粪便中大肠癌相关基因突变体与表达量的数字化微球芯片检测新方法研究

项目名称： 粪便中大肠癌相关基因突变体与表达量的数字化微球芯片检测新方法研究

项目编号： No.21275161

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 数理科学和化学

项目作者： 周国华

作者单位： 中国人民解放军南京军区南京总医院

项目金额： 80万元

中文摘要： 大肠癌是最常见的消化道恶性肿瘤，其发生与发展涉及多个基因在多阶段突变的积累过程。因此，检测粪便中与肿瘤相关基因的突变体和表达量将能实现大肠癌的早期无创诊断。然而在肿瘤发生早期，从癌组织脱落的细胞数量极少，粪便中含有大量来源于正常组织的脱落细胞，严重干扰着肿瘤组织来源脱落细胞的检测。为此，本课题首先基于免疫反应原理，建立无臭抗体微球粪便脱落细胞提取法；再利用微乳液PCR技术，发展微球介导的单分子扩增模板制备新方法；然后结合三维水凝胶微球芯片法，探讨凝胶中微球表面靶标与无标记探针高特异性杂交条件；最后采用Cy3-dUTP介导的杂交探针延伸反应，建立高效标记微球芯片中各待测微球的新方法。在此基础上，将多重单分子扩增技术和单层微球芯片技术相结合，创建粪便中微量肿瘤相关基因突变体和基因表达量的检测新方法，解决高背景下微量待测靶标难以准确检测的难题，为大肠癌早期无创诊断提供一个有力工具。

中文关键词： 大肠癌；基因突变；基因甲基化；无创检测；数字化检测

英文摘要： Colorectal cancer (CRC) is one of the most common malignant neoplasms in gastrointestinal tract, but can be effectively treated by surgical excision if detected at an earlier stage. Carcinogenesis and progression of CRC relate to the accumulation of DNA mutations in many genes at different stages; hence, early diagnosis of CRC could be achieved by using both DNA mutants and gene expression levels as the detection markers. However, at the early stage of CRC, exfoliated cells from tumor in stool is sparse comparing to those from normal tissues, significantly interfering the detection of the minority tumor-source exfoliated cells. To solve this problem, a new odorless method based on immunoreactions is proposed to extract tumor-source exfoliated cells from stool by using bead-immobilized antibodies. By employing emulsion PCR, a new way is also proposed to efficiently prepare the templates qualified for bead-mediated single molecule amplification. With the use of a 3-dimensional hydrogel chip, conditions for specifically hybridizing label-free probes with the targets coated on the beads immobilized in the gel pores will be studied. In addition, the approach based on hybridized probe extension with Cy3-dUTP will be developed for specifically labeling the beads in the gel-array. Finally, the novel method will be devel

英文关键词： colorectal cancer；gene mutation；gene methylation；noninvasive detection；digital detection