MG-132激活SHG-44细胞株自噬途径的体外实验

目的 探讨抑制蛋白酶体功能对人胶质瘤SHG-44细胞内自噬途径的激活作用.方法 采用5.0μmol/L蛋白酶体特异性抑制剂MG-132抑制人胶质瘤细胞SHG-44蛋白酶体功能.采用HE染色光镜观察细胞形态变化,AO染色荧光显微镜观察胞质内酸性囊泡细胞器的形成,锇铀铅染色透射电镜观察亚细胞结构变化.差速离心及蛋白印迹分析自噬关联蛋白Beclin及LC3的表达.结果 蛋白酶体功能被抑制后,HE染色光镜下可见SHG-44细胞的胞核呈深蓝色,大小无明显变化,部分染色质轻度浓缩,胞质内可见空泡,胞核附近胞质呈嗜酸性红染;荧光显微镜下见胞核呈黄绿色荧光,细胞核周围的胞质呈点状红色荧光;透射电镜下可见胞核无明显变化,胞质内出现自噬前体、自噬体及自噬溶酶体等亚细胞结构;蛋白印迹分析显示胞质内的Beclin、LC3-Ⅰ、LC3-Ⅱ显著增加.结论 抑制蛋白酶体功能可以有效地激活人胶质瘤SHG-44细胞内自噬途径.

In vitro study on activating autophagy pathway of SHG-44 glioma cells by MG-132

Objective To investigate the phenomenon of activating cellular autophagy pathway in glioma SHG-44 cells by dysfuctioning proteasome. Method 5.0μmol/L proteasome specific inhibitor MG-132 was used to inhibit proteasome activity in the SHG-44 glioma cells. HE staining and light microscopy was used to observe cellular alterations in morphology. OA staining and fluorescence microscopy was used to observe the formation of acidic vacuolar organelle. Transmission electronic microscopy was used to observe the subcellular alterations in morphology. Differential centrifuge and western blotting were used to examine the expressional alterations of autophagy-related protein in different cellular fractions. Results After the proteasome activity was inhibited, HE staining and light microscopy showed the nucleus presented deep blue and multi figures with light condensed chromatin and without changes in size, many vacuoles formed in the cytosol and the cytosol around nucleus became acidophilic red. AO staining and fluorescence microscopy showed the nucleus became green and the cytosol around the nucleus became red. Transmission Electronic microscopy showed autophagosome and autophagolysosome presented in the cytosol in the experimental group. Western blotting analysis revealed significantly increased expression of Beclin, LC3-Ⅰ and LC3-Ⅱ in the cells of experimental group. Conclusions Inhibiting proteasome activity could effectively activate autophagy pathway in the glioma SHG-44 cells.