【作者】 何慧；

【导师】 杨向东；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 目的:研究MG132对HepG2细胞UPP和MDR基因表达的影响,探讨MG132诱导肝癌细胞HepG2凋亡的机制。 方法:采用流式细胞术、Hoechst33258染色、吖啶橙(AO)染色检测HepG2细胞凋亡;采用RT-PCR方法检测UPP相关基因E1、E2、E3、26S proteasome、p53、Caspase3及MDR mRNA的表达;采用免疫细胞化学方法观察p53、Caspase3及P-gp蛋白表达的变化;采用Western-Blot检测P-gp蛋白的表达;采用高效液相色谱分析法检测细胞内表阿霉素(EPI)的浓度。 结果:经流式细胞术检测,蛋白酶体抑制剂MG132(2μmol/L,5μmol/L)处理24h后HepG2细胞凋亡率增加。细胞荧光染色出现核浓缩、碎裂等凋亡特征。MG132处理后HepG2细胞UPP的相关基因E1、E2、E3、26S proteasome mRNA表达下降,p53、Caspase3 mRNA水平和蛋白水平增高。1μmol/L MG132与1μg/ml表阿霉素联合应用可增强对HepG2细胞的杀伤作用,二者合用可达到单用2μg/ml表阿霉素的致凋亡效应;MG132能够下调表阿霉素处理后HepG2细胞MDRmRNA转录水平及P-gp蛋白的表达。与单用表阿霉素组相比,联合应用小剂量的表阿霉素和MG132处理组细胞内表阿霉素含量增高。更多还原

【Abstract】 Aim The study was undertaken to investigate the effects of MG132 on UPPand MDR expression, and we explore the mechanism of HepG2 cell apoptosis induced by MG132.Methods HepG2 cell apoptosis was determined by flow cytometryanalysis,Hoechst 33258 and Acridine orange staining. RT-PCR was used to detect the levels of E1,E2,E3,26S proteasome,p53,Caspase3,MDR mRNA. Immunohistochemistry was used to detect p53,Caspase3,P-gp protein expression. Western Blot was performed to detect P-gp expression. Intracellular EPI concentration was detected by HPLC.Results After 24 hours treatment with 2 μ mol/L,5 μ mol/L MG132respectively, apoptotic rate was increased with typical morphological features of apoptosis such as nuclear condensation and fragmentation in HepG2 cells. MG132 up-regulated p53,Caspase3 mRNA and protein expression, whereas E1,E2,E3,26S proteasome mRNA levels were down-regulated. The data of FCA and AO staining indicated that the cytotoxicity of 1 μ g/ml EPI to HepG2 cells was enhanced by 1 μ mol/L MG132, and similar effect was obtained as using 2μg/ml EPI separately. MG132 attenuated MDR mRNA and P-gp expression. Compared with EPI group, intracellular EPI concentration was increased by applying relatively low dose EPI and MG132 together.更多还原