基于去唾液酸糖蛋白受体的循环肝癌细胞磁性分选检测法及其改良

Detection of circulating tumor cells in patients with hepatocellular carcinoma using an improved asialoglycoprotein receptor-based separation method

目的:介绍一种肝细胞癌(hepatocellular carcinoma,HCC)循环肿瘤细胞(circulating tumor cells,CTCs)富集和检测系统,并对该系统进行改良,以提高循环肿瘤细胞的检出率.方法:去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)是一种特异表达于肝实质细胞膜的跨膜蛋白.我们建立了一种基于ASGPR的磁性分选和Heppar1免疫鉴定的循环肝癌细胞富集和计数系统.首先以生物素化去唾液酸胎球蛋白作为配体与肝癌细胞结合,然后用抗生物素抗体磁珠进行间接磁性标记,从而磁性捕获循环肝癌细胞,接着用Heppar1进行免疫荧光染色鉴定,并对阳性细胞进行计数.文献报道和实际操作中发现,检测过程中采用肝素抗凝会引起细胞悬液中出现凝胶,影响细胞通过分离柱,降低分离效率.我们将部分步骤改用乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)替代肝素抗凝,采用Hep3B肝癌细胞掺入试验对两种方法回收情况进行比较.结果:部分步骤改用EDTA抗凝后,细胞悬液中未出现凝胶现象.细胞掺入试验显示,5mL健康志愿者外周血中分别掺入10、30、90、270和810个Hep3B细胞,在每个掺入水平,改良法Hep3B细胞的平均回收率均≥70%,回收效率明显增加(P<0.05).结论:基于ASGPR的循环肝癌细胞富集和计数系统是一种具有临床应用潜能的循环肝癌细胞检测方法.经改良后能够有效消除细胞悬液中凝胶形成的现象,肝癌细胞回收效率较原检测法明显提高.

AIM： To introduce a novel magnetic cell separation system which allows for immunomorphological identification and enumeration of circulating tumor cells （CTCs） in patients with hepatocellular carcinoma （HCC）.METHODS： The asialoglycoprotein receptor （ASGPR） is a transmembrane protein expressed exclusively on the surface of hepatocytes. We have recently developed a sensitive and specific system mediated by the interaction of the ASGPR with its ligand to magnetically separate CTCs in HCC patients. In the system, HCC cells were bound by biotinylated asialofetuin, an ASGPR ligand, and subsequently labeled by anti-biotin antibody-coated magnetic beads, followed by magnetic separation. The separated HCC cells were then identified by immunofluorescence staining using the hepatocyte-specific antibody Hep Par 1. In this study, we used EDTA instead of heparin for anticoagulation because heparin could cause the presence of gels in cell suspension, which affected the passage of cells through the separation column and reduced the separation efficiency. The recovery, specificity and sensitivity of the HCC CTC separation and detection system were determined by performing Hep3B cell spiking experiments. RESULTS： Calcium chelating agent EDTA was used for anticoagulation instead of heparin in some steps of the original method and gel phenomenon no longer appeared in the cell suspension. The cell spiking experiments showed that when there were 10, 30, 90, 270 and 810 Hep3B cells spiked into five milliliters of peripheral blood from healthy volunteers, the average re- covery was ≥ 70% at each spiking level and the recovery of the modified method was higher than that of the original method （P 0.05）.CONCLUSION： We have developed a new tool that allows for highly sensitive and specific separation and detection of CTCs in HCC patients. It may be clinically useful for diagnosis and monitoring of HCC. The cell spiking experiments showed that the recovery of the modified method was higher than that of the original method.