高糖刺激对雪旺细胞增殖与凋亡机制的探讨

发布时间：2018-03-27 14:23

本文选题：糖尿病周围神经病变　切入点：lipin-1　出处：《山东大学》2014年硕士论文

【摘要】：研究背景糖尿病周围神经病变(diabetic peripheral neuropathy, DPN)是2型糖尿病(type2diabetic mellitus, T2DM)患者最为常见、最为复杂的慢性并发症之一,统计发现约70%～80%的患者会出现不同程度的感觉和运动神经功能的损害,从而影响患者的生存及生活质量。到目前为止糖尿病周围神经病变的发病机制尚未完全阐明,其可能机制是多方面因素综合作用的结果,既有代谢因素又有血管因素的影响；如氧化应激、多元醇通路的代谢异常、糖基化终末产物(AGEs)的积聚等。糖尿病周围神经病变至今尚无有效的治疗方法,因此如何有效的遏制其发生发展成为目前众学者们研究的热点问题。脂素lipin家族是新发现的一种可调控机体脂肪储存及代谢方式的脂肪因子,它是由LPIN基因表达产生的,包括lipin-1、lipin-2、 lipin-3。其中lipin-1具有两方面作用,一是作为磷脂酸磷脂酶(phosphatidic acid phosphate ester enzyme, PAP)1,催化磷脂酸的去磷酸化作用,产生甘油三酯和无机磷酸盐；二是作为转录协同刺激因子联系肝过氧化物酶增殖物活化受体(PPAR)γ协同刺激因子1α(PGC1α)和PPARα,调节脂肪酸利用和脂肪合成基因的表达。近年来研究发现,lipin-1缺乏可导致成熟神经外膜脂肪垫和雪旺细胞中PAP活性丧失,磷脂合成减少,进而影响外周神经髓鞘的形成,导致神经传导速度减慢；并且PAP底物磷脂酸的积聚还可以激活MEK-Erk旁路,诱发异常信号的产生,促进雪旺细胞的退化及凋亡,加重周围神经病变。神经生长因子(nerve growth factor, NGF)是其家族中发现最早、研究最深入的一类生长因子,它对于神经递质的合成,蛋白磷酸化及甲基化所需酶的合成,神经元正常功能的维持以及损伤后的修复过程是必不可少的。研究发现,DPN时周围神经修复功能障碍,,而这些改变与NGF的缺乏、合成减少及受体蛋白的表达减少密切相关,从而加重周围神经病变。研究目的本研究旨在观察高浓度葡萄糖刺激对雪旺细胞增殖及凋亡的影响并初步探讨其可能机制；以及高糖刺激时雪旺细胞中lipin-1、NGF蛋白表达水平的变化。研究方法将体外培养的RSC96雪旺细胞分为正常对照组(25mmol/L)和高糖组(100mmol/L),分别应用MTT法及流式细胞技术观察高糖刺激对RSC96雪旺细胞增殖及细胞凋亡的影响；应用Western-blotting观察高糖对lipin-1、NGF蛋白表达的影响；同时采用免疫荧光技术进一步验证在高糖刺激时雪旺细胞lipin-1蛋白表达变化。数据的处理均采用SPSS18.0软件进行统计分析,结果以均数±标准差(x±s)表示,以P0.05为差异具有统计学意义。结果 1.MTT法检测结果显示：与正常对照组比较,高糖组对RSC96细胞增殖具有显著抑制作用,差异具有统计学意义(P0.05)。 2.流式细胞术检测发现：高糖组细胞晚期凋亡率(12.4±0.65)%较正常对照组晚期凋亡率(1.56±0.72)%明显增加,差异有显著性(P0.05)。 3. Western-blotting及细胞免疫荧光结果显示：持续的高糖刺激时雪旺细胞中lipin-1和NGF蛋白表达显著降低(P0.05)。结论高浓度葡萄糖刺激可显著抑制雪旺细胞增殖并促进细胞凋亡；同时显著降低了lipin-1和NGF的蛋白表达。提示高糖对神经细胞的损伤作用可能存在多种机制。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R741

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