【作者】 徐敏；

【导师】 陈诗鸿；

【作者基本信息】 山东大学 ， 内分泌与代谢病， 2014， 硕士

【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P<0.05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(P<0.05)。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(P<0.05)。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(P<0.05)。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。更多还原

【Abstract】 BackgroundDiabetic peripheral neuropathy is one of the most common and complicated complication of type2diabetes. About70%to80%of patients occur to different damage of sensory and motor nerve function, which can affect the quality of life of patients. Currently the pathogensis of diabetic peripheral neuropathy has not been fully elucidated, the possible mechanism is the result of the combine effects of many factors, which results from not only metabolic factors but also vascular factors, such as oxidative stress, metabolic abnormalities of polyol pathway, accumulation of advanced glycationend products. At present time there is not yet efficient treatment to therapy diabetic peripheral neuropathy, therefore how to effectively curb the occurrence of this disease becomes the hot pot.Lipin family which is produced by the gene LPIN expression has physiological effect not only on regulating lipid metabolism; but also on maintaining normal fatty storage. Lipin family includes at least three members lipin-1, lipin-2, lipin-3. Lipin-1plays dual roles in lipid metabolisms. On one hand lipin-1acts as an Mg2+-dependent phosphatidate type-1(PAP-1), catalyzing a key step in the synthesis of glycerolipids, on the other hand it also acts as a transcriptional coactivator through its direct interaction with peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) and PPAR α, catalyzing the gene expression of fatty synthesis and consume. Lipin-1deficiency can decrease the activity of nerve fat pad of epineurium and PAP activity of Schwann cells. Therefore synthesis of phosphatidic acid reduces, which will affect the synthesis of myelin sheath of peripheral nerves, and make nerve conduction velocity slow. Accumulation of phosphatidic acid can stimulate the MEK-Erk bypass pathway to emit abnormal signal, which can increase the degradation and apoptosis of Schwann cells and peripheral neuropathy. Nerve growth factor is one of the growth factors family that discovered earlier than others. It is very essential for neurotransmitter synthesis、the enzyme of protein phosphorylation and methylation, maintaining of neurons normal function and process of repair after injury. Study demonstrate that peripheral nerve repair dysfunction is respectively related to NGF deficiency, synthesis and expression of receptors decrease.ObjectiveTo observe the effect of the high concentration of glucose on rat Schwann cells in vitro, and explore the possible mechanism. To observe the expression level of lipin-1and NGF protein.MethodsRSC96cells were cultured in vitro divided into normal control group(25mmol/L) and high glucose group(100mmol/L). The proliferation and apoptosis of Schwann cell were respectively detected by MTT and flow cytometry. The expression of lipin-1, nerve growth factor were detected by western blotting; and the expression of lipin-1were further tested by immuno fluorescence under the high concentration of glucose. Statistical software SPSS18.0was used for data analysis. All the measurement data was expressed as x±s deviation. The difference was statistically significant if P<<0.05.Result1. MTT assay:Compared with normal control group, the proliferation of RSC96Schwann cells were inhibited by high glucose, the difference was significant between the two group (P<0.05).2. Flow cytometry include:Compared with the late apoptosis rate of RSC96Schwann cells in normal control group (1.56±0.72)%, the rate increased significantly in high glucose group (12.4±0.65)%, the difference in the two groups was significant (P<0.05).3. The expression of lipin-1and NGF in Schwann cells cultured in high glucose medium were reduced detected by western-blotting (P<0.05), and the expression of lipin-1was further detected by flow cytometry(P<0.05).ConclusionThe high concentration of glucose decreased the proliferation of Schwann cells.更多还原