本文選題：尿酸 + 帕金森病(PD)��； 參考：《蘇州大學(xué)》2016年碩士論文

【摘要】：第一部分尿酸對(duì)PC12細(xì)胞內(nèi)α-synuclein蛋白影響的實(shí)驗(yàn)研究目的評(píng)價(jià)尿酸對(duì)PC12細(xì)胞中α-synuclein(α-syn)蛋白的作用。方法建立慢病毒轉(zhuǎn)染的過(guò)表達(dá)α-syn的WT-PC12細(xì)胞株(WT細(xì)胞)。在過(guò)表達(dá)α-syn的WT-PC12細(xì)胞中加入尿酸(0-200μmol/L),檢測(cè)細(xì)胞活力及α-syn蛋白。建立魚藤酮誘導(dǎo)的PD細(xì)胞模型,0-500n M的魚藤酮作用12小時(shí)后檢測(cè)α-syn蛋白。在魚藤酮模型中,尿酸預(yù)處理1h,觀察α-syn蛋白。在高分化PC12細(xì)胞中測(cè)定尿酸作用6h后,α-syn轉(zhuǎn)錄情況。為了觀察尿酸對(duì)α-syn半衰期的影響,使用蛋白抑制劑CHX單獨(dú)作用以及與尿酸共同作用0-12h,對(duì)比尿酸對(duì)α-syn半衰期的影響。結(jié)果200umol/L尿酸可以有效降低α-syn蛋白,這一結(jié)果在魚藤酮模型中進(jìn)一步得到驗(yàn)證。PCR檢測(cè)到尿酸對(duì)α-syn m RNA無(wú)明顯。對(duì)比蛋白合成抑制劑CHX(cycloheximide)單獨(dú)作用,加入尿酸之后,α-syn的降解速度增加,進(jìn)一步證明尿酸可以促進(jìn)α-syn降解。結(jié)論尿酸可以促進(jìn)PC12細(xì)胞中α-syn的降解。第二部分尿酸對(duì)PC12細(xì)胞內(nèi)自噬的調(diào)控作用及機(jī)制研究目的探討尿酸在PC12細(xì)胞中對(duì)自噬的調(diào)節(jié)機(jī)制。方法首先在PC12細(xì)胞中加入尿酸(0-200μmol/L),12小時(shí)后用Western blot檢測(cè)p62、LC3蛋白。瞬時(shí)轉(zhuǎn)染e GFP-LC3質(zhì)粒,尿酸100μmol/L、200μmol/L作用后,對(duì)比空白對(duì)照組觀察LC3點(diǎn)狀熒光表達(dá)分布的狀況。魚藤酮作用PC12細(xì)胞,建立帕金森病細(xì)胞模型,觀察LC3含量。在魚藤酮模型中,分為對(duì)照組、魚藤酮組(50nmol/L)、尿酸(100μmol/L)+魚藤酮(50nmol/L)組、尿酸(200μmol/L)+魚藤酮(50nmol/L)組,觀察p62、LC3含量以及e GFP-LC3點(diǎn)狀熒光。分別檢測(cè)對(duì)照組、氯喹(30μmol/L)組、尿酸(200μmol/L)組、氯喹(30μmol/L)+尿酸(200μmol/L)組LC3含量。采用Western blot觀察m TOR依賴通路蛋白m TOR和p70S6K及其磷酸化水平變化。結(jié)果尿酸(0-200μmol/L)作用之后,可以觀察到胞內(nèi)LC3含量呈劑量依賴性增加,同時(shí)胞內(nèi)p62的含量也逐漸減少。在e GFP-LC3轉(zhuǎn)染的PC12細(xì)胞中,與空載組相比,作用200μM尿酸組,在熒光顯微鏡下明顯觀察到綠色點(diǎn)狀熒光增加。50 nmol/L魚藤酮作用12h后可以發(fā)現(xiàn)LC3II水平明顯下降,同時(shí)對(duì)細(xì)胞活力未有明顯影響。在魚藤酮模型中,100μmol/L及200μmol/L尿酸預(yù)給藥1小時(shí),尿酸可以逆轉(zhuǎn)魚藤酮導(dǎo)致的LC3下降及p62增加的現(xiàn)象。免疫熒光實(shí)驗(yàn):與單獨(dú)魚藤酮作用組相比,預(yù)給藥尿酸組e GFP-LC3熒光點(diǎn)的數(shù)量增加。30μmol/L CQ作用明顯增加了胞內(nèi)LC3II的水平,尿酸作用之后,LC3II含量較前明顯增高。磷酸化的m TOR以及其下游的p70s6k含量在尿酸作用15分鐘后開始增加,在30分鐘時(shí),增加最為明顯。結(jié)論尿酸通過(guò)m TOR介導(dǎo)的自噬通路激活自噬。
[Abstract]:Part I the effect of uric acid on 偽 -synuclein protein in PC12 cells objective to evaluate the effect of uric acid on 偽 -synuclein (偽 -synin) protein in PC12 cells.Methods A lentivirus-transfected WT-PC12 cell line expressing 偽 -syn was established.The activity of 偽 -syn and 偽 -syn protein were detected by adding uranate (0-200 渭 mol 路L ~ (-1)) into the over-expressed 偽 -syn WT-PC12 cells.The model of PD cells induced by rotenone was established. After 12 hours of rotenone treatment, 偽 -syn protein was detected.In rotenone model, 偽-syn protein was observed after pretreatment with uric acid for 1 hour.偽 -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 偽 -syn half-life, the effect of uric acid on 偽 -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 偽 -syn protein. This result was further verified in rotenone model. The results showed that uric acid had no significant effect on 偽 -syn m RNA in rotenone model.The degradation rate of 偽 -syn was increased after the addition of uric acid. It was further proved that uric acid could promote the degradation of 偽 -syn.Conclusion uric acid can promote the degradation of 偽-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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R742.5

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