Development of genetic diagnosis system for pancreatic cancer using modified telomeric repeat amplification protocols and electrochemical array chip

为了建立高灵敏度的胰腺癌诊断检测方法，我们开发了HPA/TRAP检测、实时TRAP检测、胰腺液中mRNA的定量检测方法以及k-ras突变的电化学分析。端粒重复扩增联合杂交保护法（HPA/TRAP）检测胰液端粒酶活性具有较高的灵敏度和快速性。HPA/TRAP检测有助于胰腺癌与IPMN或胰腺炎的鉴别。实时聚合酶链反应（real time TRAP）检测端粒酶活性也更灵敏、更快速。此外，我们定量测量了端粒酶亚基hTERT在胰液中的mRNA表达。我们发现hTERT在胰腺癌、IPMN和慢性胰腺炎中的表达有显著差异。胰液中还定量检测了几种胰腺癌相关基因的mrna。MUC1和MUC5AC在胰腺癌中高表达，定量评估胰腺液中MUC1和MUC5AC mRNA对胰腺癌的术前诊断具有很高的潜力。S100家族成员S100A6在胰腺癌微解剖细胞中的表达明显升高。胰腺液中S100A6 mRNA的定量检测也是一种很有前景的胰腺癌诊断工具。为了验证电化学阵列（ECA）芯片的有效性，我们用ECA芯片检测了胰腺癌组织中k-ras基因的突变。在85%的胰腺癌中鉴定出K-ras突变，这与pcr依赖的优先同源双工形成试验的结果相同。我们发现ECA芯片是一种灵敏、快速、可靠的临床样本点突变筛选方法。

To establish highly sensitive assays for the diagnosis of pancreatic cancer, we developed HPA/TRAP assay, real time TRAP assay, methods for quantitative measurements of mRNA in pancreatic juice, and electrochemical analysis of k-ras mutations.Telomeric repeat amplification protocol combined with hybridization protection assay (HPA/TRAP) was highly sensitive and rapid to detect telomerase activity in pancreatic juice. HPA/TRAP assay was useful to differentiate pancreatic cancer from IPMN or pancreatitis. TRAP with real-time PCR (real time TRAP) was also sensitive and more rapid to detect the telomerase activity. Futhermore, we quantitatively measured mRNA of hTERT, subunits of telomerase, in pancreatic juice. We found significant difference in hTERT expression among pancreatic cancer, IPMN and chronic pancreatitis.Several mRNAs of pancreatic cancer related genes were also quantitatively measured in pancreatic juice. MUC1 and MUC5AC were highly expressed in pancreatic cancer and quantitative assessment of MUC1 and MUC5AC mRNA in pancreatic juice has high potential for preoperative diagnosis of pancreatic cancer. S100A6,a member of S100 family, was significantly higher in microdissected pancreatic cancer cells. Quantification of S100A6 mRNA in pancreatic juice is also a promising tool for diagnosis of pancreatic cancer.To investigate the validity of the electrochemical array (ECA) chip, k-ras muations were detected with ECA chip in pancreatic cancer tissues. K-ras mutations were identified in 85% of pancreatic cancer, which was identical to the results obtained by PCR-dependent preferential homoduplex formation assay. We found that ECA chip is a sensitive, rapid, and reliable for screening point mutations in clinical samples.