【作者】 马彩玲；

【导师】 王国荃； 郑玉建； 张富春；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2004， 博士

【摘要】 目的:人乳头瘤病毒16型(human papillomavirus,HPV16)是诱发妇女宫颈癌的启动因子,并与人类多种恶性肿瘤的发生密切相关。它的早期基因E6、E7是致癌的关键基因,在宫颈细胞中持续表达并为其癌化表型的维持所必需。新疆是我国宫颈癌高发区,新疆南部维吾尔妇女宮颈癌与HPV16感染密切相关,为进行新疆妇女宫颈癌病因研究及宫颈癌的预防及早期诊断,本文对新疆维吾尔妇女宫颈癌组织进行了HPV16 E6及E7基因的分子检测研究。为进一步了解新疆妇女宫颈癌组织中HPV16E6基因的结构特点,本研究对来自新疆的宫颈癌患者手术或活检标本中的HPV16E6基因进行了克隆及一级结构分析;对新疆地区宫颈不同病变组织进行了HPV16E6、E7基因检测;荧光定量聚合酶链反应(FQ-PCR)分析,探索新疆妇女宫颈不同病变组织中HPV16E6、E7基因的相对含量与病情变化的关系,为宫颈癌的筛查、早期诊断、预防及愈后评估提供新的分子生物学的方法。HPV16E6、E7蛋白是肿瘤特异性排斥抗原,是设计HPV疫苗公认的靶抗原。本研究开展新疆维吾尔妇女宫颈癌组织中HPV16E6、E7基因的克隆,融合蛋白的原核表达及准备抗血清的制备,为HPV16相关疾病的血清学诊断及HPV16E6、E7的疫苗研究提供物质基础。近年来,有关纳米材料的合成和应用方面的研究工作进展迅速,特别是纳米金,在分子领域的应用已成为研究的热点。研究采用固定于硅烷化片基上的捕获探针,特异性结合单链人乳头瘤病毒16型新疆株E6基新猎医科大学博士学位论文因(HPV 1 6E6)扩增片断,再与互补的纳米金颗粒标记的探针结合,通过银染加强显色,初步建立了金标银染法快速检测HPv 1 6E6 DNA的技术。从而为新疆妇女宫颈癌病因研究及宫颈癌的预防、诊断及治疗的研究提供科学的理论依据。 方法:从浸泡在福尔马林的宫颈癌组织(10例)及福尔马林浸泡后石蜡包埋的宫颈癌(59例)、宫颈上皮内瘤变(eervical intraepithelialneoplasia,CIN65例)及宫颈炎(33例)组织中提取DNA;另外对一批妇科门诊患者(96例)随机取宫颈管的脱落细胞及分泌物,作为对照,用聚合酶链式反应(PCR)检测各组宫颈组织中人乳头瘤病毒16型E6基因(HPV 1 6E6)。从19份新疆维吾尔族妇女宫颈癌活检组织标本中提取组织DNA,作为模板,PCR扩增HPV 1 6E6全长基因,PCR产物直接测序或克隆后测序,分析新疆维吾尔族妇女宫颈癌组织HPV16E6基因的突变。以新疆妇女宫颈不同病变组织为源材料,以人乳头瘤病毒16型(新疆株)E6基因(HpV 1 6E6,AF327851)及HPvl6E7基因作为扩增的靶基因,同时以人p一actin基因片段作为内参照,借助于设计的目的基因引物,两个特异的荧光探针,在筛选的FQ一PCR优化反应体系中,同时对两个基因片段进行扩增,得到HPV 1 6E6及HPV 1 6E7基因的相对含量。使用本实验室从新疆维吾尔族妇女宫颈癌组织中克隆的人乳头瘤病毒16型的E7基因(HPV16E7),以pGEX一ZT载体和大肠杆菌BL21(DE3)原核蛋白表达系统表达HPV16E7融合蛋白(GST,HPV16E7),经亲和纯化定量后得到大量的GsT一HPv16E7融和蛋白。根据HPV 16(新疆株)E6基因编码序列设计引物,从含有HPV 1 6E6基因的质粒中扩增出含HPV 1 6E6基因的DNA片段,将所得片段与pMD 18一T载体连接,转化到JM109大肠杆菌中,筛选的阳性克隆扩增后,提取质粒DNA,酶切,回收目的片段,定向克隆到PET28a中,转化JM109受体菌,从JM109受体菌中提出质粒,再转化到BLZI(DE3)中,筛选出转化体,用IPTG诱导表达,在PAGE上见到所表达的18kD的特异性的HPv16(新疆株)E6蛋白条带。从而为人乳头瘤病毒16型的诊断、检测、疫苗研制等方面的研究提供了物质基础。研究采用固定于硅烷化片基上的捕获探针,特异性结合单链人一2一中文摘要乳头瘤病毒16型新疆株E6基因(HPV 1 6E6)扩增片断,再与互补的纳米金颗粒标记的探针结合,通过银染加强显色,初步建立了金标银染法快速检测HPV16E6 DNA的技术,同时辅以Real一time定量PCR、地高辛斑点杂交进行平行比较和评价,确认了金标银染法检测的有效性。 结果:PCR结果宫颈癌蜡块组织59例中HPv16E6阳性37例(阳性率62.7%);宫颈上皮内瘤变(cIN)组织65例中HPv16E6阳性37例(阳性率56.92%);石蜡包埋宫颈炎组织共33例,HPV16E6阳性22例(阳性率“.67%);而宫颈管脱落细胞及分泌物共%例,HPV16E6阳性3例(阳性率3.13%)。荧光定量PCR结果宫颈癌蜡块组织59例p一actin均阳性,证实DNA提取方法可靠。从19份新疆维吾尔族妇女宫颈癌活检组织标本中提取组织DNA,作为模板,P CR扩增HPV 16E6全长基因,PCR产物直接测序或克隆后测序,14个E6分离片断的测序和序列分析表明,7个(50%)分离株E6基因与原型相同,5个(35.71%)分离株发生了L83V突变;2个(14.29%)分离株发生了L83V/D63E突变,HPV16E6原型和L83V突变型在宫颈癌组织中的分布与其在北美洲的分布接近。对FQ·PCR反应结果中p一actin阳性的宫颈不同病变组织中HPV16E6阳性率及其相对含量进行统计分析,宫颈癌蜡块组织59例,HPV 1 6E6阳性率83 .05%;宫颈上皮内瘤变(CIN)蜡块组织65例,HPV16E6阳性率75.68%;宫颈炎蜡块组织33例,HPV 1 6E6阳性率93 .33%,宫颈脱落细胞标本%例,HpV 1 6E6阳性率3 .29%。pCR及F更多还原

【Abstract】 Objective: Cervical cancer is associated with infections by "high-risk" human papillomavirus(HPVs), HPV16 is the most frequent type. In order to detect HPV16 DNA in cervical carcinomas specimens in Xinjiang uygur. Established a method for detection of HPV16 E6 DNA in paraffin-embedded cervical carcinomas specimens. HPV16E6 has some variants, each with a different geographic distribution. The southern of Xinjiang is one region of the high incidence of cervical cancer, we have reported the variant of HPV16E6 from cervical carcinoma biopsies in Xinjiang Uygur women. This study was designed to investigate distribution of the variant in the cervical cancer of Xinjiang Uygur women, and the relationship between the variant and the high incidence of cervical cancer in Xinjiang. To establish a method of FQ-PCR (fluorescence quantitative PCR ) for detection of HPV16E6 and HPV16 E7 gene in cervical carcinomas and other cervical specimens. To study the relationship between the quantities of HPV16E6 and HPV16 E7 in cervical tissues and the course of cervical disease in Xinjiang. Utilizing HPV16E7 and HPV16E6 gene of a Chinese Uygur patient of cervical carcinoma in Xinjiang, we induced HPV16E7 and HPV16E6 protein expressed in a prokaryotic system and have obtained thispurified protein by affinity chromatography and quantification. The worldwide results have clearly shown that virtually all cervical cancer is caused by human papillomavirus infection. In order to establish fast, sensitive and cost effective method to detect HPV 16 sequences in cervical cancer, we used a visualization of aggregating gold-labeled nanoparticle probe enhanced with silver staining after sequence specific hybridizations.Methods: DNA extraction from archival formalin-fixed and paraffin-embedded specimens was performed by using a standard protocal. HPV16E6 gene was detected by PCR in tissues of 59 cervical cancer, 65 cervical intraepithelial neoplasia (CIN), 33chronic cervicitis in paraffin-embedded specimens and 96 cervical smear samples of vaginitis and cervicitis. DNA was extracted from 19 cervical carcinoma biopsies in Xinjiang Uygur women. HPV16E6 genes were amplified by PCR from the cervical carcinoma tissues. Nucleotide sequences of the HPV16E6 genes were determined by direct or cloning sequencing methods. Analysis the mutations of HPV 16 type E6 gene. The number of copies of HPV16E6 gene and β -actin was detected in parallel by FQ-PCR in tissues of 69 cervical cancer, 65 cervical intraepithelial neoplasia (CIN), 33chronic cervicitis and 96 cervical smear samples of vaginitis and cervicitis. The variation in HPV copies per genomic DNA equivalent can be estimated by dividing the HPV copy number by the β -actin copy number. The number of copies of HPV 16 E7 gene and β -actin was detected in parallel by FQ-PCR in the same tissues. In this experiment, we used the pGEX vetor of 2T-HPV16E7 to transform into E.Coli BL21(DE3), and induced by 0.1 mM IPTG to express GST-HPV16E7 fusion protein. And then the extract bacterial supernatants were purified by affinity chromatography. A450bp HPV16E6 DNA fragment was amplified by using primers designed from the coding sequence of HPV16E6 gene by PCR. The fragment containing the coding sequence of HPV16E6 proteinwas cloned into vector pMD-18T (pMD-T-HPV16E6) and transformed into E.coli JM109. Positive clone was identified successfully by enzyme digestion. Detection of HPV 16 sequences in cervical cancer with visualizing nanoparticle probes technology.Gold nanoparticle conjugated at 3’-end of SH-derivative oligonucleotide served as a detection probe, and a 5’-end NH2-derivative oligonucleotide immobili zed on glass surface as a capturing probe. Target DNA was hybridized with both of the capturing probe and the detection probe visualized based on highly sensitive "nano-amplification" and silver staining.Results: The detecting efficiency depended on DNA quality. Of 59 cervical carcinoma cases, all showed positive result of β -actin .The HPV16E6 positive rate was 83.1% (The 49 in 59 cases can be detected possitivel更多还原