达水平的最高值是134.95,最低值是22.34,平均表达水平为64.79±42.71。而5例相对应的癌旁组织中mRNA的表达水平最高值为300.16,最低值为49.69,平均表达水平为128.29±88.77,经方差齐性检验,进行两样本均数t检验,t=-2.497,p=0.019,表明子宫颈癌患者组织与相应子宫颈癌旁组织IFITM1的mRNA表达水平有显著性差异。(3)成功的构建了IFITM1-pcDNA3.1重组载体,转染HeLla细胞24小时a. RT-PCR检测目的基因在Hela细胞中的mRNA的表达量高于其他两组。b.免疫组化结果显示:IFITM1-pcDNA3.1重组质粒组蛋白表达明显高于对照组及空载体组,而对照组及空载体组蛋白表达无差异。c. MTT结果显示:空载体组、对照组MTT结果分别为:0.4323±0.0224﹡,0.4236±0.0287﹡;重组质粒组:0.3307±0.0398﹡。重组质粒组吸光度值均明显低于空载体组及对照组,而后两组无显著差异。AnnexinV/PI双色法流式细胞仪检测显示:对照组、空载体组和重组质粒组的凋亡率(%)分别为:13.1607±2.4171,13.4664±3.6965,27.3445±10.0532。表明IFITM1基因转染入人子宫颈癌HeLa细胞后可以抑制细胞生长,促进细胞凋亡。结论:IFITM1基因在维吾尔族子宫颈癌组织mRNA及蛋白质表达水平均降低;IFITM1基因可抑制子宫颈癌HeLa细胞的生长促进HeLa细胞凋亡。更多还原

【Abstract】 Objective: To explore the transcription level and translation level of expression and clinical significance of IFITM1 gene in Uigur Cervical Squamous Cell Carcinomas tissues and normal Uigur Cervical tissues, to study of IFITM1gene on the growth of HeLa cells , apoptosis of HeLa cell by using gene transfection.Methods: IFITM1 protein expression level in Uigur cervical tissues was detected by immunohistochemistry(S-P Method).To detect IFITM1gene transcription level in 5 cervical squamous cell carcinoma and corresponding normal cervical tissues by real time PCR(RT-PCR).Construction the eukaryotic expression vector of IFITM1 gene,this recombinant was transfected into HeLa cells by using liposome transfection,to observe the number of cell growth by MTT Method and the cell apoptosis were examined by Annexinⅴ/PI double stainning flow cytometry.Results: (1)The Strong positive percentage of IFITM1 protein in Uigur cervical squamous cell carcinomas tissues was 15.0%(13/23)and 56.5% in chronic cervicitis immunohistochemistry method, it was significant difference (P<0.05). IFITM1 protein expression level in the hierarchical pathology of the cervical squamous cell carcinoma of Uigur women were not significant difference(Z=3.344,P>0.05) by non- parameter analysis. It indicates the expression of IFITM1 protein were not correlated with the hierarchical pathology of the cervical squamous cell carcinoma of Uigur women .(2) In 5 cervical squamous cell carcinoma tissues of the Uigur women, the maxlmum of IFITM1 gene expression at mRNA level is 134.95,the minimum is 22.34,the average expression level is 64.79±42.71. However, among 5 corresponding cervical normal tissues, the maximum of IFITM1 gene expression at mRNA level is 300.16,the minimum is 49.69 and the average expressed level is 128.29±88.77. There were significant difference(P<0.05) between Uigur Cervical Squamous Cell Carcinomas tissues and corresponding normal Uigur Cervical tissues by test of homogeneity of varianc and mean square interclass analysis (t=-2.497,p=0.019).(3) To construct the eukaryotic expression vector of IFITM1gene and transfected into HeLa cells. mRNA expression level were examined by RT-PCR method and protein expression level were detected by immunohistochemistry to ensure transfected successfully. The survial of cells after IFITM1 gene of pcDNA3.1 constructs transfected of HeLa cells were lower than pcDNA3.1 plasmid transfected in HeLa cells and non-transfected HeLa cells. With transfection experiment, the apoptosis rate of HeLa cells in non-transfected HeLa cells, pcDNA3.1 plasmid in HeLa cells and in IFITM1 gene of pcDNA3.1 recombinant plasmid in HeLa cells were 0.3307±0.0398, 0.4323±0.0224 and 0.4236±0.0287. Among IFITM1 gene of pcDNA3.1 constructs, pcDNA3.1 plasmid and non-transfected HeLa cells transfected into HeLa cells, the apoptosis rate were 27.3445±10.0532, 13.4664±3.6965 and 13.1607±2.4171, respectively. There were significant difference among three groups.Conclusion:IFITM1 gene expression at mRNA and protein level were decreased in cervical squamous cell carcinoma of Xinjiang Uigur women; IFITM1 gene may inhibit the growth of HeLa cell and inducing HeLa cell apoptosis.更多还原