表没食子儿茶素没食子酸酯对Hela细胞的诱导凋亡作用及靶点蛋白质研究(1)
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摘 要：目的：观察表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对Hela细胞的诱导凋亡作用，寻找其可能的靶点蛋白质。方法：用50、100、200、400μg/mL梯度浓度的EGCG孵育Hela细胞24h和48h，荧光显微镜下观察Hela细胞凋亡形态，用MTT检测其生长抑制率，用Annexin V-FITC/PI染色观察其凋亡率，鬼笔环肽染色观察其肌动蛋白聚合体(F-actin)降解程度。然后利用偶联EGCG的微球与Hela细胞裂解液孵育，结合靶点蛋白，靶点蛋白经考马斯蓝染色后，切下目标条带进行质谱鉴定。结果：EGCG能诱导Hela细胞凋亡和F-actin的破坏，并呈时间和剂量依赖性。EGCG结合的靶带蛋白为actin类蛋白。结论：直接结合于actin类蛋白并破坏细胞骨架可能是EGCG诱导Hela凋亡的途径之一。
关键词：表没食子儿茶素没食子酸酯；宫颈癌细胞；肌动蛋白；微球
中图分类号：R285.5 文献标识码：A 文章编号：1673-7717(2012)01-0194-04
Research on Apoptosis of Hela Cells Induced by
Epigallocatechin Gallate and Binding Proteins
ZHOU Xiao,WANG Yekai,ZHOU Jihang,ZENG Fang,ZHOU Shiquan,HUANG Yanyan,LIU Xiaoguang
(Zhoushan Hospital,Zhoushan,Hangzhou 316004,Zhejiang,China)
Abstract:Objective:To investigate the apoptosis of Hela cells induced by epigallocatechin gallate(EGCG) and to seek for the direct target proteins of EGCG in Hela cells. Methods: The Hela cells were incubated with different concentrations of EGCG (50、100、200、400μg/mL) for 24h and 48h. The morphologies of apoptotic and F-actin's structure were observed by fluorescence microscope.The inhibition rates of cell proliferation were detected by MTT.The apoptosis rates were detected by flow cytometry. The microballoons of coulping EGCG were incubated with the lysis solution of Hela cells to combine the specific target protein. The band of target protein was cut off after SDS-PAGE electrophoresis and identified by the mass spectrum. Results: With the increasing of drug concentration and incubating time,it showed:the quantity of apoptosis cells increased under the fluorescence microscope;the inhibitions rates of cell proliferation increased; the apoptosis rates of Hela cells increased;the structure of F-actin was destroyed. Family of actin proteins was directly combined to EGCG.Conclusions: EGCG directly combines family of actin proteins and destroys F-actin in Hela cells.It may be one of pathways to promote cell apoptosis ......
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