外周血捕获循环肿瘤细胞的有效方法研究

发布时间：2018-06-05 11:17

本文选题：循环肿瘤细胞 + 免疫磁珠法　；参考：《中国现代医学杂志》2017年19期

【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Di I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(Onco Quick)、淋巴细胞分离术(Ficoll)、"玫瑰花富集"(Rosette Sep)、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(q RT-PCR)技术分析单细胞的CD45 m RNA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(P0.05);SNK-q检验表明,除Onco Quick组和Ficoll组(P0.05)、CELLection组和Ficoll+CELLection组(P0.05)外,其他各组之间的差异均有统计学意义(P0.05)。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。q RT-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 establish an effective method for obtaining circulating tumor cells from peripheral blood of tumor patients. Methods SW480 cells stained with Di I were premixed into the whole anticoagulant blood of healthy adults. These tumor cells were enriched by density gradient centrifugation Onco Quickco, Ficollus, Rosette Septte, CELLection. and Ficoll CELLection. the recovery rate was analyzed and the purity was compared. The single NCI-H460 HCT-116DLD-1 SW480 TF-1 and CTCs enriched in peripheral blood of patients with liver cancer were captured by micromanipulation. The expression of CD45 m RNA in single cells was analyzed by real-time quantitative polymerase chain reaction (RT-PCR). Results the recoveries of tumor cells in the five groups were 46.6 卤1.9 and 14.1 卤3.1% and 8.8 卤1.4%, respectively. The results of ANOVA showed that there was a significant difference in the recovery rates between the five groups (P 0.05SNK-q test), except for the Onco Quick group and the Ficoll group (P0.05 CELLection group and Ficoll CELLection group P0.05). The differences among other groups were statistically significant (P 0.05). The enriched tumor cells were observed under fluorescence microscope. The results showed that the purity of CELLection group and Ficoll CELLection group was higher than that of Ficoll CELLection group. The mean value of GAPDH Ct was 29.94 卤0.59. except for the CD45 Ct of a single TF-1 cell, there was no expression of CD45 in any single detected cell or CTC. Conclusion CELLection (immunomagnetic bead method combined with micromanipulation) can effectively obtain single CTC from peripheral blood of patients with liver cancer and retain its original molecular characteristics. It is suitable for subsequent single-cell analysis and research. This method can be used to analyze the characteristics of tumor molecules, evaluate the efficacy of targeted drugs and monitor the recurrence and metastasis of tumor.
【作者单位】：四川省医学科学院(四川省人民医院)消化内科;地奥九泓制药厂前沿生物技术研究室;
【基金】：四川省科技厅资助项目(No:2014TD0028)
【分类号】：R73-3

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