【作者】 罗远材；

【导师】 糜若然； 瞿全新；

【作者基本信息】 天津医科大学 ， 妇产科学， 2010， 博士

【摘要】 目的探讨HPV(16／18)E6蛋白对PKR/EIF2a信号传导通路中PKR、p-PKR. p-EIF2a在各级别宫颈病变组织的定位及表达的影响,四者的表达差异与宫颈病变级别的相关性及对宫颈癌患者预后的影响；设计并构建以HPV18E6癌基因及无关序列(NC序列)为靶点的shRNA干扰序列的慢病毒载体(HPV18E6-RNAi-LV, NC-GFP-LV);以HPV18E6-RNAi-LV及NC-GFP-LV为工具转染HeLa细胞,研究在干扰HPV18E6癌基因表达的基础上,筛选出可能参与调节PKR/EIF2a信号传导通路活性的靶分子,为进一步探索高效激活PKR/EIF2a信号传导通路的方法提供实验依据。方法第一部分：采用免疫组化法检测HPV (16/18) E6、PKR、p-PKR、p-EIF2a在63例宫颈癌、114例宫颈上皮内瘤样病变(CINⅠ～CINⅢ)、15例正常宫颈组织中的定位及表达,分析HPV (16/18) E6蛋白对PKR/EIF2a信号传导通路活性的影响,四者在宫颈癌发生、发展中的作用,对宫颈癌患者预后的影响。第二部分：根据目的基因的相关信息,构建含有shRNA的重组质粒载体并鉴定；提取和纯化高质量的不含内毒素的重组质粒；将重组质粒载体和病毒包装质粒共转染293T细胞,进行病毒包装,收集病毒液并进行浓缩、纯化；所得病毒液感染293T细胞,测定病毒滴度。第三部分：使用HPV18E6-RNAi-LV及NC-GFP-LV慢病毒载体转染HeLa细胞,以RT-PCR、Western Blot法分别在核酸、蛋白水平(包括磷酸化型)检测转染前后HPV18E6、PKR、EIF2a、NF-KBp65、STAT1、MAPK、JNK1的表达,筛选出可能参与调节PKR/EIF2a信号传导通路活性的靶分子；以MTT法、Transwell侵袭实验检测转染前后宫颈癌HeLa细胞活性及侵袭能力的变化。结果第一部分1、HPV (16/18) E6蛋白主要定位于细胞核中,PKR、p-PKR、p-EIF2a蛋白主要定位于细胞质中；2、HPV (16/18) E6蛋白与PKR在各组的阳性表达率随宫颈病变级别升高而升高,并与宫颈病变级别呈正相关(P<0.05); p-PKR、p-EIF2a在各组的阳性表达率随宫颈病变级别升高而先升高、后下降；在宫颈癌组,PKR的阳性表达率明显高于p-PKR(P<0.01)；3、宫颈癌患者临床分期晚者病情进展快(P<0.01), HPV (16/18) E6阳性表达者病情进展快(P<0.05), p-PKR、p-EIF2a阴性表达者病情进展快(P<0.05,P<0.05)。第二部分1、经BLASTER同源性检索,HPV18E6的shRNA干扰序列与人类基因组没有同源性。HPV18E6、NC序列的shRNA干扰序列的正反义链转录后得到的RNA二级结构能形成环状发夹状结构；2、携带HPV18E6、NC序列的shRNA序列阳性克隆经测序鉴定比对后,阳性克隆即为构建成功的慢病毒载体；3、成功进行慢病毒包装、收获及浓缩,经慢病毒滴度测定,HPV18E6-RNAi-LV滴度：3E+8 TU/ml; NC-GFP-LV滴度：2E+9TU/ml。第三部分1、转染组HPV18E6、NF-κBp65 mRNA表达量明显低于空白对照组及阴性对照组(p<0.05,p<0.05)；转染组PKR mRNA表达量明显高于空白对照组及阴性对照组(p<0.05); EIF2a、STAT1、MAPK、JNK1 mRNA表达量在各组间的比较无显著性差异(p>0.05)2、转染组HPV18E6、NF-KBp65蛋白的表达量明显低于空白对照组及阴性对照组(p<0.05,p<0.05)；转染组p-STAT1、PKR蛋白的表达量明显高于空白对照组及阴性对照组(p<0.05,p<0.05)；转染组p-PKR、p-EIF2a蛋白的表达量显著高于空白对照组及阴性对照组(p<0.01,p<0.01)；STAT1、MAPK、p-MAPK、JNK1、p-JNK1蛋白的表达量在各组间的比较无显著性差异(p>0.05)3、转染组细胞活性抑制率39.6%,细胞侵袭抑制率56.2%,明显高于空白对照组及阴性对照组(p<0.05,p<0.05),空白对照组与阴性对照组比较,无显著性差异(p>0.05)结论1、HPV(16/18)E6蛋白能通过使p-PKR、p-EIF2a去磷酸化的方式抑制PKR/EIF2a信号传导通路激活,阻止病变细胞凋亡,促进宫颈病变进展；PKR/EIF2a信号传导通路有效激活,能够抑制宫颈癌患者病情进展,改善其预后；2、成功构建了HPV18E6-RNAi-LV和NC-GFP-LV慢病毒载体,为后续研究工作提供了实验工具。3、降低HPV18E6癌基因的表达水平可增加PKR的表达,恢复PKR/EIF2a信号传导通路活性,抑制HeLa细胞活性及侵袭能力,促进其凋亡,证明HPV18E6蛋白能使PKR/EIF2a信号传导通路失活；核因子NF-κBp65有望成为调节PKR/EIF2a信号传导通路活性的候选因子,为进一步探索高效激活PKR/EIF2a信号传导通路的方法提供了实验依据。更多还原

【Abstract】 ObjectiveTo identify the effects to the expressions and location of PKR、p-PKR、p-EIF2a in PKR/EIF2a signal transduction passage by HPV (16/18) E6 protein in every grades of cervical lesions tissue,the relations between the difference of their expressions and grades of cervical lesions, their effects on the prognosis of cervical carcinoma paitients; Design and construct two lentiviral vectors which contain shRNA interfering sequence aiming at the target of HPV18E6 oncogene and NC sequence (HPV18E6-RNAi-LV、NC-GFP-LV); Based on the transduction with HPV18E6-RNAi-LV and NC-GFP-LV into HeLa cell to interfere the expression of HPV18E6 oncogene and NC sequence, the target molecule which may regulate the activation of PKR/EIF2a signal transduction passage is determined, which can provide the basis of exploring methods to activate effectly PKR/EIF2a signal transduction passage in next experiments.MethodsPart 1:The expressions and location of HPV(16/18) E6, PKR, p-PKR, p-EIF2a in human cervical carcinoma tissue of 63 cases, cervical intraepithelial neoplasia (CINⅠ～CINⅢ) of 114 cases and normal cervical epithelium of 15 cases are detected by immunohistochemical technique, analyze the effects on the activation of PKR/EIF2a signal transduction passage by HPV(16/18)E6 protein, their roles in generation and progression of cervical carcinoma,their effects to prognosis of cervical carcinoma patients.Part 2:Based on the information of target gene,recombination plasmid vector which contains shRNA is constructed and apprased;Extract and purify recombination plasmid;Recombination plasmid and packaging plasmid co-transduce the 293T cell to package the lentiviral vector,collects the lentiviral vector solution,concentrates and purifies it; The lentiviral vector solution after concentrating and purifying transducer 293T cellto determine the concentration of the lentiviral vector solution.Part 3:Before and after the transduction into HeLa cell with HPV18E6-RNAi-LV and NC-GFP-LV, the expressions of mRNA and protein (including phosphating pattern) of HPV18E6、PKR、EIF2a、NF-κBp65、STAT1、MAPK、JNK1 are measured with RT-PCR and Western Blot, the target molecule which may regulate the activation of PKR/EIF2a signal transduction passage is determined; Before and after the transduction, the activation and invading ability of HeLa cell are determined with MTT and Transwell cell methods.ResultsPart 11.HPV (16/18) E6 protein mainly locates in nuclei.PKR、p-PKR、p-EIF2aprotein mainly locate in cytoplasm;2.With the rise of the grades of cervical lesions,the positive-expression rate of HPV (16/18) E6 oncoprotein and PKR increase and correlated significantly with the grades of cervical lesions (p< 0.05); With the rise of the grades of cervical lesions,the positive-expression rates of p-PKR、p-EIF2a increase firstly,then descend; In cervical carcinoma group,the positive-expression rate of PKR is much higher than that of p-PKR (p<0.01);3.The development and progression of later clinical stages cervical carcinoma is quicker than that of earlier clinical stages cervical carcinoma (p<0.01), the development and progression of cervical carcinoma which has positive-expression of HPV (16/18) E6 oncoprotein is quicker than that of negative-expression of HPV (16/18) E6 oncoprotein (p< 0.05). the development and progression of cervical carcinoma which has negative-expression of p-PKR、p:EIF2a is quicker than that of positive-expression of p-PKR、p-EIF2a (p<0.05, p<0.05).Part 21.Indexing shRNA of interfering sequence aiming at the target of HPV18E6 oncogene with human genes in BLASTER,there is no homogeneous.The second constructure of the transcripts of interfering sequence aiming at the target of HPV18E6 oncogene and NC sequence can conform short hairpin;2. The sequencing of positive clones of containing shRNA interfering sequence aiming at the target of HPV18E6 oncogene and NC sequence prove that the positive clones are the lentiviral vectors;3. After packaging and collecting and concentrating the lentiviral vector successfully, the concentration of lentiviral vector solution is determined, the concentration of HPV18E6-RNAi-LV is 3E+8 TU/ml,the concentration of NC-GFP-LV is 2E+9 TU/ml.Part 3 1.The expressions of HPV18E6、NF-κBp65 mRNA in HPV18E6-RNAi-LV group are lower than those in NC-GFP-LV group and control group (p<0.05, p <0.05); The expression of PKR mRNA in HPV18E6-RNAi-LV group are higher than that in NC-GFP-LV group and control group (p<0.05); The expression of EIF2a、STAT1、MAPK、JNK1 mRNA in every group are no significant difference (p>0.05);2.The expressions of HPV18E6、NF-κBp65 protein in HPV18E6-RNAi-LV group are lower than those in NC-GFP-LV group and control group (p<0.05, p <0.05); The expression of p-STAT1、PKR protein in HPV18E6-RNAi-LV group are higher than those in NC-GFP-LV group and control group (p<0.05, p <0.05); The expression of p-PKR、p-EIF2a protein in HPV18E6-RNAi-LV group are higher than those in NC-GFP-LV group and control group (p<0.01, p <0.01); The expression of STAT1、MAPK、p-MAPK、JNK1、p-JNK1 protein in every group are no significant difference (p>0.05);3. The restraining rate of cell activation and aggression are 39.6% and 56.2% in HPV18E6-RNAi-LV group, which are higher than those in NC-GFP-LV group and control group(p<0.05, p<0.05), there are no significant difference between NC-GFP-LV group and control group (p>0.05)Conclusions1.HPV(16/18) E6 can dephosphorylate p-PKR、p-EIF2a to restrain activation of PKR/EIF2a signal transduction passage,prevent apoptosis of abnormal cells, promote the development and progression of cervical lesions; Effective activation of PKR/EIF2a signal transduction passage can restrain the development and progression of cervical carcinoma,improve the prognosis of cervical carcinoma paitients;2. The lentiviral vectors of HPV18E6-RNAi-LV and NC-GFP-LV are constructed successfully,which provides practical tool for later study;3、That reduce the expression of HPV18E6 oncogene can increase the expression of PKR and recover the activation of PKR/EIF2a signal transduction passage, restrain the activation and aggression of HeLa cell and promote apoptosis of HeLa cell which prove that HPV (16/18) E6 protein can inactivate PKR/EIF2a signal transduction passage; It is possible that ruclear factor NF-κBp65 becomes the candidate to regulate the activation of PKR/EIF2a signal transduction passage,which provides experimental basis for exploring the methods of activating effectively PKR/EIF2a signal transduction passage later.更多还原