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目的探讨姜黄素(curcumin)诱导成骨肉瘤细胞凋亡前后,Prohibitin(PHB)蛋白的定位与表达变化,及PHB与凋亡相关基因产物的共定位关系。方法选择性抽提经姜黄素诱导处理前后的人成骨肉瘤MG-63细胞核基质蛋白,以蛋白质组学、免疫印迹法和激光扫描共焦显微技术对PHB在核基质中存在、分布及其与凋亡相关基因产物在MG-63细胞中的共定位关系进行了研究。结果双向凝胶电泳、质谱鉴定和免疫印迹法结果显示,PHB存在于MG-63细胞核基质蛋白组分中,细胞凋亡后在细胞核基质蛋白中表达下调。免疫荧光显微镜观察显示,PHB定位于MG-63细胞核基质纤维上,经姜黄素处理后出现分布位置与表达水平的变化。激光扫描共焦显微镜观察结果显示,PHB在MG-63细胞凋亡过程中与Bax、Bcl-2、Fas、P53、c-Myc和Rb等基因产物具有共定位关系,且共定位区域发生了变化。结论 PHB是一种核基质结合蛋白;PHB在MG-63细胞凋亡过程中的表达与分布变化,及其与细胞凋亡相关基因的共定位关系,对MG-63细胞凋亡具有重要影响。
Objective To investigate the localization and expression of Prohibitin (PHB) protein in curcumin-induced osteosarcoma cells and the co-localization of PHB and apoptosis-related gene products. Methods The nuclear matrix proteins of human osteosarcoma MG-63 cells before and after curcumin induction were selectively extracted. The presence and distribution of PHB in the nuclear matrix and their correlation with proteome and proteomic methods were analyzed by proteomics, immunoblotting and laser scanning confocal microscopy The co-localization of death related gene products in MG-63 cells was studied. Results The results of two-dimensional gel electrophoresis, mass spectrometry and Western blotting showed that PHB was present in the nuclear matrix protein of MG-63 cells and down-regulated in nuclear matrix proteins after apoptosis. Immunofluorescence microscopy showed that PHB was located on the nuclear matrix fibers of MG-63 cells, and the location and the expression level of PHB changed after treatment with curcumin. Confocal laser scanning microscopy showed that PHB had a co-localization relationship with the gene products of Bax, Bcl-2, Fas, P53, c-Myc and Rb in the process of MG-63 cell apoptosis and the colocalization region changed . Conclusion PHB is a nuclear matrix-associated protein. The changes of PHB expression and distribution in MG-63 cells and its co-localization with apoptosis-related genes have an important effect on the apoptosis of MG-63 cells.