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

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 developed for digital detection of both mutation rates and relative gene expression levels of CRC-biomarkers in stool by combining the multiplex single-molecule amplification with the hydrogel chip in which a single layer of beads is immobilized, and this method will enable the detection of minority targets of interest from a high background; non-invasive diagnosis of CRC will, thus, be achieved based on the detection results.

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。

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的积累变异过程。由于粪便中存在来源于肿瘤组织的脱落细胞与肿瘤DNA片段，因此，检测粪便中与大肠癌相关的基因标志物能实现大肠癌的无创筛查。.为建立基于粪便中核酸标志物检测的大肠癌早期筛查方案，本课题首先研究了粪便脱落细胞及粪便DNA的高效提取方法。采用包被有抗体的免疫磁珠，建立了高效率的无臭抗体微球粪便脱落细胞提取方法；通过改进粪便样本的预处理过程，利用聚乙烯吡络烷酮（PVP）对粪便样本中的杂质进行吸附沉淀，从而对其中的DNA样本进行浓缩，显著提高了粪便DNA提取试剂盒的提取效率；并建立了基于锁核酸（LNA）探针的粪便DNA靶标捕获方法，采用磁珠分离可高效获得粪便中人DNA靶标片段，为后期进行大肠癌核酸标志物检测提供了可靠的材料。.其次，我们对粪便核酸中的大肠癌相关基因标志物检测方法进行了研究。采用微球水凝胶芯片检测平台结合微乳液单分子扩增，发展了大肠癌相关基因表达量差异及基因突变的多重连接微球水凝胶芯片检测新方法，实现了对粪便中大肠癌相关基因表达量差异标志物与突变标志物的同时检测；此外，发展了可定量检测粪便DNA中微量基因甲基化标志物的微球水凝胶数字化检测新方法，实现了在大量非甲基化DNA背景下检测出0.05%含量的甲基化DNA，可用于大肠癌及腺瘤的无创检测。.为了简化检测流程，将核酸侵入反应与延伸链置换反应、纳米金颗粒显色反应相偶联，建立了系列基因突变检测新方法，实现了对0.1%的突变DNA可视化检测，避免对昂贵的荧光检测设备的依赖。此外，将高灵敏PCR扩增与高特异核酸侵入反应偶联，创建了高灵敏、高特异性的粪便DNA甲基化水平定量检测方法，实现了对低至0.01%含量的甲基化DNA检测。.最终，以粪便中大肠癌相关基因甲基化与突变标志物为检测对象，采用所建立的大肠癌无创检测方案对临床样本进行检测，验证方法的实用性，结果表明，对大肠癌的检测灵敏度和特异性均达到85%以上，为大肠癌早期无创筛查提供了一个有力工具。