蛋白酶体抑制剂MG132诱导人血管平滑肌细胞凋亡及其机制研究

Study on the apoptosis of human vascular smooth muscle cells induced by proteasome inhibitor MG132 and its mechanisms

目的:观察蛋白酶体抑制剂MG132对人脐静脉血管平滑肌细胞(vascular smooth muscle cells,VSMC)的致凋亡作用及其对凋亡相关的天冬氨酸特异的半胱氨酸蛋白酶3(caspase 3)表达的影响。方法:采用多个浓度(2.5、5、10μmol/L)的蛋白酶体抑制剂MG132处理人脐静脉VSMC 24 h和48 h;流式细胞术分析细胞凋亡率;RT-PCR检测泛素-蛋白酶体途径(ubiquitin-proteasome pathway,UPP)相关基因泛素、泛素活化酶(E1)、泛素缀合酶(E2)、泛素连接酶(E3)、26S蛋白酶体和caspase3基因转录水平;免疫细胞化学检测细胞caspase 3蛋白表达。结果:蛋白酶体抑制剂MG132处理48 h后细胞凋亡率增加;细胞内UPP相关的基因mRNA表达降低,而caspase 3 mRNA表达增高;免疫细胞化学检测细胞caspase 3蛋白表达水平升高。结论:蛋白酶体抑制剂MG132能够诱导人脐静脉VSMC凋亡,其作用呈量-效关系;MG132诱导VSMC凋亡与下调UPP相关基因和上调caspase3基因及蛋白的表达有关。

AIM： To study the effect of proteasome inhibitor MG132 on the expression of caspase 3 and the apoptosis in cultured human umbilical vascular smooth muscle cells. METHODS： Human umbilical vascular smooth muscle cells were treated with MG132 （2.5, 5, 10 μmol/L） for 24 hours and for 48 hours. The apoptotic cells were determined by flow cytometfic analysis. The levels of Ub, El, E2, E3, 26S and caspase 3 mRNA expression were detected by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of caspase 3 protein was detected by immunocytochemistry. RESULTS： The results showed that the expressions of Ub, El, E2, E3, 26S genes were decreased in treatment group and the apoptosis rates were increased obviously. MG132 could up-regulate the gene/protein expression of caspase 3. CONCLUSION：The results implicate that proteasome inhibitor MG132 could dose-dependently induce human umbilical vascular smooth muscle cells apoptosis, the apoptosis of human umbilical vascular smooth muscle cells induced by MG132 probably is relates to the decreases of Ub, El, E2, E3 and 26S expressions and the increase of caspase 3 expression.