本文選題：HPV　切入點：宮頸癌　出處：《第三軍醫(yī)大學》2006年碩士論文　論文類型：學位論文

【摘要】： 背景 人類乳頭狀瘤病毒(Human papillomavirus, HPV)屬于Papovaviridae家族,是一種雙鏈環(huán)狀DNA病毒,大小約8000 bp,目前已發(fā)現(xiàn)了200多種亞型。HPV感染是宮頸癌發(fā)生的主要病因,持續(xù)高危型HPV的感染是癌前病變和浸潤性宮頸癌發(fā)展的必要因素。由于不同亞型HPV的致病機制和致癌危險性各不相同,因此對HPV各亞型進行分型檢測有重要的臨床意義。目前,臨床上對HPV進行分型檢測主要依靠熒光實時PCR技術,但該方法檢測通量低,一個反應管中只能檢測一個亞型,難以滿足臨床上同時檢測多個亞型的需要。本研究探討了利用寡核苷酸芯片技術同時檢測HPV多個亞型的可能。 目的 建立帶有不同亞型HPV L1基因片段的哺乳動物細胞克隆;在此基礎上初步探討利用寡核苷酸芯片技術對HPV的分型檢測的可能。 方法 1、建立帶有不同亞型HPV L1基因片段的哺乳動物細胞克隆用做標準陽性對照。 2、收集NCBI數(shù)據(jù)庫中的HPV各亞型的序列信息,設計相應的探針。 3、利用基因芯片技術及熒光定量PCR方法對HPV進行分型檢測。 結果 1、成功克隆了30個宮頸癌相關亞型HPVL1基因片段,成功構建并篩選出6個攜帶有HPV各亞型L1基因片段的細胞株。 2、初步制備出可以分型檢測HPV的寡核苷酸芯片并建立了相應的檢測方法。 結論 應用重疊延伸PCR的方法,成功克隆了30個宮頸癌相關亞型HPVL1基因片段并構建6個攜帶有HPV各亞型L1基因片段的細胞株;初步初步制備出可以分型檢測HPV的寡核苷酸芯片并建立了相應的檢測方法。
[Abstract]:Background. Human papillomavirus (HPVs) belongs to the Papovaviridae family and is a double-stranded circular DNA virus of about 8000 BP in size. At present, more than 200 subtypes of HPV infection have been found to be the main etiology of cervical cancer. Persistent and high risk HPV infection is a necessary factor for the development of precancerous lesions and invasive cervical cancer. Because different subtypes of HPV have different pathogenetic mechanisms and carcinogenic risks, it is of great clinical significance to detect the subtypes of HPV subtypes. Clinical typing and detection of HPV mainly rely on fluorescence real-time PCR technology, but the detection flux of this method is low, only one subtype can be detected in a reaction tube. It is difficult to detect multiple subtypes simultaneously in clinic. In this study, the possibility of simultaneous detection of multiple subtypes of HPV using oligonucleotide chips was discussed. Purpose. The mammalian cell clones with different subtypes of HPV L1 gene were established and the possibility of using oligonucleotide microarray to detect HPV typing was preliminarily discussed. Method. 1. Mammalian cell clones with different subtypes of HPV L1 gene were used as standard positive control. 2. The sequence information of HPV subtypes in NCBI database was collected and the corresponding probes were designed. 3. Genotyping of HPV was detected by gene chip technique and fluorescence quantitative PCR. Results. 1. 30 HPVL1 gene fragments of cervical cancer related subtype were cloned successfully, and 6 cell lines carrying L1 gene fragments of HPV subtype were successfully constructed and screened. 2. The oligonucleotide chip which can be typed to detect HPV was prepared and the corresponding detection method was established. Conclusion. By means of overlapping extension PCR, 30 HPVL1 gene fragments of cervical cancer related subtypes were cloned and 6 cell lines carrying L1 gene fragments of HPV subtypes were constructed. The oligonucleotide chip which can be used to type and detect HPV was preliminarily prepared and the corresponding detection method was established.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R373

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本文編號：1575247