尿酸对PC12细胞内α-synuclein降解和自噬的调控作用及机制研究

发布时间：2018-04-14 18:46

本文选题：尿酸 + 帕金森病(PD)　；参考：《苏州大学》2016年硕士论文

【摘要】：第一部分尿酸对PC12细胞内α-synuclein蛋白影响的实验研究目的评价尿酸对PC12细胞中α-synuclein(α-syn)蛋白的作用。方法建立慢病毒转染的过表达α-syn的WT-PC12细胞株(WT细胞)。在过表达α-syn的WT-PC12细胞中加入尿酸(0-200μmol/L),检测细胞活力及α-syn蛋白。建立鱼藤酮诱导的PD细胞模型,0-500n M的鱼藤酮作用12小时后检测α-syn蛋白。在鱼藤酮模型中,尿酸预处理1h,观察α-syn蛋白。在高分化PC12细胞中测定尿酸作用6h后,α-syn转录情况。为了观察尿酸对α-syn半衰期的影响,使用蛋白抑制剂CHX单独作用以及与尿酸共同作用0-12h,对比尿酸对α-syn半衰期的影响。结果200umol/L尿酸可以有效降低α-syn蛋白,这一结果在鱼藤酮模型中进一步得到验证。PCR检测到尿酸对α-syn m RNA无明显。对比蛋白合成抑制剂CHX(cycloheximide)单独作用,加入尿酸之后,α-syn的降解速度增加,进一步证明尿酸可以促进α-syn降解。结论尿酸可以促进PC12细胞中α-syn的降解。第二部分尿酸对PC12细胞内自噬的调控作用及机制研究目的探讨尿酸在PC12细胞中对自噬的调节机制。方法首先在PC12细胞中加入尿酸(0-200μmol/L),12小时后用Western blot检测p62、LC3蛋白。瞬时转染e GFP-LC3质粒,尿酸100μmol/L、200μmol/L作用后,对比空白对照组观察LC3点状荧光表达分布的状况。鱼藤酮作用PC12细胞,建立帕金森病细胞模型,观察LC3含量。在鱼藤酮模型中,分为对照组、鱼藤酮组(50nmol/L)、尿酸(100μmol/L)+鱼藤酮(50nmol/L)组、尿酸(200μmol/L)+鱼藤酮(50nmol/L)组,观察p62、LC3含量以及e GFP-LC3点状荧光。分别检测对照组、氯喹(30μmol/L)组、尿酸(200μmol/L)组、氯喹(30μmol/L)+尿酸(200μmol/L)组LC3含量。采用Western blot观察m TOR依赖通路蛋白m TOR和p70S6K及其磷酸化水平变化。结果尿酸(0-200μmol/L)作用之后,可以观察到胞内LC3含量呈剂量依赖性增加,同时胞内p62的含量也逐渐减少。在e GFP-LC3转染的PC12细胞中,与空载组相比,作用200μM尿酸组,在荧光显微镜下明显观察到绿色点状荧光增加。50 nmol/L鱼藤酮作用12h后可以发现LC3II水平明显下降,同时对细胞活力未有明显影响。在鱼藤酮模型中,100μmol/L及200μmol/L尿酸预给药1小时,尿酸可以逆转鱼藤酮导致的LC3下降及p62增加的现象。免疫荧光实验:与单独鱼藤酮作用组相比,预给药尿酸组e GFP-LC3荧光点的数量增加。30μmol/L CQ作用明显增加了胞内LC3II的水平,尿酸作用之后,LC3II含量较前明显增高。磷酸化的m TOR以及其下游的p70s6k含量在尿酸作用15分钟后开始增加,在30分钟时,增加最为明显。结论尿酸通过m TOR介导的自噬通路激活自噬。
[Abstract]:Part I the effect of uric acid on 伪 -synuclein protein in PC12 cells objective to evaluate the effect of uric acid on 伪 -synuclein (伪 -synin) protein in PC12 cells.Methods A lentivirus-transfected WT-PC12 cell line expressing 伪 -syn was established.The activity of 伪 -syn and 伪 -syn protein were detected by adding uranate (0-200 渭 mol 路L ~ (-1)) into the over-expressed 伪 -syn WT-PC12 cells.The model of PD cells induced by rotenone was established. After 12 hours of rotenone treatment, 伪 -syn protein was detected.In rotenone model, 伪-syn protein was observed after pretreatment with uric acid for 1 hour.伪 -syn transcription was measured in well-differentiated PC12 cells after exposure to uric acid for 6 h.In order to observe the effect of uric acid on 伪 -syn half-life, the effect of uric acid on 伪 -syn half-life was compared by using protein inhibitor CHX alone and in combination with uric acid for 0-12 h.Results 200umol/L uric acid could effectively reduce 伪 -syn protein. This result was further verified in rotenone model. The results showed that uric acid had no significant effect on 伪 -syn m RNA in rotenone model.The degradation rate of 伪 -syn was increased after the addition of uric acid. It was further proved that uric acid could promote the degradation of 伪 -syn.Conclusion uric acid can promote the degradation of 伪-syn in PC12 cells.The second part is the regulation of uric acid on autophagy in PC12 cells and its mechanism. Objective to investigate the regulation mechanism of uric acid on autophagy in PC12 cells.Methods at first, uric acid was added to PC12 cells from 0 to 200 渭 mol 路L ~ (-1) for 12 hours, then Western blot was used to detect p62 ~ (LC3) protein.After transient transfection of e GFP-LC3 plasmid and treatment with 100 渭 mol / L uric acid at 200 渭 mol/L, the distribution of dot fluorescence expression of LC3 was observed in the blank control group.PC12 cells were treated with rotenone and the cell model of Parkinson's disease was established. The content of LC3 was observed.The rotenone model was divided into control group, rotenone group (50 nmol / L), rotenone (100 渭 mol / L) uric acid group (50 nmol / L) and uric acid (200 渭 mol / L) rotenone (50 nmol / L) group.LC3 levels were measured in control group, chloroquine 30 渭 mol / L group, uric acid 200 渭 mol / L group and chloroquine 30 渭 mol / L) group.The changes of m TOR and p70S6K and their phosphorylation levels were observed by Western blot.Results the intracellular LC3 content was increased in a dose-dependent manner and the intracellular p62 content was gradually decreased after the treatment of uric acid (0-200 渭 mol / L).In the PC12 cells transfected with e GFP-LC3, the green dot fluorescence increased by .50 nmol/L rotenone for 12 h after exposure to 200 渭 M uric acid group.At the same time, there was no obvious effect on cell viability.In rotenone model treated with 100 渭 mol/L and 200 渭 mol/L uric acid for 1 hour, uric acid could reverse the decrease of LC3 and increase of p62 induced by rotenone.Immunofluorescence test: compared with rotenone alone group, the number of fluorescence point of e GFP-LC3 in preadministered uric acid group increased. 30 渭 mol/L CQ significantly increased the level of intracellular LC3II, and the content of LC3II increased significantly after uric acid treatment.The content of phosphorylated m TOR and its downstream p70s6k began to increase after exposure to uric acid for 15 minutes, and the increase was most obvious at 30 minutes.Conclusion uric acid activates autophagy through m TOR mediated autophagy pathway.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742.5

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