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

使用改良端粒重复扩增方案和电化学阵列芯片开发胰腺癌基因诊断系统

基本信息

批准号：
15390399
负责人：
MIZUMOTO Kazuhiro
金额：
$ 9.34万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390399/
关键词：
Pancreatic cancer pancreatic juice TRAP HPA hTERT real time PCR ECA chip

项目摘要

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.

为了建立诊断胰腺癌的高灵敏度检测方法，我们开发了HPA/TRAP检测、实时TRAP检测、胰液中mRNA的定量测量方法以及k-ras突变的电化学分析方法。端粒重复扩增方案结合杂交保护测定（HPA/TRAP）可高度灵敏且快速地检测胰液中的端粒酶活性。 HPA/TRAP 测定可用于区分胰腺癌与 IPMN 或胰腺炎。 TRAP 实时 PCR（实时 TRAP）检测端粒酶活性也更灵敏且更快速。此外，我们定量测量了胰液中端粒酶亚基 hTERT 的 mRNA。我们发现胰腺癌、IPMN和慢性胰腺炎之间hTERT表达存在显着差异。还在胰液中定量测量了胰腺癌相关基因的几种mRNA。 MUC1和MUC5AC在胰腺癌中高表达，胰液中MUC1和MUC5AC mRNA的定量评估对于胰腺癌的术前诊断具有很高的潜力。 S100A6是S100家族的成员，在显微解剖的胰腺癌细胞中显着升高。胰液中 S100A6 mRNA 的定量也是诊断胰腺癌的一种有前景的工具。为了研究电化学阵列 (ECA) 芯片的有效性，使用 ECA 芯片检测了胰腺癌组织中的 k-ras 突变。在 85% 的胰腺癌中发现了 K-ras 突变，这与 PCR 依赖性优先同源双链体形成测定获得的结果相同。我们发现ECA芯片对于筛查临床样本中的点突变敏感、快速、可靠。