乳腺癌循环肿瘤细胞分离鉴定试剂盒的建立

doi:10.3969/j.issn.1673-8640.2015.10.012

检验医学 ›› 2015, Vol. 30 ›› Issue (10): 1011-1016.DOI: 10.3969/j.issn.1673-8640.2015.10.012

• 技术研究与评价·论著 • 上一篇下一篇

乳腺癌循环肿瘤细胞分离鉴定试剂盒的建立

张健¹, 冯晓燕², 孙亭亭³, 王凯⁴, 张希锐³, 高杰锋³, 梁晓飞⁴, 康向东⁵, 彭俊杰⁶, 张贺秋², 苗朝良⁷, 沈鹤柏³

1.上海市临床检验中心,上海 2001262
2.军事医学科学院基础医学研究所生物诊断研究室,北京 100850
3.上海柏慧康生物科技有限公司,上海 201900
4.上海交通大学医学院附属仁济医院上海市肿瘤研究所,上海 200032
5.上海市普陀区中心医院检验科,上海 200062
6.复旦大学附属肿瘤医院大肠科,上海 200032
7.聊城市肿瘤医院,山东聊城 252000

收稿日期:2015-04-07 出版日期:2015-10-30 发布日期:2015-11-04
作者简介:null
作者简介:张健,男,1970年生,主任技师,博士,主要从事免疫检验及质量控制。
基金资助:
上海市科委生物医药产业处产学研项目(12DZ1941702);上海市中小企业技术创新基金资助项目(1302H159400);第2届中国创新创业大赛上海张江扶持资金资助项目

Esablishment on isolation and identification kit for circulating tumor cells in breast cancer

ZHANG Jian¹, FENG Xiaoyan², SUN Tingting³, WANG Kai⁴, ZHANG Xirui³, GAO Jiefeng³, LIANG Xiaofei⁴, KANG Xiangdong⁵, PENG Junjie⁶, ZHANG Heqiu², MIAO Chaoliang⁷, SHEN Hebai³

1.Shanghai Center for Clinical Laboratory, Shanghai 200126, China
2. Biological Diagnostic Laboratory, Institute of Basic Medicine, Academy of Military Medical Sciences, Beijing 100850, China
3.Shanghai Baihuikang Bio-Sci&Tech Co.,Ltd., Shanghai 201900, China
4. Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200032, China
5. Department of Clinical Laboratory, Putuo Central Hospital, Shanghai 200062, China
6. Department of Colorectal Surgery, Shanghai Cancer Center, Fudan University, Shanghai 200032, China
7. Liaocheng Cancer Hospital, Shandong Liaocheng 252000, China

Received:2015-04-07 Online:2015-10-30 Published:2015-11-04

摘要/Abstract

摘要： 目的

探讨乳腺癌循环肿瘤细胞(CTC)分离鉴定试剂盒中上皮细胞黏附分子(EpCAM)免疫脂质磁球及人乳腺珠蛋白(hMAM)单克隆抗体在乳腺癌CTC分离鉴定中的应用价值。

方法

将乳腺癌MCF-7细胞混于健康志愿者的新鲜血样中,采用免疫磁球分离技术分析EpCAM免疫脂质磁球对乳腺癌细胞的分离效果。在强生CellTracks^® AutoPrep^® CTC检测仪(简称强生Cellsearch^TM系统)上进行EpCAM免疫脂质磁球和强生磁球对MCF-7细胞捕获效率的模拟实验及乳腺癌患者血液的临床检测。

结果

荧光显微镜的观察结果显示EpCAM免疫脂质磁球的细胞捕获能力明显强于无抗体修饰的纳米磁球。激光共聚焦显微镜的观察结果显示EpCAM抗体修饰可显著增强磁球捕获MCF-7细胞的效率和特异性。模拟实验结果显示EpCAM免疫脂质磁球对10、20、40、80、150、200个MCF-7细胞的捕获结果与强生磁球接近;对乳腺癌患者血液中的CTC捕获效率与强生磁球相当,部分病例要明显优于强生磁球。

结论

构建的基于EpCAM免疫脂质磁球和hMAM单克隆抗体的CTC分离鉴定试剂盒对乳腺癌患者血液中的CTC具有较高的捕获效率,这将为实现乳腺癌早期诊断、术前、术后分析及治疗效果检测提供了有力的支撑。

关键词: 循环肿瘤细胞, 上皮细胞黏附分子, 免疫磁球, 人乳腺珠蛋白

Abstract: Objective

To investigate the clinical application significance of epithelial cell adhesion molecule (EpCAM)lipid immune magnetic particles and human breast mammaglobin (hMAM) monoclonal antibody in breast cancer circulating tumor cell (CTC) isolation and identification kit.

Methods

Breast cancer MCF-7 cells were mixed in healthy fresh blood samples, and the isolation ability of EpCAM lipid immune magnetic particles on breast cancer was analyzed by immune magnetic particle isolating technique. A comparison between EpCAM lipid immune magnetic particles and Cell Tracks^® AutoPrep^® System (CellSearch^TM) magnetic particles for the capture efficiency of MCF-7 cells was performed in a simulation experiment, and the hematological items were determined.

Results

The results of fluorescence microscopy showed that the capture ability of EpCAM lipid immune magnetic particle was significantly stronger than that of nanoparticles without antibody. The results of laser scanning confocal microscopy showed that the modification of EpCAM lipid immune magnetic particle antibody significantly enhanced the efficiency and specificity of magnetic particles capturing MCF-7 cells. Simulation experiment results showed that the capturing results of EpCAM lipid immune magnetic particles of 10, 20, 40, 80, 150 and 200 MCF-7 cells were close to CellSearch^TM magnetic particles. In the blood samples from patients with breast cancer, the CTC capturing efficiencies of EpCAM lipid immune magnetic particles and CellSearch^TM magnetic particles were almost the same, in some cases, the CTC capturing efficiency of EpCAM lipid immune magnetic particles was significantly better than that of CellSearch^TM magnetic particles.

Conclusions

The research on the establishment of EpCAM lipid immune magnetic particles and hMAN monoclonal antibody based on CTC isolation and identification kit has high capturing efficiency of CTC in blood samples from patients with breast cancer. It will provide a strong support for the early diagnosis, pre-operation and post-operation analysis and treatment effect evaluation.

Key words: Circulating tumor cell, Epithelial cell adhesion molecule, Immune magnetic particle, Human breast mammaglobin

中图分类号:

R446.1

张健, 冯晓燕, 孙亭亭, 王凯, 张希锐, 高杰锋, 梁晓飞, 康向东, 彭俊杰, 张贺秋, 苗朝良, 沈鹤柏. 乳腺癌循环肿瘤细胞分离鉴定试剂盒的建立[J]. 检验医学, 2015, 30(10): 1011-1016.

ZHANG Jian, FENG Xiaoyan, SUN Tingting, WANG Kai, ZHANG Xirui, GAO Jiefeng, LIANG Xiaofei, KANG Xiangdong, PENG Junjie, ZHANG Heqiu, MIAO Chaoliang, SHEN Hebai. Esablishment on isolation and identification kit for circulating tumor cells in breast cancer[J]. Laboratory Medicine, 2015, 30(10): 1011-1016.

图/表 8

图1 磁球粒径分布

图2 纳米磁球和EpCAM免疫脂质磁球捕获MCF-7细胞能力对比

图3 纳米磁球和EpCAM免疫脂质磁球捕获MCF-7细胞的激光共聚焦显微镜分析

图4 EpCAM免疫脂质磁球捕获MCF-7细胞的流式细胞术分析

图5 EpCAM免疫脂质磁球捕获MCF-7细胞的流式荧光信号分析

图6 EpCAM免疫脂质磁球与强生磁球捕获MCF-7细胞总数的对比

图7 强生磁球与EpCAM免疫脂质磁球捕获临床血样中CTC的对比

图8 EpCAM免疫脂质磁球捕获乳腺癌临床血样中CTC的免疫荧光观察

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乳腺癌循环肿瘤细胞分离鉴定试剂盒的建立

Esablishment on isolation and identification kit for circulating tumor cells in breast cancer

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