外周血捕获循环肿瘤细胞的有效方法研究

Isolation of single circulating tumor cell from peripheral blood of liver cancer patients by immunomagnetic beads and micromanipulation

摘要:

目的建立有效获取肿瘤患者外周血中单个循环肿瘤细胞（CTC）的方法。方法预先将经DiI染色的SW480 细胞混入健康成人的抗凝全血中，分别单独采用密度梯度离心（OncoQuick）、淋巴细胞分离术（Ficoll）、“玫瑰花富集”（RosetteSep）、免疫磁珠（CELLection）及淋巴细胞分离术联合免疫磁珠联用（Ficoll+CELLection）富集这些肿瘤细胞，统计回收率并观察纯度以进行比较。再使用显微操作仪捕获单个NCI-H460、HCT-116、DLD-1、SW480、TF-1 等探测细胞及从肝癌患者外周血中富集获得的CTC，采用实时荧光定量聚合酶链反应（qRT-PCR）技术分析单细胞的CD45 mRNA 表达水平。结果5 组方法的肿瘤细胞回收率分别为（52.5±3.5）%、（46.6±1.9）%、（36.9±5.5）%、（14.1±3.1）%和（8.8±1.4）%；单因素方差分析表明，5 组方法回收率比较差异有统计学意义（p <0.05）；SNK-q 检验表明，除OncoQuick 组和Ficoll 组（p >0.05）、CELLection组和Ficoll+CELLection 组（ p>0.05）外，其他各组之间的差异均有统计学意义（p <0.05）。在荧光显微镜下观察富集的肿瘤细胞，发现CELLection 组及Ficoll+CELLection组的纯度更高。qRT-PCR结果显示，总循环数60，GAPDH Ct 均值为（29.94±0.59），除了单个TF-1 细胞的CD45 Ct 均值为（34.21±0.22），其他单个探测细胞及CTC 的CD45 均无表达。结论CELLection 即免疫磁珠法配合显微操作能有效获取肝癌患者外周血中的单个CTC 并保留其原始的分子特性，适用于后续单细胞分析与研究。该方法为CTC 运用于剖析肿瘤分子的特征、评价靶向治疗药物的药效以及监控肿瘤的复发与转移等临床问题奠定了基础。

Abstract:

Objective To isolate single circulating tumor cell (CTC) from peripheral blood of liver cancer patients. Methods Four commercial kits including OncoQuick, Ficoll-Paque PLUS (Ficoll), RosetteSep and CELLection, as well as Ficoll-CELLection-combination were used to enrich DiI-stained SW480 cells added to peripheral blood of healthy adults. In order to compare the above five methods, recovery and purity of enriched tumor cells were counted or observed. Then, detection cells including NCI-H460，HCT-116，DLD-1，SW480 and TF-1, and CTCs from the peripheral blood of liver cancer patients were isolated by micromanipulation individually. Subsequently, mRNA expression level of CD45 antigen, the leukocyte common antigen, was detected by qRT-PCR. Results Compared in pairs, the recovery of the five methods [OncoQuick (52.5 ±3.5)%, Ficoll (46.6 ±1.9)%, RosetteSep (36.9 ±5.5）%,CELLection (14.1 ±3.1)% and Ficoll-CELLection-combination (8.8 ±1.4)%] revealed significant differences (p <0.05) except for OncoQuick and Ficoll (p > 0.05), CELLection and Ficoll-CELLection-combination (p > 0.05) by one-way ANOVA test and SNK-q method. Moreover, CELLection and Ficoll-CELLection-combination enriched tumor cells with higher purity when they were observed under fluorescent microscope. In addition, CD45 mRNA expression of all single detection cells and CTC were negative except TF-1 (mean Ct within 60 cycles: TF-1 (34.21±0.22), GAPDH (29.94±0.59), others none; n= 3). Conclusions The combination of immunomagnetic beads and micromanipulation is an effective method to isolate single CTC from peripheral blood of liver cancer patients. The captured single CTC keeps its original molecular characteristics for subsequent single-cell analysis. Furthermore, our method lays a foundation for clinical application of CTC, for tumor molecular characteristic analysis, therapeutic effect evaluation and treatment monitoring.