甲基化特异性聚合酶链反应检测粪便ppENK基因甲基化对胰腺癌诊断的可行性

Feasibility of testing ppENK gene methylation in stool with methylation-specific polymerase chainreaction assay in pancreatic cancer diagnosis

目的评价甲基化特异性聚合酶链反应（MSP）检测粪便ppENK基因高甲基化用于诊断胰腺癌的可行性。方法收集胰腺癌患者24例和对照6例的新鲜粪便标本。采用MSP检测全部粪便标本中ppENK的甲基化状态；采用PCR检测全部粪便标本中野生型ppENK的阳性率。采用PCR-限制性片段长度多态性（RFLP）检测粪便K-ras的突变情况。将胰腺癌细胞PC3单细胞悬液加入同一健康者粪便标本，MSP法检测ppENK甲基化阳性，计算甲基化阳性时需掺人的胰腺癌细胞的最少数量。结果30份粪便标本的甲基化检出率为0（0／30），非甲基化检出率为10％（3／30），野生型ppENK检出率为6．7％（2／30）；PCR-RFLP可检测出所选10份野生型ppENK阴性的胰腺癌标本中的8份，其中7份有K-ras第12位密码子突变。MSP方法能够检测到粪便中ppENK甲基化条带所需胰腺癌细胞的数量至少为50个／ml。结论采用MSP方法检测胰腺癌患者粪便标本的ppENK基因甲基化状态，尚不能成为筛查和诊断胰腺癌的方法。

Objective To evaluate the feasibility of testing the high methylation of ppENK gene in stool with methylation-specific polymerase chain reaction （MSP） assay in pancreatic cancer diagnosis. Methods Twenty-four fresh stool samples of pancreatic cancer patients and six healthy control samples were collected. The methylation status of ppENK gene in all the stool samples was detected by MSP assay. The positive rate of wild-type ppENK gene in all the stool samples was determined by polymerase chain reaction （PCR）. And 10 non wild-type ppENK gene negative pancreatic cancer samples were collected, and K-ras gene mutation was detected by PCR-restriction fragment length polymorphism （RFLP）. The single cell suspension of pancreatic cancer PC3 cell line was added into stool sample from the same healthy individual, the positive rate of ppENK gene methylation was detected by MSP assay. The minimum number of pancreatic cancer cell was calculated when methylation was positive. Results The raie of methylation detection in 30 samples was 0 （0/ 30）; and the rate of non-methylation detection was 10% （3/30）. The rate of wild-type ppENK detection was 6.7% （2/30）. By PCR-RFLP assay, eight were successfully amplified and seven had mutation in 12th code of K-ras gene in 10 selected wild-type ppENK gene negative pancreatic cancer samples. The minimum number of pancreatic cancer cells needed for ppENK methylation band positive detected by MSP was 50 cell/ml. Conclusion Detecting ppENK gene methylation status instool samples of pancreatic cancer patients by MSP assay has not yet become the method of pancreatic cancer screening and diagnosis.