【作者】 张瑜；

【导师】 胡林森；

【作者基本信息】 吉林大学 ， 神经病学， 2007， 博士

【摘要】 本研究采用MTT及荧光染色法检测PSI对PC12细胞的毒性作用;首次采用荧光差异凝胶电泳(DIGE)和质谱(MS)技术分析PSI引起的动态蛋白质组学改变。本研究成功地建立了PSI诱导的PC12细胞帕金森病(PD)模型,比较全面地复制出PD细胞自噬、细胞凋亡、包涵体形成的病理特征。首次发现并鉴定了PSI诱导的PC12细胞PD模型动态蛋白质组学改变。通过DIGE和MS,鉴定出39个差异表达的蛋白点,它们是27种不同基因的表达产物。本研究已鉴定的27种蛋白中氧调节蛋白150前体、热休克蛋白105、热休克蛋白4、结构型热休克同源蛋白70拟基因蛋白1、葡萄糖调节蛋白58、葡萄糖调节蛋白75、葡萄糖调节蛋白94、酪氨酸羟化酶、26S蛋白酶体亚单位ATP酶2、类26S蛋白酶体亚单位p40.5、醛还原酶、细胞角蛋白8、血红素加氧酶-1、泛素羧基末端水解酶PGP9.5、钙激活蛋白酶共15种蛋白表达上调(p<0.05或p<0.01),外周蛋白、F1-ATP合成酶、actin cytoplasmic 2、Asnal、14-3-3蛋白、T-complex protein 1 alpha subunit、α-微管蛋白6、过氧化物还原酶2、过氧化物还原酶4、ERp29共10种蛋白表达下降(p<0.05或p<0.01)。除此之外,点936、952同为p47蛋白,但点936表达升高(p<0.05或p<0.01),点952表达下降(p<0.05);点598、1316、1351都是HSP27蛋白,但点598表达增加(p<0.01)而点1316、1351表达下降(p<0.05),这可能与同种蛋白的不同修饰状态有关。已鉴定的蛋白质中有7种蛋白表现为两种或两种以上的同功体(isform)。如蛋白点36、37、38,点174、178、183、185、186,点252、253,点275、281,点471、475,点936、952,点598、1316、1351,它们的分子量相当,等电点间隔相同,而且各点的质谱鉴定结果完全一致,提示同一种蛋白的一系列蛋白点可能代表不同的翻译后修饰。更多还原

【Abstract】 Part I. Establishment of the model of Parkinson’s disease induced by proteasome inhibitor PSI in PC12 cellsParkinson’s disease (PD) is the second most common neurodegenerative disorder. Ubiquitin-proteasome system (UPS) is the primary enzymes involved in the degradation of proteins within cells. It can clear mutated, damaged and misfolded proteins in eukaryotic cells and maintain cell homeostasis. Recently, investigations have shown that UPS dysfunction may be the core pathogenesis of PD, but the details of the pathogenetic mechanism of UPS dysfunction in PD is still unknown.Objective To study the effects of proteasome inhibition on PC12 cells. Methods PC12 cells were treated with different concentrations of PSI (1, 2.5, 5, 10, 20μM) for 12h, 24h, 36h, 48h, 60h, 72h, respectively. Cell viability was calculated by MTT conversion; apoptotic percentage was measured by fluorescent staining; morphological changes were determined by HE staining. Result 10μM and 20μM PSI significantly decreased the cell viability to 85.46% and 84.08% after 24 hours, compared with that of control groups (p<0.05 or p<0.01). The cell viability further decreased with time. After 72 hours the viability of PC12 cells decreased significantly to 18.27% and 21.09% in 10μM and 20μM PSI treated groups, compared with control group (p<0.01). At all time points, except 12th hour, the viability of PC12 cells treated with 10μM or 20μM PSI lowered significantly, compared with that with 1μM PSI (P<0.05 or P<0.01). Apoptotic percentage of PC12 cells exposed to vehicle or increasing concentrations of PSI was measured by fluorescent DNA-binding dyes. The apoptotic nuclei of PC12 cells increased in groups exposed to increasing concentrations of PSI. In 10μM PSI treated groups, the apoptotic rate of PC12 cells increased with time. The apoptotic rates at 12h, 24h, 36h, 48h in 10μM PSI treated groups were 5.13±1.74%, 12.80±3.92%, 23.64±5.79%, 13.37±4.02%, respectively, measured by Hoechst33342 stain and 5.21±1.78%, 14.47±4.55%, 25.53±5.45%, 14.86±2.98%, respectively, measured by AO and EB stains, significantly higher than that in control groups (p<0.01), except that at 12h. In PSI treated groups, the population of dead cells increased from 36h, the cell membrane was destroyed from 48h, cell structure was severely damaged and cell loss obviously at 60h and 72h. Thus the apoptotic rate can’t be calculated at 60h and 72h. Control and 10μM PSI treated PC12 cells were fixed and stained with HE. In cells of control groups, the nucleus was stained with blue and the cytoplasm was stained with pink. There were no inclusions and vacuoles in cells of control groups. Eosinophilic cytoplasmic inclusions appeared in cells exposed to PSI for 12 hours and presented in almost all cells from 24h. Cytoplasmic vacuoles, putative autophagic vacuoles, became apparently at 24h and 36h after PSI treatment. At 48h, the cell membrane was damaged or broke down and eosinophilic inclusions remained intact. Conclusion Proteasome inhibitor PSI has obvious toxicity to PC12 cells, which decreases the cell viability, induces apoptosis and promotes eosinophilic inclusion formation, mimics the pathological hallmarks of Parkinson’s disease. Significance A widely accepted cell model of Parkinson’s disease was established, which set up a reliable basis for the study of mechanisms of proteasome dysfunction in the pathogenesis of Parkinson’s disease at proteomic level.Part II Dynamic proteomic studies of model of Parkinson’s disease induced by proteasome inhibitor PSI in PC12 cells Previous investigations have shown that UPS dysfunction may be the core pathogenesis of PD,but the details of the pathogenetic mechanism of UPS dysfunction in PD is still unknown. We have established the model of Parkinson’s disease induced with PSI in PC12 cells, which can mimic the primary hallmarks of PD, including cell autophagy, cell apoptosis, and cytoplasmic inclusions.Objective To investigate the dynamic proteomic changes in PC12 cells treated with proteasome inhibitor PSI. Methods 2D-DIGE and MALDI-TOF MS techniques were used to detect dynamic proteomic changes in PC12 cells treated with 10μM PSI for 3h, 6h, 12h, 24h, 36h, and 48h. Results About 2000 protein spots were seen in 2D-DIGE images. In PSI treated groups, 6 protein points were significantly changed at 3h, 9 protein points were significantly changed at 6h, 49 protein points were significantly changed at 12h, 46 protein points were significantly changed at 24h, 122 protein points were significantly changed at 36h, 97 protein points were significantly changed at 48h, compared with those in control groups. 39 protein points were successfully identified by MALDI-TOF-MS and they are the products of 27 different genes. 7 proteins had two or more isforms. Protein point 36, 37, 38, protein point 174, 178, 183, 185, 186, protein point 252, 253, protein point 275, 281, protein point 471, 475, protein point 936 and 952, and protein point 598, 1316, 1351 showed almost identical molecular mass but different pI, suggesting different post-translation modifications. In PSI treated groups, ORP150, HSP105, HSP4, HSC70-ps1, GRP58, GRP75, GRP94, TH, PSMC2, 26S proteasome subunit p40.5, AR, CK8, HO-1, UCH-L1 PGP9.5, calpain were all significantly up-regulated (p<0.05 or p<0.01) and PRP, F1-ATPase, actin cytoplasic 2, Asnal, tyrosine-3-mono -oxygenase/ryptopha5-monooxygenase activation protein, TCP-1α,α-tubulin 6, Prx2, Prx4, ERp29 were all significantly down-regulated, compared with those in control groups (p<0.05 or p<0.01). Both of protein spot 936 and 952 were p47, but point 936 was up-regulated and point 952 was down-regulated in PSI treated groups, compared to those in control groups (p<0.05). All of protein spot 598, 1316, 1351 were HSP27, but spot 598 was significantly up-regulated (p<0.01) and spot 1316 and 1351 were significantly down-regulated (p<0.05). Significance For the first time, present study identified 39 protein points of 27 proteins which significantly changed in Parkinson’s disease model induced by proteasome inhibitor PSI in dopaminergic PC12 cells and revealed the dynamic proteomic changes. These findings provide new valuable clues for further study of the pathogenesis of Parkinson’s disease and present new candidates of treatment targets of Parkinson’s disease.更多还原