肝細(xì)胞癌（HCC）是一種預(yù)后很差的惡性腫瘤,亞洲是全球發(fā)病率最高的地區(qū)。許多生物和化學(xué)物質(zhì)是HCC的主要病原,如乙型肝炎病毒（HBV）、丙型肝炎病毒（HCV）和黃曲霉素。我國(guó)是HBV感染和HCC的高發(fā)地區(qū),約10%的人群感染HBV,其慢性攜帶者部分最終發(fā)展成肝細(xì)胞癌。HBV的感染與肝細(xì)胞癌（HCC）的發(fā)生發(fā)展密切相關(guān),HBV DNA與肝細(xì)胞基因組DNA的整合在HCC的發(fā)病過(guò)程中具有十分重要的意義。HBV DNA整合機(jī)制和后果的研究將進(jìn)一步闡明HCC發(fā)生的分子生物學(xué)機(jī)制,為探索新型的治療技術(shù)和治療方法奠定基礎(chǔ)。本實(shí)驗(yàn)首先運(yùn)用普通聚合酶鏈反應(yīng)（PCR）技術(shù),確定乙肝病毒基因整合位點(diǎn)的大致位置,并據(jù)此在病毒基因鄰近整合斷端處設(shè)計(jì)兩對(duì)反向引物,然后使用內(nèi)切酶ApaⅠ和HNcoⅠ分別對(duì)病毒基因組左側(cè)和右側(cè)進(jìn)行酶切。對(duì)酶切片斷進(jìn)行自身環(huán)化后,運(yùn)用反向聚合酶鏈反應(yīng)（IPCR）對(duì)人高轉(zhuǎn)移潛能肝癌細(xì)胞株MHCC97-H基因組中整合序列進(jìn)行擴(kuò)增。然后將PCR產(chǎn)物轉(zhuǎn)入pGEM-T載體、測(cè)序。測(cè)序結(jié)果顯示整合的HBV DNA片段位于左側(cè)C基因區(qū)的2220 nt到右側(cè)X基因區(qū)的1590nt,整合的HBV DNA... 

【文章來(lái)源】：中南大學(xué)湖南省 211工程院校 985工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：67 頁(yè)

【學(xué)位級(jí)別】：碩士

【部分圖文】：

普通PCR確定HBV整合斷端的大概位置

圖5MHCC97一HgDNA整合片斷擴(kuò)增M.DL1500Marker1.X端整合片斷2.C端整合片斷3重組質(zhì)粒的鑒定結(jié)果.3.1重組質(zhì)粒的pcR鑒定結(jié)果C端與X端整合片斷經(jīng)質(zhì)粒重組，PCR鑒定結(jié)果與3.2結(jié)果相符。M日f(shuō)kerl231000bP500bP300bP圖6整合片斷重組質(zhì)粒鑒定M.DLIO00Marker1，2質(zhì)粒重組x端整合片斷

Mar晚r1500bpI000bpSOObP300bp10Obp圖5MHCC97一HgDNA整合片斷擴(kuò)增M.DL1500Marker1.X端整合片斷2.C端整合片斷重組質(zhì)粒的鑒定結(jié)果.1重組質(zhì)粒的pcR鑒定結(jié)果C端與X端整合片斷經(jīng)質(zhì)粒重組，PCR鑒定結(jié)果與3.2結(jié)果相符。

【參考文獻(xiàn)】：
期刊論文
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