顺序特异性引物法快速检测胰腺癌K-ras基因点突变

Rapid detection of K-ras gene point mutation in pancreatic adenocarcinonia by sequence special primer PCR

目的 研究简捷、特异、敏感的检测胰腺癌K-ras基因点突变的方法及其在胰腺疾病中定性诊断的价值。方法 采用针对K-ras基因点突变方式（CGT、GAT、GTT）设计的顺序特异性引物（SSP）,先后对胰腺癌石蜡包埋组织、冰冻新鲜组织、细针穿刺组织及胰液进行多聚酶链反应（PCR）,扩增产物借助常规电泳和染色检测有无K-ras基因突变及突变方式。结果 胰腺癌石蜡包埋组织、冰冻新鲜组织、细针穿刺组织及胰液中K-ras基因点突变率分别为74.2％、95.1％、91.4％及94.1％,而所有被检测的慢性胰腺炎、胰岛素瘤、壶腹癌、胆管癌、十二指肠乳头癌及外伤胰腺的组织标本和胰液标本均无K-ras基因突变发生。结论 该检测法简便、快速、特异、敏感,具有临床实用性,可以作为鉴别胰腺肿块良恶性和诊断胰腺癌的一种方法。

Objective To explore a simple, rapid, specific and sensitive method of detecting K-ras gene point mutation and evaluate its qualitative diagnostic value in pancreatic adenocarcinoma. Methods According to the K-ras point mutant type (CGT, GTT and GAT) in pancreatic adenocarcinoma, three kinds of sequence special primer (SSP) for ploymerase chain reaction were designed, where K-ras gene fragment was amplified from frozen or paraffin embedded pancreatic adenocarcinoma tissues, fine needle aspiration sample from pancreatic adenocarcinoma and pancreatic juice of patients with pancreatic carcinoma. K-ras point mutation of the amplified product was detected by gel electrophoresis. Results K-ras gene point mutation rate was 74.2% (23/31) in paraffin embedded pancreatic adenocarcinoma tissues, 95.1%(39/41) in frozen pancreatic adenocarcinoma tissues, 91.4%(31/35) in fine needle aspiration sample and 94.1%(16/17) in pancreatic juice. No mutant K-ras gene was found in tissues and pancreatic juice of patients with chronic pancreatitis, insulinoma, ampullary carcinoma, bile duct carcinoma, duodenum papillary adenocarcinoma and injured pancreas. Conclusions Detecting the mutant K-ras with PCR-SSP is rapid, convenient, specific and as sensitive. It may serve as a practical method for distinguishing pancreatic benign masses from malignant ones, and can make a definitive diagnosis of pancreatic adenocarcinoma.