【作者】 王薇；

【导师】 马丁；

【作者基本信息】 华中科技大学 ， 妇产科学， 2007， 博士

【摘要】 研究背景与目的:宫颈癌是严重威胁妇女生命健康最常见的恶性肿瘤之一,在全球女性恶性肿瘤中其发病率仅次于乳腺癌,在发展中国家则居于首位。近年来,早期宫颈癌的发生,特别是年轻化趋势十分明显,这被证实与人乳头瘤病毒(Human papillomavirus, HPV)感染密切相关。因此,深入探索HPV的致病关键及其与宫颈癌的内在联系,从而靶向病源对宫颈癌发生发展进行干预阻遏,成为宫颈癌防治攻关的重要研究方向。目前研究认为,高危HPV DNA的整合是宫颈癌变的重要机制,在E2基因DNA结合区和转录调节区之间为不稳定的铰链区,被认为是HPV整合入宿主细胞最常见的缺失和断裂部位,而宫颈癌组织中HPV致癌基因E6/E7转录产物的增加是HPV整合导致E2基因缺失的结果。另外,细胞毒性T淋巴细胞(CTL)通过结合Fas/FasL系统诱发靶细胞凋亡是机体免疫系统杀伤病毒感染及肿瘤的重要途径,在HPV相关宫颈癌的发生过程中,其地位不容忽视。近期研究提示HPVE2蛋白可能通过活化caspase-8而激活Fas/FasL外源性凋亡通路诱导细胞凋亡,而cFLIP(cellular FLICE inhibitory protein)由于在结构与序列上与caspase-8相似且能抑制其功能,从而对Fas/FasL介导的凋亡信号转导产生调控作用。本项目选择从高危HPV整合的角度,以HPV16 E2、cFLIP以及caspase-8活化作为研究对象,探讨Fas/FasL系统的下游分子与高危HPV整合、HPVE2缺失引起的宫颈癌发生发展之间的相互作用机制,开发宫颈癌新型防治干预靶点,并利用选择增殖性腺病毒建立新型基因-病毒治疗策略,为宫颈癌的生物治疗奠定实验基础。方法:1.选取宫颈病变组织存档蜡块,包括正常宫颈、CIN1,2,3及宫颈癌病例。采用改良的蛋白酶K消化法提取蜡片组织DNA,HC-Ⅱ和免疫组化法确定HPV16感染,多重PCR扩增法确定宫颈癌组织中HPV16感染状态并分析其相关临床意义。2.构建HPV16 E2全长正义cDNA及其N端反式激活域和C端DNA结合域真核表达载体,RT-PCR和Western Blot检测各载体对SiHa细胞中HPV16 E6/E7基因表达的调控作用。光镜、荧光显微镜和共聚焦显微镜观察转染前后细胞形态变化;MTT、流式细胞仪PI法以及Hoechst33258/PI双染法检测转染前后细胞生长和凋亡变化;细胞黏附实验、Transwell实验检测转染前后细胞侵袭转移能力的变化。3.用抗人CD95单抗处理宫颈癌细胞,观察其促凋亡效应以了解宫颈癌细胞中Fas/FasL凋亡通路受抑制状况;构建带有GFP和His标签的HPV16 E2全长正义真核表达载体;激光共聚焦显微镜和流式细胞仪观察转染HPV16 E2真核表达载体对抗人CD95单抗诱导宫颈癌细胞凋亡效应的影响;RT-PCR、Western Blot和免疫组化法检测转染前后宫颈癌细胞内Fas基因及DISC成员基因的表达变化;Western Blot和caspase-3/8活性试剂盒检测转染前后caspase-3/8活性改变;CO-IP法检测HPV16 E2蛋白与FLIP蛋白之间的相互作用。4.采用基因重组法构建复制选择性腺病毒载体Adv5/dE1A-HPV16 E2(简称E2-Ad),体外转染E2-Ad观察其对宫颈癌细胞的CPE效应、复制裂解效应;Western Blot检测E2-Ad对E6/E7基因表达的调控作用;流式细胞仪检测E2-Ad对宫颈癌细胞的放化疗增敏作用;细胞黏附实验、Transwell实验检测E2-Ad对宫颈癌细胞侵袭转移能力的影响;体内裸鼠接种实验观察E2-Ad的抗肿瘤效应;免疫组化和TUNNEL法检测裸鼠皮下瘤组织中E6/E7表达和凋亡比率。结果:1.HPV16整合感染率随着宫颈病变级别增高(CIN1-CIN2-CIN3-Cancer)而逐渐增高,两两之间比较均存在显著性差异;HPV16整合感染的早期宫颈病变更容易出现病程持续和进展。2.HPV16 E2可以在mENA和蛋白水平显著降低SiHa细胞内E6/E7基因的表达,并抑制细胞生长、促进细胞凋亡、降低细胞的侵袭转移能力。3.带有GFP和His标签的HPV16 E2融合蛋白能在宫颈癌细胞内成功表达,并下调SiHa细胞中HPV16 E6/E7基因的表达;有一定量Fas表达的SiHa细胞和无Fas表达的C33A细胞对抗Fas抗体诱导的凋亡存在抵抗,而HPV16 E2直接诱导其产生凋亡并提高SiHa细胞对抗Fas抗体的敏感性;HPV16 E2诱导细胞内caspase-3/8活化;HPV16 E2降低SiHa细胞内FADD、FLIPS基因表达,而对C33A细胞无明显影响;FLIP可能通过直接作用阻遏HPV16 E2诱导细胞凋亡。4.体外转染宫颈癌细胞后,复制选择性腺病毒E2-Ad能在细胞中大量复制,裂解细胞产生CPE效应,特异性地下调E6/E7基因表达,并对宫颈癌细胞的放化疗具有显著的增敏效应;体内实验结果显示E2-Ad能显著抑制SiHa细胞成瘤和生长,并逆转瘤细胞的恶性表型。结论:1.高危型HPV整合状态的存在是宫颈病变恶性转化过程中的高危因素,在HC-Ⅱ的基础上联合应用高危HPV感染状态的检测,有利于提高宫颈癌筛查的靶向性并早期预测宫颈病变的转归。2.HPV16 E2对宫颈癌细胞具有强大的抗增殖和促凋亡作用,可望成为宫颈癌治疗的有效靶标。3.HPV16 E2通过激活caspase-3/8诱导对Fas凋亡通路存在抵抗的宫颈癌细胞产生凋亡,一方面可能与下调E6/E7基因表达有关,一方面也可能由于HPV16 E2直接与FLIP蛋白相互作用而产生。4.复制选择性腺病毒E2-Ad在体内外均具有显著的抗肿瘤效应。更多还原

【Abstract】 Background and objective: In developing countries, cervical cancer is one of the most frequent neoplastic diseases among women for poor screening and therapeutics procedures. Cervical carcinomas are frequently associated with infection by human papillomaviruses (HPVs). It would be an important work to study the relationship between HPV infection and cervical carcinogenesis (CC) and to reverse CC’s development by targeting HPV oncogenic keypoint. Integration of HPV DNA into the host genome is a major mechanism of cervical carcinogenesis. The HPV E2 open reading frame (ORF) has been identified as the preferential site of integration because it has been found to be disrupted or deleted more frequently than other sites. Therefore, the disruption of E2 dependent negative feedback controlling E6 and E7 transcription is considered a selective event in tumor development and progression. Furthermore, several observations have suggested that other E2 functions are necessary to mediate cellular growth arrest. Recent studies have shown that HPVE2 can induce apoptosis through activation of caspase 8, the Fas/FasL extrinsic pathway effector, but its exact mechanism has not been explored. Since c-FLIP has shown to act as dominant negative inhibitors of FADD, a crucial mediator of Fas/FasL signaling, it was suggested to inhibit death receptor-mediated apoptosis by displacing DED-containing caspase-8 from the death-inducing signaling complex (DISC). This study is designed to explores the relationship among HPV16E2, cFLIP and caspase8 and the regulation between HPV16E2 and the Fas/FasL immune monitoring mediated by CTL,and to set up a series of selective proliferating adenovirus to validate the aforesaid mechanisms in vivo. The aim of this study is to realize efficacious biotherapy for cervical cancer targeting the key carcinogenic factor of HPV and to make a great contribution to cut down the incidence and mortality of cervical cancer.Methods:1. A series of archival samples, including squamous cervical carcinomas, cervical intraepithelial neoplasia (CIN) lesions and normal cervical tissues, were subjected to for HPV HC-Ⅱanalysis. Typing HPV-16 infection was analysed by the polymerase chain reaction (PCR) and immunohistochemical staining, and its infection status was assessed with the integrity and disruption of the HPV-16 E2 gene, which was amplified in three overlapping fragments.2. Three vectors, which express HPV16 E2, its DNA binding domain and transcriptional domain respectively, were constructed. RT-PCR、WB、Confocal、MTT、FCM、Transwell methods were used to study the role of these vectors in reversing the malignant phenotype of cervical carcinoma cells.3. Construct HPV16 E2 expression vector containing GFP or His tag. RT-PCR、WB、Confocal、MTT、FCM methods were used to study the role of these two vectors in inducing apoptosis and the relationship between HPV E2 and FLIP.4. Construct selective replicating adenovirus E2-Ad with gene recombination method, transfect it into cervical carcinoma cells to observe CPE effect, adenovirus replication and apoptosis after treated with DDP and irradiation. In vivo antitumor activity of E2-Ad was investigated in human SiHa xenografts.Results:1. HPV16 E2 disruption was associated closely with cervical lesion severity and its progression.2. HPV16 E2 can decrease the expression of E6/E7 gene in SiHa, and inhibit cell growth, promote cell apoptosis, decrease cell transfer activity.3. HPV16 E2 can induce apoptosis not only in HPV(+) SiHa cell, but also in HPV(-) C33A cell through activating caspase-8 and caspase-3. In SiHa, other than C33A, HPV16 E2 can decrease the expression of E6/E7、FADD、FLIPS gene. And there exits a direct interaction between HPV16 E2 and FLIP protein.4. Transfect E2-Ad into different cervical carcinoma cell can induce selective replication and CPE effect, block E6/E7 expression, and excessively increase the role of irradiation and DDP. In vivo, E2-Ad can significantly retard the growth of the SiHa xenografts.Conclusions:1. The loss of HPV16 E2 function during viral integration has been an activation mechanism for progression from high grade CINs to invasive CC.2. HPV16 E2 would be an effective target for cervical carcinoma treatment.3. HPV16 E2 can induce apoptosis through activating caspase-8/3. The mechanism is not only related to the bloke of E6/E7 expression, but also associated with the direct interaction between HPV16 E2 and FLIP protein.4. E2-Ad has significant antitumor effect in vivo and in vitro.更多还原