胃癌手术前后血浆中p16基因的异常甲基化检测  

Promoter hypermethylation of the p16 gene in pre- and post-gastrectomy plasma of patients with gastric adenocarcinoma

作　　者：刘亚航[1] 张连海[1] 任晖[1] 张桂国[1] 秦斐[2] 孔广忠[2] 邓国仁[1] 季加孚[1] 

机构地区：[1]北京大学临床肿瘤学院,北京肿瘤医院外科 [2]内蒙古医学院第一附属医院外科

出　　处：《北京大学学报（医学版）》2005年第3期257-260,共4页Journal of Peking University:Health Sciences

基　　金：北京大学肿瘤学院基金项目(00 01; 04 22)资助~~

摘　　要：目的:研究胃癌患者手术前后外周血浆、对应原发肿瘤以及癌旁组织中p16基因启动子区异常甲基化的检出状况,探讨其在胃癌疗效评估中的应用.方法:采用甲基化特异性PGR方法,扩增84例胃癌患者的原发肿瘤、癌旁组织、手术前血浆中pl6基因启动子区,以及上述患者中30例行胃癌根治性手术患者术后14～21天血浆中p16基因启动子区,以15例健康人血浆作为正常对照.采用凝胶电泳-溴乙锭显色以及高效液相色谱两种方法检测产物.结果:84例患者样品中,原发肿瘤中存在pl6基因异常甲基化26例(31.0%),癌旁组织中阳性2例(0.02%),术前血浆中阳性12例(14.3%),15例健康人血浆检测均为阴性.患者血浆阳性者同时也存在对应肿瘤组织阳性.30例同时有手术前后血浆标本的胃癌根治手术患者中,原发肿瘤组织中p16异常甲基化14例,其中6例术前血浆阳性,术后5例转阴.在检测样本中,高效液相色谱的检测结果与凝胶电泳-溴乙锭显色结果一致.结论:胃癌患者外周血浆中可以检测到与原发肿瘤一致的p16异常甲基化,手术后血浆中p16甲基化状态的变化与手术治疗有关,高效液相色谱作为一种便捷的技术可以应用于PCR产物的检测.Objective: To detect promoter hypermethylation of the p16 gene in pre- and post-operative plasma, matched cancer tissues and para-tumor non-cancerous tissues of patients with gastric adenocarcinoma for evaluating the effectiveness of therapeutic intervention. Methods: Primary tumor tissues and para-tumor tissues and preoperative plasma samples of 84 patients with gastric adenocarcinoma were collected, and 14-21 days’ post-operative plasma of 30 of the 84 patients who underwent curative gastectomy was available. Plasma of 15 healthy people was also collected as control. After sodium-bisulfite treatment, extracted DNA was amplified for p16 promoter hypermethylation by methylation-specific polymerase chain reaction (MSP). The PCR products were detected by both gel-ethidium bromide electrophoresis and high performance liquid chromatogram (HPLC). Results: Among the samples from 84 patients, p16 hypermethylation was detected in 26 (31.0%) cancer tissues and 2 (0.02%) para-tumor non-cancerous tissues and 12 (14.3%) preoperative plasma, while plasma of the healthy people was negative. In all positive plasma, the paired primary tumor tissues were also confirmed to be methylated. As far as samples from 30 patients with post-operative plasma were concerned, 14 cancer tissues and 6 preoperative plasma samples were positive, and only 1 of 6 post-operative plasma remained hypermethylated. The results detected by HPLC exactly matched those by gel-ethidium bromide electrophoresis. Conclusion: Promoter hypermethylation of the p16 gene detected in plasma consists with that in primary cancer tissues of patients with gastric adenocarcinoma. The alteration of status of hypermethylation of p16 in post-operative plasma is considered the consequences of surgical intervention. HPLC can be used as an efficient tool in detecting the product of MSP.

关 键 词：手术前后 P16基因启动子区 P16基因异常甲基化 高效液相色谱 原发肿瘤 胃癌患者 癌旁组织 甲基化特异性 外周血浆 手术患者 凝胶电泳 肿瘤组织 PCR方法 健康人血浆 人血浆检测 甲基化状态 PCR产物 疗效评估 正常对照 患者血浆 

分 类 号：R654.2[医药卫生—外科学] R735.9[医药卫生—临床医学]