本文選題：帕金森病　切入點(diǎn)：Trim27　出處：《吉林大學(xué)》2015年博士論文

【摘要】：目的： 細(xì)胞凋亡被認(rèn)為是帕金森病病理機(jī)制中多巴胺能神經(jīng)元的主要死亡方式，但是其中具體的分子機(jī)制和信號通路還有待研究。Trim27是Trim家族成員，有顯著的促凋亡作用。本文旨在對Trim27在帕金森病中的功能及其可能機(jī)制進(jìn)行探討，以期為臨床帕金森病的早期診斷及有效治療提供新的有效靶位點(diǎn)。 方法： 采用實(shí)時(shí)熒光定量PCR（qPCR）方法對帕金森病患者及正常人外周血中Trim27的表達(dá)進(jìn)行檢測；采用qPCR和免疫蛋白印跡（western blot）方法檢測Trim27在1-甲基-4-苯基吡啶離子（1-methyl-4-phenylpyridinium，MPP+）誘導(dǎo)的大鼠嗜鉻細(xì)胞瘤細(xì)胞（PC12）和1-甲基-4-苯基-1,2,3,6-四氫吡啶（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP）誘導(dǎo)的帕金森病小鼠黑質(zhì)致密區(qū)的表達(dá)情況；采用基因敲除技術(shù)結(jié)合western blot方法分析探討Trim27敲除對PC12細(xì)胞和帕金森病小鼠神經(jīng)元凋亡的影響。 結(jié)果： 帕金森病患者外周血中Trim27表達(dá)顯著高于正常人。50μM MPP+刺激24h能誘導(dǎo)PC12細(xì)胞凋亡，細(xì)胞凋亡的標(biāo)志物活化型Caspase-3的表達(dá)上升，酪氨酸活化酶TH表達(dá)顯著下降，，與此同時(shí)Trim27表達(dá)升高。在PC12細(xì)胞中轉(zhuǎn)染Trim27siRNA后，Trim27mRNA和蛋白的表達(dá)被有效抑制；MPP+誘導(dǎo)的細(xì)胞凋亡明顯減少，活化型Caspase-3表達(dá)降低。機(jī)制研究表明Trim27通過激活NF-κB信號通路來促進(jìn)細(xì)胞凋亡的發(fā)生。MPTP成功誘導(dǎo)的帕金森病小鼠模型中，黑質(zhì)TH陽性多巴胺能神經(jīng)元顯著減少，western blot檢測顯示誘導(dǎo)后活化型Caspase-3的表達(dá)顯著上升，且伴隨著Trim27表達(dá)升高；Trim27-/-小鼠MPTP誘導(dǎo)后，神經(jīng)元損失減少，凋亡蛋白Caspase-3表達(dá)降低。 結(jié)論： Trim27在帕金森病患者、凋亡的PC12細(xì)胞和帕金森病模型小鼠中表達(dá)均上升。Trim27的基因沉默使MPP+誘導(dǎo)的PC12細(xì)胞凋亡受到抑制，Trim27基因敲除小鼠黑質(zhì)多巴胺能神經(jīng)元數(shù)量顯著高于野生型小鼠，與未誘導(dǎo)組無顯著差異，說明，Trim27在帕金森病發(fā)生發(fā)展過程中發(fā)揮了重要作用；Trim27基因敲除顯著改善了小鼠帕金森病的狀況，提示Trim27可能成為帕金森病治療的有效新靶點(diǎn)。
[Abstract]:Objective:. Apoptosis is thought to be the main death mode of dopaminergic neurons in the pathophysiology of Parkinson's disease, but the specific molecular mechanisms and signaling pathways remain to be studied. Trim27 is a member of the Trim family. The purpose of this study is to investigate the function and possible mechanism of Trim27 in Parkinson's disease, in order to provide a new effective target for early diagnosis and effective treatment of Parkinson's disease. Methods:. The expression of Trim27 in peripheral blood of patients with Parkinson's disease and normal subjects was detected by real-time fluorescence quantitative PCR. The expression of Trim27 in the substantia nigra compact region induced by 1-methyl-4-phenylpyridinium-MPP in rat pheochromocytoma cells (PC12) and 1-methyl-4-methyl-4-phenyl-1-methyl-4-phenyl-1-tetrahydropyridine (MPP) induced by 1-methyl-4-phenylpyridine (1-methyl-4-phenylpyridyl) and 1-methyl-4-phenyl-6-tetrahydropyridine (MPP) in the substantia nigra of Parkinson's disease mice was detected by qPCR and Western blot. The effects of Trim27 knockout on apoptosis of PC12 cells and neurons in Parkinson's disease mice were studied by gene knockout technique and western blot method. Results:. The expression of Trim27 in peripheral blood of patients with Parkinson's disease was significantly higher than that of normal controls (.50 渭 M MPP) for 24 hours. The expression of activated Caspase-3, a marker of apoptosis, was increased, and the expression of tyrosine activase th was significantly decreased. At the same time, the expression of Trim27 was increased. After transfection of Trim27siRNA in PC12 cells, the expression of trim27 mRNA and protein was effectively inhibited. The expression of activated Caspase-3 was decreased. The mechanism of Trim27 promoting apoptosis by activating NF- 魏 B signaling pathway. MPTP successfully induced Parkinson's disease mice model. Substantia nigra th positive dopaminergic neurons significantly decreased the expression of activated Caspase-3 after induction by western blot. With the increase of Trim27 expression, the loss of neurons and the expression of apoptotic protein Caspase-3 decreased after MPTP induction. Conclusion:. Trim27 in Parkinson's disease, The gene silencing of apoptotic PC12 cells and Parkinson's disease model mice increased the number of dopaminergic neurons in substantia nigra of PC12 cells induced by MPP, and the number of dopaminergic neurons in substantia nigra of MPP knockout mice was significantly higher than that in wild-type mice. There was no significant difference between the two groups, which suggested that Trim27 gene knockout played an important role in the pathogenesis and development of Parkinson's disease. The knockout of Trim27 gene significantly improved the status of Parkinson's disease in mice, suggesting that Trim27 might be an effective new target for the treatment of Parkinson's disease.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R742.5

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