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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.

为建立高灵敏度的胰腺癌诊断方法，我们建立了HPA/TRAP法、真实的TRAP法、胰液中端粒酶mRNA定量检测方法和k-ras基因突变电化学分析法，其中端粒重复序列扩增结合杂交保护法（HPA/TRAP）是一种快速、灵敏的检测胰液中端粒酶活性的方法。HPA/TRAP检测有助于胰腺癌与IPMN或胰腺炎的鉴别诊断。TRAP结合实时荧光定量PCR（真实的TRAP）检测端粒酶活性也更灵敏、快速。同时，我们定量检测了胰液中端粒酶亚单位hTERT的mRNA。我们发现hTERT在胰腺癌、IPMN和慢性胰腺炎中的表达有显著性差异，并定量检测了胰腺癌相关基因的mRNA。MUC 1和MUC 5AC在胰腺癌组织中高表达，定量检测胰液中MUC 1和MUC 5AC mRNA对胰腺癌的术前诊断具有重要意义。S100家族成员S100 A6在显微切割的胰腺癌细胞中显著升高。为探讨电化学芯片（electrochemical array，ECA）检测胰腺癌组织中K-ras基因突变的有效性，采用ECA芯片检测胰腺癌组织中K-ras基因突变。85%的胰腺癌组织中存在K-ras基因突变，这与PCR依赖的优先同源双链形成试验的结果一致。我们发现ECA芯片是一种敏感、快速、可靠的点突变筛查方法。