运用反向聚合酶链反应技术检测MHCC97-H肝癌细胞整合的乙肝病毒序列

发布时间：2021-08-11 04:12

　　肝细胞癌（HCC）是一种预后很差的恶性肿瘤,亚洲是全球发病率最高的地区。许多生物和化学物质是HCC的主要病原,如乙型肝炎病毒（HBV）、丙型肝炎病毒（HCV）和黄曲霉素。我国是HBV感染和HCC的高发地区,约10%的人群感染HBV,其慢性携带者部分最终发展成肝细胞癌。HBV的感染与肝细胞癌（HCC）的发生发展密切相关,HBV DNA与肝细胞基因组DNA的整合在HCC的发病过程中具有十分重要的意义。HBV DNA整合机制和后果的研究将进一步阐明HCC发生的分子生物学机制,为探索新型的治疗技术和治疗方法奠定基础。本实验首先运用普通聚合酶链反应（PCR）技术,确定乙肝病毒基因整合位点的大致位置,并据此在病毒基因邻近整合断端处设计两对反向引物,然后使用内切酶ApaⅠ和HNcoⅠ分别对病毒基因组左侧和右侧进行酶切。对酶切片断进行自身环化后,运用反向聚合酶链反应（IPCR）对人高转移潜能肝癌细胞株MHCC97-H基因组中整合序列进行扩增。然后将PCR产物转入pGEM-T载体、测序。测序结果显示整合的HBV DNA片段位于左侧C基因区的2220 nt到右侧X基因区的1590nt,整合的HBV DNA... 

【文章来源】：中南大学湖南省 211工程院校 985工程院校 教育部直属院校

【文章页数】：67 页

【学位级别】：硕士

【部分图文】：

普通PCR确定HBV整合断端的大概位置

图5MHCC97一HgDNA整合片断扩增M.DL1500Marker1.X端整合片断2.C端整合片断3重组质粒的鉴定结果.3.1重组质粒的pcR鉴定结果C端与X端整合片断经质粒重组，PCR鉴定结果与3.2结果相符。M日fkerl231000bP500bP300bP图6整合片断重组质粒鉴定M.DLIO00Marker1，2质粒重组x端整合片断

Mar晚r1500bpI000bpSOObP300bp10Obp图5MHCC97一HgDNA整合片断扩增M.DL1500Marker1.X端整合片断2.C端整合片断重组质粒的鉴定结果.1重组质粒的pcR鉴定结果C端与X端整合片断经质粒重组，PCR鉴定结果与3.2结果相符。

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