【作者】 任剑珍；

【导师】 霍继荣；

【作者基本信息】 中南大学 ， 消化内科， 2007， 硕士

【摘要】 目的：探讨E-cadherin在结肠癌HT-29细胞系中的蛋白表达及其甲基化状况，分析二者的关系以及甲基化抑制剂对其影响，了解DNA甲基化在结肠癌发生发展中的作用。方法：利用人结肠癌细胞系HT-29；用免疫细胞化学染色检测甲基化转移酶抑制剂5-氮杂脱氧胞苷(5-Aza-CdR)干预前后的HT-29细胞系E-cadherin的表达变化；采用甲基化特异性聚合酶链式反应(MSP)检测HT-29细胞中E-cadherin基因启动区域甲基化状态及去甲基化作用后甲基化的变化。结果：(一)*5-Aza-CdR处理前后HT-29细胞形态学出现明显改变：未经5-Aza-CdR处理的HT-29细胞呈梭形或多边形，不规则，有时成团生长；经5-Aza-CdR处理后，部分细胞于出现细胞体积缩小、核固缩，染色质边集，核质比例减少；呈分裂相的细胞数目减少，部分核细胞出现核仁消失，提示HT-29细胞系在5-Aza-CdR干预作用下生长情况同对照组比较有显著性差异(P＜0.01)；且随着药物浓度的增加，细胞生长速度呈现减缓的趋势。*免疫细胞化学染色检测结果示，在5-Aza-CdR干预作用下，24h后E-cadherin表达较干预前增强，干预前后相比具有明显的差异(P＜0.01)；细胞E-cadherin阳性表达率由1μM药物处理时的21％±7％提高到5μM药物处理时的39％±13％；积分光密度值检测结果示5-Aza-CdR对HT-29细胞株E-cadherin的表达有明显的上调作用。(二) MSP检测结果示，未经5-Aza-CdR处理的HT-29细胞中E-cadherin基因启动子区域甲基化阳性；而经1μM和5μM 5-Aza-CdR各干预24h后HT-29细胞中E-cadherin基因启动子区域均为甲基化阴性。结论：1)结肠癌中E-cadherin的蛋白表达下调，且E-cadherin低表达与其启动区域异常甲基化相关；2)去甲化作用对HT-29细胞株E-cadherin的表达有明显的上调作用，提示寻找特异性抑制甲基化酶活性的药物是肿瘤治疗的一个方向；3) E-cadherin基因启动区域异常甲基化可能是与结肠癌发生有密切关系的重要分子事件，为结肠癌的临床早期诊断、检测和治疗提供一定的分子理论与实验依据。更多还原

【Abstract】 Objective To explore the expression and the methylation status ofE-cadherin in colon carcinoma cell line HT-29, to analyze the relationbetween the expression and DNA methylation of E-cadherin, and tounderstand the role of DNA methylation during the development of coloncarcinoma.Materials and method Colon carcinoma cell line HT-29 fromhomo were examined; Immunocytochemistry dicho-step method wasused to detect the expression change of E-cadherin protein in HT-29 cellswith 5-Aza-CdR and without 5-Aza-CdR; Methylation specific-PCR(MSP) was used to detect the methylation status at promoter ofE-cadherin gene and the change of this status after demethylation in cellline HT-29.Results (一) * There were much morphological change in HT-29cells: the shape of HT-29 cells without 5-Aza-CdR was fusiform orpolygon, irregular, sometimes agglomerate growth; to some of HT-29cells with 5-Aza-CdR, whose change included cell shrinkage, nucleuspycnosis, chromatin marhgination and so on. These change showed therewere significant differences between the treated group and the controlgroup(P＜0.01), and the cell growth velocity slowed down gradually with the increasing drug concentration. * Immunocytochemistry stainingshowed the expression of E-cadherin protein, enhanced obviously withthe role of 5-Aza-CdR contrast to the control after 24 hours, there wereobvious differences between the treated group and the control group(P＜0.01); the positiveexpression ratio of E-cadherin rised from 21%±7%(1μM) to 39%±13% (5μM); 5-Aza-CdR had made the expression ofE-cadherin protein in HT-29 cells up-regulated significantly. (二) MSPshowed that the promoter methylation of E-cadherin gene in the controlgroup was positive, and it was negative in the treated.Conclusion 1) The expression of E-cadherin protein was downregulation in colon carcinoma; there was significant correlation betweenthe low expression of E-cadherin protein and the aberrant promotermethylation of E-cadherin gene; 2) Demethylation had an obviousup-regulation role to the expression of E-cadherin in HT-29 cells, whichshowed that drugs with controlling DMT were a direction of tumortreatment; 3) Aberrant promoter methylation of E-cadherin gene might bean inchoate molecular event; which offered some molecular theory andexperiment evidence for clinical early diagnosis, detection and treatmentof colon carcinoma.更多还原