热休克蛋白70在人食管癌EC9706细胞凋亡过程中的表达与定位变化  

Changes in expression and localization of heat shock protein 70 during curcumin-induced apoptisis of human esophageal cancer cell line EC9706

作　　者：刘凡[1] 陈兰英[2] 黄建[3] 刘用金[1] 石松林[1] 

机构地区：[1]厦门大学医学院基础医学部,福建厦门361005 [2]河南城建学院生物医学教研室,河南平顶山467036 [3]桂林医学院生物技术学院,桂林541004

出　　处：《解剖学报》2012年第4期490-499,共10页Acta Anatomica Sinica

基　　金：福建省自然科学基金资助项目(2011J01256);中央高校基本科研业务费专项资金(2011121061)

摘　　要：目的探讨姜黄素对人食管癌EC9706细胞凋亡的诱导作用,热休克蛋白70(HSP70)在肿瘤细胞凋亡过程中在核基质上的变化及其与凋亡调控相关蛋白的关系。方法用细胞计数和流式细胞仪检测姜黄素对人食管癌EC9706细胞的增殖抑制作用,以光学显微镜和透射电镜观察姜黄素诱导人食管癌EC9706细胞凋亡前后的细胞结构变化,琼脂糖凝胶电泳观察人食管癌EC9706细胞凋亡前后的DNA结构变化。双向凝胶电泳和质谱鉴定分析HSP70在核基质中的存在与变化;并以Western blotting进行确证;激光扫描共焦显微镜观察HSP70在EC9706细胞凋亡过程中的定位及其与Bax、Bcl-2等基因产物的共定位关系。结果姜黄素能显著抑制人食管癌EC9706细胞增殖并诱导人食管癌EC9706细胞凋亡,双向凝胶电泳、质谱鉴定和结果发现并证实,HSP70在姜黄素处理前后的EC9706细胞核基质蛋白中的存在及其表达下调变化。激光扫描共焦显微镜观察结果显示,HSP70在EC9706细胞凋亡过程中与Bax、Bcl-2等基因产物具有共定位关系,且其共定位区域发生了变化。结论姜黄素对人食管癌EC9706细胞具有显著的凋亡诱导作用;HSP70作为一种新发现的核基质蛋白,在姜黄素诱导人食管癌EC9706凋亡过程中的表达与分布发生了显著变化。HSP70与凋亡相关基因的关系对EC9706细胞凋亡具有重要影响。Objective To investigate the effect of curcumin on human esophageal cancer EC9706 cells and explore the role of heat shock protein（HSP70） in cell apoptosis by examining changes in the nuclear matrix and its relationship with apoptosis-related proteins. Methods Cell counting and flow cytometry were performed to probe the inhibitory effect of curcumin on cellular proliferation. Transmission electron microscopy and optical microscopy were used to observe the structural changes in EC9706 cells before and after apoptosis. Agarose gel electrophoresis was conducted to investigate the DNA structure of EC9706 ceils before and after apoptosis. Two-dimensional polyacrylamide gel electrophoresis （2-D PAGE） and mass spectrometry （MS） analysis were performed to investigate the presence and changes of HSP70 in the nuclear matrix of EC9706 cells before and after eurcumin treatment, which was further corroborated by Western blotting assay. Laser confocal scanning microscopy was used to observe the colocalization of HSP70 with Bax and Bel-2 during apoptosis. Results The results indicated that curcumin could markedly inhibited EC9706 cell proliferation and finally induced apoptosis. Data from 2-D PAGE, MS, and Western blotting showed that HSP70 was involved in the nuclear matrixproteins and expression of HSP70 was downregulated after curcumin treatment. Laser confocal microscopy showed that HSPT0 colocalized with Bax and Bcl-2, and the colocalized regions were altered by the curcumin treatment. Conclusion Our work proves that curcumin could definitely induce EC9706 cells into apoptosis. As a new found nuclear matrix protein, the expression and distribution of HSP70 are altered during the apoptosis of EC9706 cells. The colocalization of HSP70 with apoptosis-related genes evidently affects the apoptosis of EC9706 cells.

关 键 词：姜黄素 热休克蛋白70 EC9706细胞 食管癌 流式细胞术 琼脂糖凝胶电泳 人 

分 类 号：R735.1[医药卫生—肿瘤] Q291[医药卫生—临床医学]