固定后乳腺癌细胞雌激素受体和孕激素受体的FCM和Westernblot检测

Western Blot Analysis and Flow Cytometric Analysis of Estrogen and Progesterone Receptors in Fixed Breast Cancer Cells

背景与目的:雌激素受体(estrogenreceptor,ER)和孕激素受体(progesteronereceptor,PR)水平的定性、定量检测,对乳腺癌患者预后判断和内分泌治疗效果的评价具有重要意义。Westernblot法和流式细胞术(flowcytometry,FCM)是蛋白质定性、定量分析的重要手段,但常规Westernblot法要求提取新鲜样本的蛋白质。本研究拟建立固定后乳腺癌细胞ER和PR的Westernblot检测方法,探索Westernblot法和FCM对同批固定样本ER、PR进行同步分析的可行性。方法:取不同乳腺癌细胞株对数生长期新鲜细胞和固定细胞的蛋白提取物,分别用ERα单克隆抗体1D5和PR单克隆抗体PgR636以Westernblot法对ER、PR的表达情况进行检测,并与同期固定细胞的FCM检测结果进行比较。结果:经Westernblot法检测,T-47d、MCF-7、ZR-75-1细胞可见分子量正确的ERα清晰条带,固定T-47d和ZR-75-1细胞的ERα条带较新鲜细胞的条带浓,MM231细胞ERα检测为阴性;T-47d和ZR-75-1细胞可见清晰且分子量正确的PR条带,固定细胞的PR条带较新鲜细胞的条带浓,MM231和MCF-7细胞PR检测为阴性;同期固定细胞ER、PR阳性表达的FCM检测结果与Westernblot检测结果一致。结论:不同乳腺癌细胞在经0.25%多聚甲醛(paraformaldehyde,PFA)和75%乙醇固定后,可用于ER、PR的FCM定量检测。

BACKGROUND & OBJECTIVE: Quantitative and qualitative detection of estrogen receptor (ER) and progesterone receptor (PR) levels are very importent for predicting prognosis and evaluating the outcome of endocrine therapy of brea st cancer patients. Western blot analysis and Flow cytometry (FCM) are important means for quantitative and qualitative analysis of proteins, but conventional W estern blot analysis need to extract proteins from fresh cells. The present stud y was designed to establish Western blot analysis of human estrogen receptor (ER ) and progesterone receptor (PR) in fixed breast cancer cells, and to explore th e 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 ERα and PR, respectively. T he 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. RES ULTS: Clear and correct ERα 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 a nd ZR-75-1 was higher than that of fresh cells. The expression of ERα in MM2 31 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 wa s 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 Wester n 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.