双抗体捕获结合量子点多色成像系统捕获并鉴定异质性循环肿瘤细胞  

Capture and identification of heterogeneous circulating tumor cells using double antibodies capture system and quantum dots-based multiplexed imaging

作　　者：王林伟[1] 程勃然 王舒艺[1] 陈芳芳[1] 王振盟 方远[1] 熊斌[1] 

机构地区：[1]武汉大学中南医院肿瘤科肿瘤生物学行为湖北省重点实验室湖北省肿瘤医学临床研究中心,430071

出　　处：《中华实验外科杂志》2015年第11期2669-2672,共4页Chinese Journal of Experimental Surgery

基　　金：国家高技术研究发展计划863资助项目（2012AA02AS02、2012AA02AS06）

摘　　要：目的 构建一个循环肿瘤细胞（CTCs）捕获和检测系统,实现对上皮和间质表型CTCs的捕获和鉴定.方法 制备基于羟基磷灰石-壳聚糖的透明纳米薄膜芯片,耦联上皮细胞黏附分子（EpCAM）和CD133抗体,利用免疫亲和原理捕获CTCs,采用量子点多色成像技术分别标记CD45（1：100）、角蛋白（CK,1：600）和波形蛋白（Vimentin,1：100）,对捕获的CTCs进行识别及表型鉴定.结果 双抗体捕获芯片对EpCAM中表达和低表达细胞株的捕获效率要显著高于单抗体芯片捕获（P＜0.05）.量子点多色成像证实,32例肿瘤患者血液中存在CK阳性、Vimentin阳性和CK/Vimentin均阳性的CTCs.此外,Vimentin阳性及CK/Vimentin均阳性的CTCs在进展期（Ⅲ/Ⅳ）肿瘤中所占的比例（68.4％,52/76;78.3％,47/60）远高于早期（Ⅰ/Ⅱ）肿瘤（31.6％,24/76;21.7％,13/60）.结论 双抗体捕获系统和量子点多色成像技术,可同时捕获并鉴定上皮和间质表型的CTCs,提高CTCs的捕获效率.Objective A new capture and identification system was established to capture and identify epithelial and mesenchymal circulating tumor cells （CTCs）.Methods Transparent nanofilm chips based on hydroxyapatite-chitosan nanomaterials were coupled with double antibodies of epithelial cell adhesion molecule （EpCAM） and CD133 to capture CTCs using imnune affinity reaction.Quantum dots-based multiple imaging of CD45 （1：100）, cytokeratin （1：600） and vimentin （1： 100） was applied to recognize and identify the captured CTCs.Results The captured chips with double antibodies had higher capture efficiency than chips with single antibody for EpCAM-middle and EpCAM-low expression cell lines （P 〈 0.05）.Quantum dots-based multiple imaging demonstrated cytokeratin-positive, vimentin-positive and cytokeratin/vimentin-positive CTCs existed in 32 cancer patients.In addition, percentage of CTCs with cytokeratin-/vimentin ＋ and cytokeratin ＋/vimentin ＋ in advanced stage （Ⅲ/Ⅳ） cancer （68.4%, 52/76;78.3%, 47/60） was higher than that in early stage （Ⅰ / Ⅱ） cancer （31.6%, 24/76;21.7%, 13/60）.Conclusion This study demonstrated a reliable capture and detection system that combined double antibodies capture system with quantum dots-based multiplexed imaging, for epithelial and mesenchymal CTCs, which improved the capture efficiency of CTCs.

关 键 词：循环肿瘤细胞 双抗体 量子点 上皮-间充质转化 

分 类 号：R739.91[医药卫生—肿瘤]