HPV基因分型筛查方法及HIF-1α和c-myc癌变标记物检测

发布时间：2018-04-24 13:22

本文选题：HPV自动基因分型 + 限制性片段多态性　；参考：《福州大学》2014年硕士论文

【摘要】：宫颈癌是威胁女性生命健康的第二大杀手,却也是至今病因明确的唯一癌症。超过99%的宫颈癌与人乳头状瘤病毒(HPV)的感染有因果关系。因此,对HPV感染情况的筛查成为重要而有效的预防宫颈癌的早期筛查手段。但是HPV的不同亚型的致病风险程度及其在不同地域人群中的分布都有所差异,直接对HPV进行分型检测显然更具有实际意义和价值。然而,目前仍然缺乏灵敏且经济适用的分型方法。宫颈癌的发展不仅仅是HPV感染的结果,还是因其影响导致宿主细胞中发生一系列基因及其表达功能改变的结果。研究这些改变并找到关键的癌变相关标志物,例如基因或蛋白,不仅有利于印证宫颈癌的筛查效果,也为治疗宫颈癌的治疗提供了新的目标。因此,本课题研究了可用于宫颈癌筛查的HPV分型方法并探索了宫颈癌变的可能标志物,研究成果在宫颈癌的早期诊断上具有实际应用价值,为宫颈癌的治疗提供新的可能。第一章综述了HPV与宫颈癌的相关背景,介绍了HPV分型检测的方法,论述了其优缺点。详述了HPV影响致癌的主要分子机制,由此引出本文选用标志物的可能性猜想,并对其进行了初步验证。在最后,提出了本文的思路和研究方案。第二章建立了基于聚合酶链式反应(PCR),限制性片段长度多态性(RFLP)分析结合芯片电泳(MCE)的方法对HPV进行分型。以47种HPV类型为对象,与细胞学检查和DNA测序方法对比,考察了其在宫颈脱落细胞样品中的检测效果。结果显示阳性检出率是传统的细胞形态学检查的4倍,检测出单一感染占2/3,共测出11种HPV类型,分型准确度大于90%。证明了 PCR-RFLP-MCE自动分型方法的优越性以及用于辅助宫颈癌早期诊断的HPV筛查的可能性。第三章初步验证了所选标志物的变化规律。选取HIF-1α及c-myc为目标癌变标志物,猜想了 HIF-1α与c-myc在宫颈癌细胞中的相互作用方式,利用基因启动子上游存在能形成G-四联体序列的特征,研究了HIF-1αα,c-myc,p21和VEGF的基因表达变化。采用G-四联体配体化合物TMPyP4刺激,获得基本与预测相符的结果,即HIF-1α,c-myc,VEGF表达均下降,p21表达增加,细胞周期停滞在G1期。以上结果为进一步研究监测标志物变化和基因治疗的靶标奠定了基础。最后,论文对本研究课题进行了总结,并对所建立的HPV分型方法的更广泛应用以及癌变标志物的应用和在基因治疗上的前景进行了预测和展望。
[Abstract]:Cervical cancer is the second-biggest killer of women's lives, but it is the only cancer with a clear etiology. More than 99% of cervical cancer is causally associated with HPV infection. Therefore, screening for HPV infection has become an important and effective early screening method to prevent cervical cancer. However, different subtypes of HPV have different pathogenicity risk and their distribution in different geographical population, so it is of great significance and value to detect HPV directly. However, there is still a lack of sensitive and economical classification method. The development of cervical cancer is not only the result of HPV infection, but also the result of a series of gene and expression changes in host cells due to its influence. Studying these changes and finding key markers, such as genes or proteins, not only help to confirm the screening effect of cervical cancer, but also provide a new goal for the treatment of cervical cancer. Therefore, this paper studies the HPV typing method which can be used for cervical cancer screening and explores the possible markers of cervical cancer. The research results have practical application value in the early diagnosis of cervical cancer, and provide a new possibility for the treatment of cervical cancer. The first chapter summarizes the background of HPV and cervical cancer, introduces the method of HPV typing and discusses its advantages and disadvantages. In this paper, the main molecular mechanism of HPV affecting carcinogenesis is described, and the possibility of selecting markers in this paper is deduced, and it is preliminarily verified. In the end, the paper puts forward the train of thought and research scheme. In chapter 2, a method of HPV typing based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis combined with microarray electrophoresis (HPV) was established. Compared with cytological examination and DNA sequencing, 47 HPV types were used to detect cervical exfoliated cells. The results showed that the positive rate was 4 times higher than that of the traditional cell morphological examination. The detection rate of single infection was 2 / 3. Eleven types of HPV were detected, and the accuracy of typing was more than 90%. The advantages of PCR-RFLP-MCE automatic typing method and the possibility of HPV screening for early diagnosis of cervical cancer were demonstrated. The third chapter preliminarily verifies the rule of change of the selected markers. HIF-1 伪 and c-myc were selected as target markers of carcinogenesis. The interaction between HIF-1 伪 and c-myc in cervical cancer cells was conjectured. The gene expression of HIF-1 伪 -c-mycp21 and VEGF was studied by using the G- tetraad sequence in upstream of gene promoter. G- tetraad ligand (TMPyP4) was used to stimulate the cell cycle, and the results were in good agreement with the prediction. The expression of HIF-1 伪 -c-mycton-VEGF decreased and the expression of p21 increased, and the cell cycle stagnated in G1 phase. These results lay a foundation for further study on the changes of markers and the target of gene therapy. Finally, the thesis summarizes the research topic, and forecasts the wider application of the established HPV typing method, the application of cancer markers and the prospect of gene therapy.
【学位授予单位】：福州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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