基于cell-SELEX核酸适配体功能化微流控芯片的循环肝癌细胞捕获、识别及分子表征谱鉴定的研究

Circulating tumor cells (CTCs) with EMT phenotype and tumor-like characteristics are the metastatic origin of carcinoma. CTCs counting and/or detection of CTCs molecule expression profiling are of great importance for detecting tumor metastasis in early stage and individualized diagnosis and treatment. Due to the lack of specific targeting molecules, clinical detection of circulating HCC cells or its molecular expression profiling processed slowly. In our previous study, we have screened aptamer which is specifically binding to HCC cells with highly metastatic potential by cell-SELEX. When conjugated with fluorescent nanoparticles QDs, the aptamer - QD conjugates could specifically recognize highly metastatic HCC cells. Furthermore, the aptamer - magnetic particles conjugates could effectively detect and extract metastatic HCC cells from complex mixture whole blood. So, in this project, we plan to establish a new diagnostic approach for detecting circulating HCC cells by using a combination of triple steps- - -specific capture by microfluidic chip, target recognition by aptamers, and precisely labeling by quantum dots for sensitive imaging. In other words, circulating HCC cells can be captured and counted by aptamers conjugated on the nanostructure that is coated on the surface of microfluidic chip. Then, in order to further identify their molecular expression profiling, these captured cells will be released and cultured on chips. Either way, our research will provide useful evidence for developing aptamer functionalized microfluidic chip; moreover, it will also serve to identify molecular expression profiling of CTCs and further diagnosis. Of great importance, our work will also provide strong support for developing HCC "liquid biopsy" based on detecting circulating hepatocarcinoma cells in an aptamer-and-quantum-based integrated microchip system.

具有EMT表型或肿瘤样干性的循环肿瘤细胞（CTCs）是肿瘤转移根源。CTCs计数或CTCs分子表征谱的检测对早期转移预警和个体化诊治具有重要价值。由于缺乏特异性靶向分子，肝癌CTCs或其分子表征谱的临床检测进展缓慢。我们前期应用cell-SELEX 技术筛选出特异结合高转移肝癌细胞的适配子LY-1，该适配子耦联量子点的分子探针可识别高转移肝癌细胞，该适配子耦联磁颗粒的复合物可从外周血模拟环境中捕获肝癌CTCs。本项目拟以适配子靶向识别、微流控芯片细胞捕获和量子点荧光标记成像，建立肝癌CTCs检测新方法。适配子修饰在微流控芯片纳米结构表面，高灵敏、高特异的捕获肝癌CTCs并计数，捕获的细胞释放后在芯片上培养以进一步鉴定CTCs 的分子表征谱。该研究将为开发适配子功能化微流控芯片提供佐证，为CTCs分子表征谱鉴定和基于分子表征谱的肝癌CTCs检测以及基于CTCs的肝癌"液体活检"提供实验依据。

由于肝癌EpCAM表达率低下、EMT表型转化及缺乏特异性靶向膜表面分子，肝癌CTCs及其分子表征谱临床研究进展缓慢。肝癌CTCs的研究报道主要如下：密度梯度离心法、ISET法、qRT-PCR法、基于CD90和 ICAM-1的流式细胞法、CellSearch法、基于CD45的阴性富集法、基于去唾液酸糖蛋白受体的磁性分选和微流控芯片法等。我们以前期筛选出的特异结合高转移肝癌细胞的核酸适配体LY1为循环肝癌细胞靶向识别分子、表面三维纳米柱阵列的微流控芯片为循环肝癌细胞捕获载体和纳米材料量子点耦联核酸适体的复合物为循环肝癌细胞分子识别荧光成像，建立循环肝癌细胞检测的新方法。适配子修饰在微流控芯片表面三维纳米柱阵列上，高灵敏、高特异的捕获循环肝癌细胞并计数。同时，以捕获后释放的循环肝癌细胞为研究对象，进一步鉴定出了循环肝癌细胞关联性的分子表型谱。核酸适体LY1所识别肝癌细胞EpCAM阴性，未识别的肝癌细胞EpCAM阳性表达。核酸适体LY1所识别肝癌细胞具有MET和干性样表型，Twist、Vimentin、CK19、CD44、PIK3、CD133、ICAM-1、TLR4高表达，E-cadherin、 CD90、CXCR4、PTEN低表达。 基于核酸适体LY1功能能纳米材料捕获的肝癌病人外周血CTCs，经DAPI、CD45、CKs(CK8/18/19)、Vimentin 荧光鉴定，有四种荧光模式: DAPI + CD45 – CKs+ 、DAPI + CD45– Vimentin+、DAPI + CD45 + CKs+、DAPI + CD45– CKs –。其中还发现有细胞核很小的肝癌CTCs，捕获到比单个CTC更具侵袭力的循环肝癌细胞簇CTM。发现不同肝癌病人和同一肝癌病人的不同时期，CTC分子表型异质性较高。病人外周血循环肝癌细胞团CTM(CTC-clusters)内部有异质性，且预示肝癌病人预后较差。核酸适体LY1功能化纳米材料捕获的外周血模拟环境中肝癌CTCs，经核酸外切酶无创释放后，可体外培养、裸鼠体内成瘤，捕获的临床肝癌病人外周血CTCs，经核酸外切酶可无创释放，但体外培养、裸鼠体内成瘤还未成功。本项研究结合我国肝癌防治研究的实际需要，以循环肝癌细胞为研究对象，建立基于核酸适配子靶向识别、微流控芯片捕获及量子点多色成像的循环肝癌细胞检测新方法。同时，鉴定出循环肝癌细