PCR-SSP法和基因测序检测人胰腺癌细胞株Patu 8988 K-ras基因第12位密码子点突变及其方式  

Detection of K-ras Gene Point Mutation and Its Style at Codon 12 in Human Pancreatic Cancer Cell Line Patu 8988 by PCR-SSP and Sequence Analysis

作　　者：宋妙丽[1] 杨晓春[2] 申咏梅[1] 张梅花[2] 孙亦晖[3] 沈钧康[2] 

机构地区：[1]苏州大学附属第二医院放免中心 [2]苏州大学附属第二医院磁共振室,江苏苏州215004 [3]苏州大学附属第二医院普外科,江苏苏州215004

出　　处：《苏州大学学报（医学版）》2008年第4期545-548,共4页Suzhou University Journal of Medical Science

基　　金：苏州市社会发展基金资助项目(SZD0614)

摘　　要：目的检测人胰腺癌细胞株Patu 8988 K-ras基因点突变及其突变方式,明确基因治疗靶点的碱基序列。方法针对K-ras基因第12位密码子点突变方式(CGT、GTT、GAT)设计顺序特异性引物(SSP),对人胰腺癌细胞株Patu 8988进行聚合酶链反应(PCR),扩增产物经12%非变性聚丙烯酰胺凝胶电泳后判断该细胞株有无K-ras基因点突变及其突变方式。逆转录-聚合酶链反应(RT-PCR)扩增人胰腺癌细胞株Patu 8988 K-ras基因,扩增产物直接进行基因序列测定。结果PCR-SSP法检测人胰腺癌细胞株Patu 8988存在K-ras基因点突变,突变方式为GGT→GTT,其结果与基因序列测定完全相符。结论明确了人胰腺癌细胞株Patu 8988 K-ras基因第12位密码子点突变方式(GGT→GTT),为胰腺癌的下一步基因治疗研究打下了坚实的基础;PCR-SSP方法检测K-ras基因点突变简便、快速、经济且特异性高,有望在临床上成为早期诊断和鉴别诊断胰腺癌的实用检测方法。Objective To decide the base sequences of target position for gene therapy, the K-ras point mutation and its style at codon 12 in human pancreatic cancer cell line Patu 8988 was detected. Methods Three kinds of special sequence primers reaction （PCR） with regard to the mutation styles （CGT,GTT,GAT） used to study the human pancreatic cancer cell line Patu 8988. The studied with 12% polyacrylamine gel eletrophoresis to detect the style （SSP） for polymerase chain at codon 12 of K-ras were amplification products were of point mutation. The K-ras gene of Patu 8988 cell was amplified by RT-PCR and the amplification products were directly sequenced. Results The human pancreatic cancer cell line Patu 8988 had point mutation at codon 12 and the mutation style was GGT→GTT. The result of PCR-SSP was completely in accord with that of sequence analysis. Conclusion The experiment shows that human pancreatic cancer cell line Patu 8988 has GGT→GTT mutation at codon 12. The method of PCR-SSP is rapid, convenient, economical and high specific for detecting K-ras gene point mutation. The goal in the long term is to develop a simple and useful clinical procedure for early diagnosis and differential diagnosis of pancreatic cancer.

关 键 词：胰腺癌 K—ras基因 点突变 聚合酶链反应 

分 类 号：R735.9[医药卫生—肿瘤]