發(fā)布時間：2018-03-27 14:23

  本文選題：糖尿病周圍神經病變　切入點：lipin-1　出處：《山東大學》2014年碩士論文

【摘要】：研究背景 糖尿病周圍神經病變(diabetic peripheral neuropathy, DPN)是2型糖尿病(type2diabetic mellitus, T2DM)患者最為常見、最為復雜的慢性并發(fā)癥之一,統(tǒng)計發(fā)現(xiàn)約70%～80%的患者會出現(xiàn)不同程度的感覺和運動神經功能的損害,從而影響患者的生存及生活質量。到目前為止糖尿病周圍神經病變的發(fā)病機制尚未完全闡明,其可能機制是多方面因素綜合作用的結果,既有代謝因素又有血管因素的影響；如氧化應激、多元醇通路的代謝異常、糖基化終末產物(AGEs)的積聚等。糖尿病周圍神經病變至今尚無有效的治療方法,因此如何有效的遏制其發(fā)生發(fā)展成為目前眾學者們研究的熱點問題。 脂素lipin家族是新發(fā)現(xiàn)的一種可調控機體脂肪儲存及代謝方式的脂肪因子,它是由LPIN基因表達產生的,包括lipin-1、lipin-2、 lipin-3。其中l(wèi)ipin-1具有兩方面作用,一是作為磷脂酸磷脂酶(phosphatidic acid phosphate ester enzyme, PAP)1,催化磷脂酸的去磷酸化作用,產生甘油三酯和無機磷酸鹽；二是作為轉錄協(xié)同刺激因子聯(lián)系肝過氧化物酶增殖物活化受體(PPAR)γ協(xié)同刺激因子1α(PGC1α)和PPARα,調節(jié)脂肪酸利用和脂肪合成基因的表達。近年來研究發(fā)現(xiàn),lipin-1缺乏可導致成熟神經外膜脂肪墊和雪旺細胞中PAP活性喪失,磷脂合成減少,進而影響外周神經髓鞘的形成,導致神經傳導速度減慢；并且PAP底物磷脂酸的積聚還可以激活MEK-Erk旁路,誘發(fā)異常信號的產生,促進雪旺細胞的退化及凋亡,加重周圍神經病變。神經生長因子(nerve growth factor, NGF)是其家族中發(fā)現(xiàn)最早、研究最深入的一類生長因子,它對于神經遞質的合成,蛋白磷酸化及甲基化所需酶的合成,神經元正常功能的維持以及損傷后的修復過程是必不可少的。研究發(fā)現(xiàn),DPN時周圍神經修復功能障礙,,而這些改變與NGF的缺乏、合成減少及受體蛋白的表達減少密切相關,從而加重周圍神經病變。 研究目的 本研究旨在觀察高濃度葡萄糖刺激對雪旺細胞增殖及凋亡的影響并初步探討其可能機制；以及高糖刺激時雪旺細胞中l(wèi)ipin-1、NGF蛋白表達水平的變化。 研究方法 將體外培養(yǎng)的RSC96雪旺細胞分為正常對照組(25mmol/L)和高糖組(100mmol/L),分別應用MTT法及流式細胞技術觀察高糖刺激對RSC96雪旺細胞增殖及細胞凋亡的影響；應用Western-blotting觀察高糖對lipin-1、NGF蛋白表達的影響；同時采用免疫熒光技術進一步驗證在高糖刺激時雪旺細胞lipin-1蛋白表達變化。數(shù)據(jù)的處理均采用SPSS18.0軟件進行統(tǒng)計分析,結果以均數(shù)±標準差(x±s)表示,以P0.05為差異具有統(tǒng)計學意義。 結果 1.MTT法檢測結果顯示：與正常對照組比較,高糖組對RSC96細胞增殖具有顯著抑制作用,差異具有統(tǒng)計學意義(P0.05)。 2.流式細胞術檢測發(fā)現(xiàn)：高糖組細胞晚期凋亡率(12.4±0.65)%較正常對照組晚期凋亡率(1.56±0.72)%明顯增加,差異有顯著性(P0.05)。 3. Western-blotting及細胞免疫熒光結果顯示：持續(xù)的高糖刺激時雪旺細胞中l(wèi)ipin-1和NGF蛋白表達顯著降低(P0.05)。 結論 高濃度葡萄糖刺激可顯著抑制雪旺細胞增殖并促進細胞凋亡；同時顯著降低了lipin-1和NGF的蛋白表達。提示高糖對神經細胞的損傷作用可能存在多種機制。
[Abstract]:Research background
Diabetic peripheral neuropathy (diabetic peripheral, neuropathy, DPN) type 2 diabetes mellitus (type2diabetic mellitus, T2DM) were the most common chronic complications, one of the most complex, the statistics found that about 70% to 80% of patients may have the function of sensory and motor nerve damage in different degree, which affects the survival and quality of life of patients. So far, the pathogenesis of diabetic peripheral neuropathy has not been fully elucidated, the possible mechanism is the result of many factors, both metabolic factors and vascular factors; such as oxidative stress, polyol pathway metabolism, advanced glycation end products (AGEs) accumulation. Treatment of diabetes peripheral neuropathy has not been effective, so how to effectively curb the occurrence and development of the public has become a hot issue for scholars at present.
Visfatin lipin family is a fat factor regulating body fat storage and metabolism of the newly discovered, which is composed of LPIN genes, including lipin-1, lipin-2 lipin-3. lipin-1, which has two effects, one is as phosphatidic acid (phosphatidic acid phosphate ester phospholipase enzyme, PAP) 1, dephosphorylation effect of catalytic phosphatidic acid, triglyceride production and inorganic phosphate; two is as a transcription coactivator associated hepatic peroxisome proliferator activated receptor (PPAR) gamma coactivator 1 alpha (PGC1 alpha) and PPAR alpha, expression regulation of fatty acid utilization and lipid synthesis genes. Recent studies showed that lipin-1 deficiency can lead to the loss of mature fat pad epineurium and Schwann cell PAP activity, reduce the synthesis of phospholipids, thereby affecting the formation of peripheral nerve myelin, cause nerve conduction velocity slowed down; and the PAP substrate p The accumulation of fatty acids can also activate the MEK-Erk pathway, induced abnormal signal generation, promote the degradation of Schwann cells and apoptosis, aggravation of peripheral neuropathy. Nerve growth factor (nerve growth, factor, NGF) is the family found in the earliest, most in-depth study of a kind of growth factor, it is for the neurotransmitter synthesis. The synthesis of protein phosphorylation and methylation enzymes required for normal neuronal function, maintenance and repair process after injury is essential. The study found that the repair of peripheral nerve dysfunction, DPN, and the lack of these changes and NGF expression, reduced the synthesis and receptor protein is closely related to reduction, thereby increasing the peripheral neuropathy.
research objective
The aim of this study is to observe the effect of high glucose on the proliferation and apoptosis of Schwann cells, and to explore its possible mechanism, and the expression of lipin-1 and NGF protein in Schwann cells during high glucose stimulation.
research method
RSC96 Schwann cells cultured in vitro were divided into normal control group (25mmol/L) and high glucose group (100mmol/L), respectively using MTT method and flow cytometry to observe the effect of high glucose on the proliferation of RSC96 cells and Schwann cell apoptosis; application of Western-blotting during exposure to lipin-1, NGF protein expression by immunofluorescence technique further; validation of Schwann cell lipin-1 protein expression in high glucose stimulation. Data were analyzed with SPSS18.0 software, the mean standard deviation (x + s) said, with P0.05 as the difference was statistically significant.
Result
The results of 1.MTT assay showed that compared with the normal control group, the high glucose group had a significant inhibitory effect on the proliferation of RSC96 cells, and the difference was statistically significant (P0.05).
2. flow cytometry showed that the late apoptosis rate of the high glucose group (12.4 + 0.65)% was significantly higher than that of the normal control group (1.56 + 0.72)%, with significant difference (P0.05).
The results of 3. Western-blotting and cell immunofluorescence showed that the expression of lipin-1 and NGF protein in Schwann cells decreased significantly (P0.05) when sustained high glucose was stimulated.
conclusion
High glucose stimulation can significantly inhibit the proliferation and promote apoptosis of Schwann cells, and significantly reduce the protein expression of lipin-1 and NGF, suggesting that there may be multiple mechanisms of the damage of high glucose on nerve cells.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R587.2;R741

【參考文獻】

相關期刊論文 前1條

1 唐勝球;江青艷;楊楚芬;鄒曉庭;董小英;;Lipin家族研究進展[J];遺傳;2010年10期

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本文編號：1671896