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.

abstractsObjective 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.