發(fā)布時(shí)間：2018-04-24 13:22

  本文選題：HPV自動(dòng)基因分型 + 限制性片段多態(tài)性��； 參考：《福州大學(xué)》2014年碩士論文

【摘要】：宮頸癌是威脅女性生命健康的第二大殺手,卻也是至今病因明確的唯一癌癥。超過99%的宮頸癌與人乳頭狀瘤病毒(HPV)的感染有因果關(guān)系。因此,對(duì)HPV感染情況的篩查成為重要而有效的預(yù)防宮頸癌的早期篩查手段。但是HPV的不同亞型的致病風(fēng)險(xiǎn)程度及其在不同地域人群中的分布都有所差異,直接對(duì)HPV進(jìn)行分型檢測(cè)顯然更具有實(shí)際意義和價(jià)值。然而,目前仍然缺乏靈敏且經(jīng)濟(jì)適用的分型方法。宮頸癌的發(fā)展不僅僅是HPV感染的結(jié)果,還是因其影響導(dǎo)致宿主細(xì)胞中發(fā)生一系列基因及其表達(dá)功能改變的結(jié)果。研究這些改變并找到關(guān)鍵的癌變相關(guān)標(biāo)志物,例如基因或蛋白,不僅有利于印證宮頸癌的篩查效果,也為治療宮頸癌的治療提供了新的目標(biāo)。因此,本課題研究了可用于宮頸癌篩查的HPV分型方法并探索了宮頸癌變的可能標(biāo)志物,研究成果在宮頸癌的早期診斷上具有實(shí)際應(yīng)用價(jià)值,為宮頸癌的治療提供新的可能。第一章綜述了HPV與宮頸癌的相關(guān)背景,介紹了HPV分型檢測(cè)的方法,論述了其優(yōu)缺點(diǎn)。詳述了HPV影響致癌的主要分子機(jī)制,由此引出本文選用標(biāo)志物的可能性猜想,并對(duì)其進(jìn)行了初步驗(yàn)證。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鏈?zhǔn)椒磻?yīng)(PCR),限制性片段長(zhǎng)度多態(tài)性(RFLP)分析結(jié)合芯片電泳(MCE)的方法對(duì)HPV進(jìn)行分型。以47種HPV類型為對(duì)象,與細(xì)胞學(xué)檢查和DNA測(cè)序方法對(duì)比,考察了其在宮頸脫落細(xì)胞樣品中的檢測(cè)效果。結(jié)果顯示陽性檢出率是傳統(tǒng)的細(xì)胞形態(tài)學(xué)檢查的4倍,檢測(cè)出單一感染占2/3,共測(cè)出11種HPV類型,分型準(zhǔn)確度大于90%。證明了 PCR-RFLP-MCE自動(dòng)分型方法的優(yōu)越性以及用于輔助宮頸癌早期診斷的HPV篩查的可能性。第三章初步驗(yàn)證了所選標(biāo)志物的變化規(guī)律。選取HIF-1α及c-myc為目標(biāo)癌變標(biāo)志物,猜想了 HIF-1α與c-myc在宮頸癌細(xì)胞中的相互作用方式,利用基因啟動(dòng)子上游存在能形成G-四聯(lián)體序列的特征,研究了HIF-1αα,c-myc,p21和VEGF的基因表達(dá)變化。采用G-四聯(lián)體配體化合物TMPyP4刺激,獲得基本與預(yù)測(cè)相符的結(jié)果,即HIF-1α,c-myc,VEGF表達(dá)均下降,p21表達(dá)增加,細(xì)胞周期停滯在G1期。以上結(jié)果為進(jìn)一步研究監(jiān)測(cè)標(biāo)志物變化和基因治療的靶標(biāo)奠定了基礎(chǔ)。最后,論文對(duì)本研究課題進(jìn)行了總結(jié),并對(duì)所建立的HPV分型方法的更廣泛應(yīng)用以及癌變標(biāo)志物的應(yīng)用和在基因治療上的前景進(jìn)行了預(yù)測(cè)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 偽 and c-myc were selected as target markers of carcinogenesis. The interaction between HIF-1 偽 and c-myc in cervical cancer cells was conjectured. The gene expression of HIF-1 偽 -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 偽 -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.
【學(xué)位授予單位】：福州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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