[固定乳腺癌细胞中雌激素和孕激素受体的蛋白质免疫印迹分析及流式细胞术分析]

[Western blot analysis and flow cytometric analysis of estrogen and progesterone receptors in fixed breast cancer cells].

作者信息

Tao De-Ding, Jiang Min, Wu Jian-Hong, Feng Yong-Dong, Gong Jian-Ping

机构信息

Molecular Medicine Center, Tongji Hospital Affiliated Tongji Medical College in Huazhong University of Science and Technology, Wuhan, Hubei, 430030, PR China.

出版信息

Ai Zheng. 2004 Mar;23(3):339-41.

PMID:15025971

Abstract

BACKGROUND & OBJECTIVE: Quantitative and qualitative detection of estrogen receptor (ER) and progesterone receptor (PR) levels are very important for predicting prognosis and evaluating the outcome of endocrine therapy of breast cancer patients. Western blot analysis and Flow cytometry (FCM) are important means for quantitative and qualitative analysis of proteins, but conventional Western blot analysis need to extract proteins from fresh cells. The present study was designed to establish Western blot analysis of human estrogen receptor (ER) and progesterone receptor (PR) in fixed breast cancer cells, and to explore the possibility of ER and PR analysis in fixed cells by flow cytometry and Western blot analysis simultaneously.

METHODS

Proteins extracted from fresh and fixed cells of different exponentially growing breast cancer cell lines were labelled using 1D5 and PgR636, which were monoantibodies to ERalpha and PR, respectively. The expression of ER and PR were determined using Western blot analysis, and the results were compared with that of fixed cells measured with flow cytometry.

RESULTS

Clear and correct ERalpha bands were observed with Western blot analysis in cell lines T-47d, MCF-7, and ZR-75-1, and the band density of fixed T-47d and ZR-75-1 was higher than that of fresh cells. The expression of ERalpha in MM231 cells was negative. Clear and correct PR bands were visible in T-47d and ZR-75-1 cells with Western blot analysis, and the band density of fixed cells was higher than that of fresh cells. And the expression of PR in MM231 and MCF-7 cells were negative. In addition, positive expression of ER and PR in different cell lines measured by flow cytometry were the same with that analyzed by Western blot analysis.

CONCLUSION

After fixed with 0.25% paraformaldehyde and 75% ethanol, breast cancer cells can be used for not only quantitative measurement of ER and PR, but also Western blot analysis of ER and PR.

摘要

背景与目的

雌激素受体（ER）和孕激素受体（PR）水平的定量和定性检测对于预测乳腺癌患者的预后及评估内分泌治疗效果非常重要。蛋白质免疫印迹分析和流式细胞术（FCM）是蛋白质定量和定性分析的重要手段，但传统的蛋白质免疫印迹分析需要从新鲜细胞中提取蛋白质。本研究旨在建立固定乳腺癌细胞中人类雌激素受体（ER）和孕激素受体（PR）的蛋白质免疫印迹分析方法，并探讨通过流式细胞术和蛋白质免疫印迹分析同时对固定细胞中的ER和PR进行分析的可能性。

方法

使用分别针对ERα和PR的单克隆抗体1D5和PgR636标记从不同指数生长期乳腺癌细胞系的新鲜细胞和固定细胞中提取的蛋白质。采用蛋白质免疫印迹分析测定ER和PR的表达，并将结果与流式细胞术检测的固定细胞结果进行比较。

结果

在细胞系T-47d、MCF-7和ZR-75-1中，蛋白质免疫印迹分析观察到清晰且正确的ERα条带，固定的T-47d和ZR-75-1细胞的条带密度高于新鲜细胞。MM231细胞中ERα的表达为阴性。蛋白质免疫印迹分析在T-47d和ZR-75-1细胞中可见清晰且正确的PR条带，固定细胞的条带密度高于新鲜细胞。MM231和MCF-7细胞中PR的表达为阴性。此外，流式细胞术检测的不同细胞系中ER和PR的阳性表达与蛋白质免疫印迹分析结果一致。