摘要
目的 构建膀胱癌循环肿瘤细胞特异性筛选平台,实现对膀胱癌循环肿瘤细胞的高特异性筛选.方法 2015年5月至2016年4月借助微流控技术设计并制作膀胱癌循环肿瘤细胞特异性筛选芯片.通过设计芯片、制作光刻阳膜、用聚二甲基硅氧烷制作基片、修剪、打孔、清洗处理,之后封接制成芯片.将链霉亲和素包被到微通道内,对膀胱癌单克隆抗体BCMab1进行生物素化修饰.链霉亲和素与生物素化抗体结合,形成链霉亲和素-生物素-抗体系统.为研究抗体对捕获率的影响,将芯片分为包被BCMab1抗体的芯片和不包被BCMab1抗体的芯片两组,将60μl、浓度为5 000个/ml的样本液以流速为10 μl/min泵入到两组芯片中.为探讨细胞浓度及流速对捕获率的影响,将细胞浓度为500、5 000和50 000个/ml的样本液,借助注射泵分别以10、15、20、25和30 μl/min的速度泵入到通道内进行筛选.为研究流速及鱼骨结构对芯片捕获靶细胞的影响,我们设计了两种不同的芯片,即含有鱼骨结构和未含有鱼骨结构的芯片,二者均包被BCMab1抗体,将60μl细胞浓度为5 000个/ml的样本液,借助注射泵,分别以流速为10、15、20、25和30 μl/min泵人到这两种芯片中.结果 制成的特异性筛选膀胱癌循环肿瘤细胞的微流控芯片,大小为4.0 cmx0.3 cm×0.5 cm.芯片有上下两层,上层为鱼骨样结构,宽50 μm,高45 μm,相邻两个鱼骨结构之间的间距为100～300 μm;下层为微通道,微通道高50 iμm.高倍显微镜下观察,包被BCMab1抗体的芯片捕获靶细胞平均（20.8±6.3）个,未含抗体的芯片捕获靶细胞平均（1.2±1.1）个,二者差异有统计学意义（P＜0.05）.在流速为10μl/min时,含有抗体及鱼骨结构的芯片捕获率为（90.3 ±1.0）％,含有抗体而未含有鱼骨结构的芯片捕获率为（46.7±2.1）％,差异有统计学意义（P＜0.05）.结论 设计的捕获膀胱癌循环肿瘤细胞微流控芯片成本低,操�
Objective To fabricate a microfluidic chip to specifically screen bladder cancer cells.Methods Using microfluidic technology,we designed and fabricated filter chips that can specifically isolate circulating bladder cancer cells from May 2015 to April 2016.The chip was fabricated through photolithography after spin coating negative photoresist,trimming,punching and cleaning process.The surface of the microchannel was modified by biotin-avidin-BCMabl-system （BAS）.The blood sample containing T24 cells was used to test the validly of the chip.Results Chip was consisted of two layers,whose size was 4.0 cm x 0.3 cm x 0.5 cm.The upper structure was the fishbone structure that changed the fluid flow state.The fishbone structure was 45μm high and the spacing between fishbone structure was 50μm.The lower structure that captures the circulating tumor cells was micro-channel whose height was 50 μm.The whole chip modified by bladder cancer monoclonal antibody-BCMab1 could capture bladder cancer circulating tumor cells with high specificity,and capture rate was as high as 90%.Conclusions In summary,this chip might be a new way to diagnosis bladder cancer.
作者
王云超
牛海涛
李凯
门同义
梁晔
张勇
张晓明
杨吉伟
沈彬
李现铎
陈冬冬
唐冠宝
王建宁
Wang Yunchao Niu Haitao Li Kai Men Tongyi Liang Ye Zhaag Yong Zhang Xiaoming Yang Jiwei Shen Bin Li Xianduo Chen Dongdong Tang Guanbao Wang Jianning(Department of Urology, Qianfoshan Hospital, Shandong University, Jinan 250014, China)
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
2016年第12期936-939,共4页
Chinese Journal of Urology
关键词
膀胱癌
循环肿瘤细胞
特异性筛选
微流控芯片
Bladder cancer
Circulating tumor cells
Specific screening
Microfluidic device
