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目的:研究蛋白酶体抑制对体外培养的星形胶质细胞周期素D1(cyclin D1)和周期素依赖性激酶4(CDK4)表达的影响。方法:SD乳鼠皮质星形胶质细胞原代培养,并纯化鉴定;予不同浓度(2和4μmol·-1)的蛋白酶体抑制剂L(lactacystin)对第二代星形胶质细胞进行短期(12h)急性干预处理,应用免疫荧光及Western blot检测星形胶质细胞cyclinD1和CDK4表达的水平。结果:纯化传代的皮质星形胶质细胞经胶质纤维酸性蛋白(GFAP)免疫荧光鉴定,其阳性率可达99%;lactacystin2和4μmol·-1可诱导星形胶质细胞cyclin D1和CDK4表达的下降,与对照组相比差异有显著统L计学意义(P<0.01)。结论:一定程度蛋白酶体活性抑制可诱导培养的星形胶质细胞cyclin D1和CDK4表达的减少,从而影响胶质细胞细胞周期,促进胶质细胞分化。提示蛋白酶体功能障碍后可能通过影响胶质细胞细胞周期来参与阿尔茨海默病的病理改变。
Objective: To study the effects of proteasome inhibition on the expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) in cultured astrocytes. Methods: Primary cultured astrocytes from neonatal SD rats were purified and identified. The second generation of astrocytes were treated with different concentrations of lactacystin (2 and 4 μmol · L) (12h) acute intervention. The expression of cyclinD1 and CDK4 in astrocytes was detected by immunofluorescence and Western blot. Results: The positive clones of passaged cortical astrocytes were identified by GFAP immunofluorescence, and the positive rate was 99%. The expression of cyclin D1 and CDK4 in astrocytes was induced by lactacystin2 and 4μmol · l (P <0.01). There was significant difference between the control group and the control group (P <0.01). Conclusion: The inhibition of proteasome activity to a certain degree can induce the decrease of the expression of cyclin D1 and CDK4 in cultured astrocytes, thus affecting the glial cell cycle and promoting glial cell differentiation. Suggesting that proteasome dysfunction may participate in the pathological changes of Alzheimer’s disease by affecting the cell cycle of glial cells.