【摘要】：第一部分尿酸增加谷胱甘肽合成減輕6-OHDA對(duì)SH-SY5Y細(xì)胞損傷作用 目的：探討尿酸（uric acid, UA）對(duì)6-羥基多巴胺(6-OHDA)誘導(dǎo)的SH-SY5Y細(xì)胞損傷發(fā)揮保護(hù)作用的機(jī)制。 方法：采用6-OHDA制作SH-SY5Y細(xì)胞損傷的帕金森病細(xì)胞模型，分為對(duì)照（DMEM）組、6-OHDA（50μmol/L）組、尿酸組（200μmol/L）以及6-OHDA（50μmol/L）+尿酸組（200μmol/L)。預(yù)給尿酸0.5h后6-OHDA作用6h、12h、14h、18h、24h后MTT比色法檢測(cè)細(xì)胞活性；作用12h倒置顯微鏡下觀測(cè)細(xì)胞形態(tài)；比色法檢測(cè)谷胱甘肽含量；預(yù)給尿酸0.5h后6-OHDA作用12h后Western blot和RT-PCR法分別測(cè)定γ-谷氨酰半胱氨酸連接酶催化亞基（γ-GCLC）和γ-谷氨酰半胱氨酸連接酶調(diào)節(jié)亞基（γ-GCLM）蛋白和mRNA水平的表達(dá)量。 結(jié)果：200μmol/L尿酸對(duì)50μmol/L6-OHDA作用12h、14h、18h、24h的SH-SY5Y細(xì)胞活性下降有顯著的改善（78.10±2.36%vs57.44±12.02%，80.52±3.16%vs50.15±2.32%，70.82±1.81%vs39.22±0.78%，42.84±4.73%vs27.99±4.23%，P0.01，P0.001， P0.001，P0.001）；作用12h后尿酸處理組細(xì)胞形態(tài)明顯改善；谷胱甘肽含量下降有顯著的改善（9.51±0.37μmol/L vs3.88±0.78μmol/L，，P0.01）;200μmol/L尿酸+50μmol/L6-OHDA組與50μmol/L6-OHDA組相比，γ-GCLC蛋白表達(dá)水平顯著升高（139.85±11.62%vs49.20±7.09%，P0.01）；γ-GCLM.表達(dá)水平明顯下降（95.36±18.98%vs160.05±8.59%，P0.05）。mRNA水平上γ-GCLC轉(zhuǎn)錄水平顯著升高（110.99±14.41%vs49.19±6.87%，P0.001）；γ-GCLM.轉(zhuǎn)錄水平明顯下降（102.70±10.43%vs190.05±8.59%，P0.05）。 結(jié)論：尿酸對(duì)6-OHDA誘導(dǎo)的SH-SY5Y細(xì)胞損傷具有保護(hù)作用，與增加谷胱甘肽合成有關(guān)。 第二部分尿酸對(duì)Nrf2信號(hào)通路的調(diào)節(jié)作用 目的：探討尿酸增加谷胱甘肽生物合成的分子機(jī)制。 方法：采用6-OHDA損傷SH-SY5Y細(xì)胞制造帕金森病細(xì)胞模型，分為對(duì)照（DMEM）組、6-OHDA（50μmol/L）組、尿酸組（200μmol/L）以及6-OHDA（50μmol/L）+尿酸（200μmol/L)組，預(yù)先給予尿酸0.5h后，6-OHDA作用6h用共聚焦法觀察Nrf2在胞漿、胞核的分布情況，運(yùn)用胞漿胞核蛋白提取法后Western blot測(cè)定不同組別SH-SY5Y細(xì)胞上Nrf2表達(dá)情況；用基因轉(zhuǎn)染siRNA敲減Nrf2方法后再運(yùn)用比色法測(cè)定敲減組與未敲減組不同組別谷胱甘肽含量及細(xì)胞活力。 結(jié)果：與對(duì)照組相比，50μmol/L6-OHDA作用于SH-SY5Y細(xì)胞6h后Nrf2的分布無(wú)明顯變化,胞漿胞核蛋白也無(wú)明顯改變；與單獨(dú)6-OHDA組相比，200μmol/L尿酸作用6h后共聚焦示Nrf2由胞漿明顯轉(zhuǎn)入胞核，提取胞漿胞核蛋白后尿酸處理組Nrf2核蛋白含量明顯增加（196.33±35.21%vs93.11±29.25%，P0.05）；單獨(dú)尿酸與對(duì)照組比，Nrf2核蛋白含量明顯增加（231.36±55.19%vs100.00±33.01%，P0.01）；給予Nrf2-siRNA敲除Nrf2后尿酸+6-OHDA與單獨(dú)6-OHDA組相比，谷胱甘肽含量無(wú)明顯改變（4.16±0.12μmol/L vs3.64±0.50μmol/L, P0.05），細(xì)胞活力無(wú)明顯升高（53.43±2.21%vs51.37±1.69%, P0.05）。 結(jié)論：尿酸對(duì)6-OHDA誘導(dǎo)的SH-SY5Y細(xì)胞損傷具有保護(hù)作用，與其調(diào)節(jié)Nrf2信號(hào)通路有關(guān)。
[Abstract]:Part I uric acid increases glutathione synthesis in SH-SY5Y cells. Objective: to investigate the effect of uric acid (uric acid, on the injury of SH-SY5Y cells induced by 6-OHDA. UA has a protective effect on 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y cell injury. Methods: the model of Parkinson's disease (PD) induced by SH-SY5Y cells was induced by 6-OHDA. They were divided into three groups: control (DMEM) group, 6-OHDA (50 渭 mol / L) group, uric acid group (200 渭 mol / L) and 6-OHDA (50 渭 mol / L) group (200 渭 mol / L). After pretreated with uric acid 0.5 h, 6-OHDA for 6 h, 12 h, 14 h, 18 h, 24 h later, MTT colorimetric assay was used to detect cell activity, 12 h inverted microscope was used to observe cell morphology, and GSH content was measured by colorimetric method. Determination of 緯-glutamylcysteine ligase catalytic subunit (緯-GCLC) and 緯-glutamylcysteine ligase regulatory subunit (緯-GCLM) eggs by Western blot and RT-PCR after preadministration of uric acid for 12 h with 6-OHDA White and mRNA levels of expression. Results: 200 渭 mol / L uric acid significantly improved the decrease of SH-SY5Y cell activity (78.10 鹵2.36%vs57.44 鹵12.02%, 80.52 鹵3.16%vs50.15 鹵2.32%) at 12 h, 14 h, 18 h and 24 h after exposure to 50 渭 mol / L6-OHDA, respectively. 70.82 鹵1.81%vs39.22 鹵0.78%, 42.84 鹵4.73%vs27.99 鹵4.23%, P0.01, P0.001, P0.001); After treatment with uric acid for 12 hours, the morphology of the cells in the treatment group was obviously improved. The GSH content decreased significantly (9.51 鹵0.37 渭 mol / L vs 3.88 鹵0.78 渭 mol / L, P0.01). The expression of 緯-GCLC protein in 200 渭 mol / L uric acid 50 渭 mol / L6-OHDA group was significantly higher than that in 50 渭 mol / L6-OHDA group (139.85 鹵11.62%vs49.20 鹵7.09%, P0.01). 緯-GCLM. The expression level of 緯-GCLC was significantly decreased (95.36 鹵18.98%vs160.05 鹵8.59%, P0.05). The mRNA level of 緯-GCLC was significantly increased (110.99 鹵14.41%vs49.19 鹵6.87%, P0.001) and 緯-GCLM. was observed. The transcription level was significantly decreased (102.70 鹵10.43%vs190.05 鹵8.59%, P0.05). Conclusion: uric acid has protective effect on SH-SY5Y cells induced by 6-OHDA, which is related to the increase of glutathione synthesis. Part two Regulation of uric Acid on Nrf2 signaling Pathway objective: to explore the molecular mechanism of uric acid increasing glutathione biosynthesis. Methods: Parkinson's disease (PD) cells were induced by 6-OHDA injury in SH-SY5Y cells. They were divided into three groups: control (DMEM) group, 6-OHDA (50 渭 mol / L) group, uric acid group (200 渭 mol / L) and 6-OHDA (50 渭 mol / L) uric acid group (200 渭 mol / L). The distribution of Nrf2 in cytoplasm and nucleus was observed by confocal method after pretreated with uric acid 0.5 h and 6-OHDA for 6 h. The expression of Nrf2 in different groups of SH-SY5Y cells was measured by Western blot after extraction of cytoplasmic nucleoprotein. The glutathione content and cell viability in different groups of knock-down group and non-knock-down group were measured by colorimetric assay after gene transfection of siRNA knockdown Nrf2 method. Results: compared with the control group, there was no significant change in the distribution of Nrf2 and cytoplasmic nucleoprotein in SH-SY5Y cells treated with 50 渭 mol / L6-OHDA for 6 h. Compared with 6-OHDA alone group, the confocal Nrf2 was transferred from cytoplasm to nucleus after 6 h treatment with 200 渭 mol / L uric acid, and the content of Nrf2 nucleoprotein increased significantly after extraction of cytoplasmic nucleoprotein (196.33 鹵35.21%vs93.11 鹵29.25%, P0.05). Compared with the control group, the nuclear protein content of Nrf2 increased significantly (231.36 鹵55.19%vs100.00 鹵33.01%, P0.01). After Nrf2 knockout by Nrf2-siRNA, there was no significant change in glutathione content in uric acid 6-OHDA group compared with 6-OHDA alone group (4.16 鹵0.12 渭 mol / L vs3.64 鹵0.50 渭 mol / L, P0.05). There was no significant increase in cell viability (53.43 鹵2.21%vs51.37 鹵1.69%, P0.05). Conclusion: uric acid has protective effect on 6-OHDA-induced SH-SY5Y cell injury, which is related to its regulation of Nrf2 signaling 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】：2014
【分類(lèi)號(hào)】：R742.5

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