人肝癌组织中γ-谷氨酰转移酶异常表达及其基因甲基化程度的研究.docVIP

???? 人肝癌组织中γ-谷氨酰转移酶异常表达及其基因甲基化程度的研究 ???? ????　【摘要】　目的　探讨γ-谷氨酰转移酶(GGT)基因在人类肝癌发生过程中的表达及改变机制。方法　以病理组织学、生物化学和分子生物学方法，将自身配对的肝癌、癌旁组织和远癌组织中总RNA纯化，对GGT的表达与改变进行了分析；并设计两套引物，以逆转录巢式PCR法扩增GGT5′-非编码区(NC)基因和限制性内切酶消化分析了不同肝癌组织中GGT基因的甲基化程度。结果　在病理组织学证实的24例肝癌、癌旁和远癌组织中：①从肝癌灶至远癌的不同组织中，总RNA浓度呈逐步升高趋势，三种不同组织间的差异均有显著性(P0.05);②癌组织中总GGT比活性(U/g)较癌周和远癌组织明显升高(P0.05);③以自行设计建立的逆转录巢式PCR法，成功地扩增GGT的5′-NC基因片段，特异性强且区带清晰，PCR产物经酶切分析证明，GGT基因5′-NC M3位点(CCGG)，其内位C在癌灶、癌周和远癌组织中的低甲基化频率分别为70.83%,58.33%和54.17%，非常明显地高于同组中已甲基化程度。结论　肝癌组织中GGT基因甲基化程度较低，且与肝癌GGT的异常表达关系密切，对肝细胞癌变可能起促进作用。　　【关键词】　肝癌　　γ-谷氨酰转移酶　　基因　　表达　　RNA　　甲基化 Abnormal expression and methylation status of γ-glutamyl transferase (GGT) genes in human hepatoma tissue　TANG Qiyun, YAO Dengfu, LIU Yanhua, et al. 　The First Hospital, Yancheng City of Jiangsu Prouince, 224001　　【Abstract】　Objective　To explore the mechanisms of expression and alteration of γ-glutamyl transferase (GGT) genes during the development of human hepatic carcinoma.Methods　The total RNA concentration and the expressions and changs of different molecular forms of GGT in the self-controled liver cancerous, paracancerous and distal cancerous tissues were investigated by pathologic examination, biochemical and molecular biological assays. Moreover, the part GGT genes of the 5′-NC region were amplified with two sets of primers by using a nest RT-PCR assay, and methylation status of GGT genes were also studied by a restriction endonuclease RT-PCR technique.Results　Of the 24 tissue specimens confirmed by pathological examination:① An increasing tendency of total RNA concentrations was found from cancer to distal cancerous tissues, and there was a marked difference (P0.05) between those in liver cancers and paracancerous /or distal cancerous tissues; ② The specific activities (U/g) of total GGT was significantly higher (P0.05) in hepatoma tissues than in those of paracancerous/or distal cancerous tissues; ③ Analysed by using a restriction endonuclease RT-PCR assay, the inner C of M3-CCGG-site of