多肽功能化的微流芯片用于循环肿瘤细胞检测和分析的研究

Cancer metastasis is the main reason of the death of tumor patients.The appearance of circulating tumor cells (CTCs) could hint the condition of cancer metastasis and help to evaluate the effect of tumor therapy. Currently,the detection of CTCs is mainly based on the size difference between tumor cells and blood cells, or the specific markers on the epithelial tumor cells, which is not accurate enough for the diagnosis of CTCs. In our study, specific binding peptides for epithelial cell adhesion molecule(EpCAM) and the marker of epithelial-tomesenchymal transition (EMT), N-Cadherin, were screened and identified by bacteria surface display methods, and microfluidic chips functionalized by those binding peptides groups were used to detect and collect the CTCs. The collected CTCs were examined by miRNA chips to identify the valuable miRNAs which were either diffently expressed or mutant. Those valuable miRNAs would help us to identify the biological molecules and signal transduction pathways which could mediate the cancer metastasis. The key scientific questions in our study were the relationship and function of epithelial cells and cells under EMT in the CTCs; adaptation relationship among specific binding peptides for EpCAM and N-Cadherin, linker peptides and substrate peptides on the microfluidic chips functionalized by peptides; the activity of biomolecules and signal transduction pathways indicated by the change of miRNAs on the cancer metastasis. The successful conduction of the project would provide the sensitive and precise platform for the detection of CTCs and provide the novel clues for the study and therapy of metastatic cancers.

肿瘤转移是导致肿瘤致死的主要原因，循环肿瘤细胞（CTCs）的出现可以提示肿瘤转移的情况以及对肿瘤治疗进行评估，目前对于CTCs的检测多利用肿瘤细胞与血细胞之间尺寸差异或上皮来源肿瘤细胞表面的上皮细胞粘附因子（EpCAM），存在较大的局限性。本项目拟利用细菌表面展示技术筛选可以与EpCAM和上皮细胞间充质化（EMT）标志物神经型-钙粘蛋白特异结合的多肽，利用多肽组合功能化的微流芯片对CTCs进行检测和捕获，通过miRNA芯片检测的方法获知导致肿瘤转移发生的生物分子和信号转导通路。本项目要解决的关键科学问题是CTCs中上皮来源和EMT化的细胞在肿瘤转移中的作用和相互关系；多肽功能化的微流芯片上结合多肽、链接肽链和底物多肽之间的空间适配关系；受miRNA改变影响的蛋白和信号转导通路在肿瘤转移中的作用。项目的成功实施可以为CTCs的检测提供准确灵敏的检测平台，为转移性肿瘤的研究和治疗提供新的思路。

循环肿瘤细胞的检测对于转移瘤的早期诊断、药物的疗效评估、肿瘤患者的预后以及肿瘤治疗新靶点的发现都具有非常深远的研究和临床应用价值。考虑到循环肿瘤细胞的异质性（上皮类的、间充质类的和肿瘤干细胞同时存在），以及其稀少性，本项目使用了微流体系结合多价多肽的方法对循环肿瘤进行捕获和释放，并进行相应的后续研究。具体来说，本项目的研究内容在于使用细菌表面展示技术筛选获得可以与上皮类肿瘤细胞特异结合多肽序列，设计构建成功适于循环肿瘤细胞捕获的U形微流管道及细胞通过条件，初步确定用于细胞捕获的多价多肽组合及比例，构建成功小鼠的原位肺癌模型，对肿瘤患者的真实血液进行了初步的循环肿瘤细胞检测探索；设计了新的捕获多肽序列用于微流流道内的细胞无损释放。在项目的执行过程中，深入探讨了流道设计、多肽类型与循环肿瘤细胞捕获效率相关性的问题。本项目的成功实施，为循环肿瘤细胞的检测和后续研究提供了新的工具，进一步验证了循环肿瘤细胞异质性的存在，为肿瘤转移机制的研究提供了新的手段。