左卡尼汀对高糖致雪旺细胞损伤的保护作用及可能机制

发布时间：2018-03-19 13:15

本文选题：肉碱　切入点：糖尿病神经病变　出处：《天津医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:左卡尼汀(L-carnitine,LC)可用于糖尿病周围神经病变(diabetic peripheral neuropathy,DPN)的临床治疗,但其作用是否与抑制高糖致雪旺细胞(Schwan cells,SC)的损伤有关有待研究,本研究通过建立高糖诱导的大鼠雪旺细胞系RSC96的损伤模型,拟探讨左卡尼汀对高糖诱导下雪旺细胞损伤的影响及可能机制,为明确其治疗DPN的机制提供新的理论参考。方法:将离体的大鼠雪旺细胞株(RSC96),以1.5×104/ml的密度接种于96孔板(200μl/孔)或以2×105/ml的密度接种于6孔板(2ml/孔)。1、将上述细胞分别于设定的葡萄糖终浓度为25mmol/L、50mmol/L、100mmol/L的培养基中培养48h,采用甲基四唑蓝(3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-diphenytetrazoliumromide,MTT)测RSC96的细胞活性,依据结果选择合适的高糖浓度。然后分别在LC终浓度为10μmol/L、25μmol/L、50μmol/L、75μmol/L、100μmol/L培养液中处理细胞,测定48 h后细胞活性,根据MTT结果选择最佳左卡尼汀药物浓度。为排除高糖引起的渗透压对实验结果的影响,设甘露醇组作为对照组。2、根据上一步实验结果,本部分实验选择高糖刺激浓度为50mmol/L,左卡尼汀药物终浓度为50μmol/L。将RSC96细胞随机分为五组:正常对照组(C组)、高糖组(H组)、高糖+左卡尼汀组(H+L组)、单独左卡尼汀组(L组)、甘露醇组(M组),培养48h后,观察各组生长状况并分别拍照;测定各组细胞凋亡率;测定各组细胞SOD的活力以及MDA的含量;采用Western Blot方法检测各组细胞内聚腺苷二磷酸-核糖聚合酶-1(PARP(poly(ADP-ribose)polymerase-1),PARP-1)、门冬氨酸特异半胱氨酶3(Cysteine aspartate-specific protease-3,Caspase-3)的蛋白表达。结果:1、MTT结果显示,高糖处理后细胞活性降低,并且损伤效应呈现剂量依赖性。使用浓度为50mmol/L高糖培养48h会导致细胞活性降为C组活性的58%(P0.05),引起细胞增殖活性显著降低,因此选择50mmol/L作为后续实验的高糖剂量。左卡尼汀培养液对正常雪旺细胞增值活性无明显影响(P0.05),但可以显著改善高糖所致的雪旺细胞活性降低,结果显示左卡尼汀浓度为50μmol/L时的细胞活性显著高于其他剂量组,因此选择50μmol/L的左卡尼汀作为后续实验的药物剂量。2、与C组相比,H组和H+L组贴壁细胞减少,悬浮细胞增多,SOD活性降低,MDA含量和细胞凋亡率升高,细胞内活化的Caspase-3片段和PARP-1片段表达均明显升高(P0.05);H组、M组上述指标与C组的差异无统计学意义(P0.05);与H组比较,H+L组贴壁细胞增多,细胞连接增多,悬浮细胞减少,SOD活性升高,MDA含量和细胞凋亡降低率,细胞内活化的Caspase-3和PARP-1片段表达均明显降低(P0.05)。结论:高糖可诱导雪旺细胞的损伤,且与高糖引起的高渗透压无关,与雪旺细胞内氧化应激水平升高而引起的细胞毒性有关。左卡尼汀对高糖致雪旺细胞的损伤具有保护作用,其作用机制可能与降低氧化应激水平,下调PARP-1(89kDa)、Caspase-3(17kDa)的表达,减少细胞凋亡有关。
[Abstract]:Objective: L-carnitine L-carnitine may be used in the treatment of diabetic peripheral neuropathy (peripheral), but whether the effect of L-carnitine can inhibit the injury of Schwan-Schwann cells (SCS) induced by high glucose needs to be studied. In this study, we established the model of RSC96 damage induced by high glucose in rat Schwann cell line, to explore the effect of L-carnitine on the damage induced by high glucose and its possible mechanism. Methods: the isolated rat Schwann cell line RSC96 was inoculated at a density of 1.5 脳 10 4 / ml to the 96-well plate (200 渭 l / well) or 2 脳 105 / ml to the 6-well plate (2ml / pore 路1). The cell activity of RSC96 was measured by methyl tetrazolium tetrazolium romidedetron (MTT) after 48 h culture on a culture medium with a final glucose concentration of 25 mmol / L 50 mmol / L or 100 mmol / L, and methyltetrazoliumromide MTT was used to measure the cell activity of RSC96 with methyl tetrazolium tetrazolium romidedet MTT (5-diphenytetrazolium romidede MTT). According to the results, the optimal concentration of high glucose was selected, and then the cells were treated with the final concentration of 10 渭 mol / L ~ (25 渭 mol / L ~ (-1)) 50 渭 mol / L ~ (50) 渭 mol / L ~ (75) 渭 mol 路L ~ (-1) / L ~ (100) 渭 mol/L, and the cell activity was measured after 48 h. In order to exclude the effect of osmotic pressure caused by high glucose on the experimental results, the mannitol group was used as the control group. In this part of the experiment, 50 mmol / L high glucose stimulation concentration and 50 渭 mol / L L-carnitine final drug concentration were selected. The RSC96 cells were randomly divided into five groups: normal control group (C group), high glucose group (group H), high glucose group (L-carnitine group), high glucose group (L-carnitine group) and single L-carnitine group (L-carnitine group). After 48 hours of culture, mannitol group and mannitol group were cultured for 48 hours. The growth status of each group was observed and photographed, the apoptosis rate of each group was measured, the activity of SOD and the content of MDA in each group were measured. Western Blot method was used to detect the protein expression of polyadenosine diphosphate-ribose polymerase-1 (PARP-1) and aspartate specific cysteine aspartate-specific protease-3 (Caspase-3) in the cells of each group. Results the cell activity was decreased after high glucose treatment, and the protein expression of PARP-1 and aspartate specific cysteine aspartate-specific protease-3caspase-3 were detected by Western Blot. The damage effect was in a dose-dependent manner. After 48 hours of high glucose concentration of 50 mmol / L, the cell activity decreased to 58% of group C activity, and the proliferation activity decreased significantly. Therefore, 50 mmol / L was chosen as the high glucose dose in the follow-up experiment. The L-carnitine medium had no significant effect on the proliferation activity of normal Schwann cells, but could significantly improve the decrease of Schwann cell activity induced by high glucose. The results showed that the cell activity of levocarnitine at 50 渭 mol/L was significantly higher than that of other dose groups. Therefore, when 50 渭 mol/L levocarnitine was selected as the drug dose of the follow-up experiment, the adherent cells in H and H L groups were decreased compared with those in C group. Superoxide dismutase (SOD) activity in suspension cells decreased MDA content and apoptosis rate. The expression of activated Caspase-3 fragments and PARP-1 fragments in HL-H group was significantly higher than that in group C, but the number of adherent cells and cell junctions in HL-treated group was higher than that in group C, but there was no significant difference between group C and group C in the expression of Caspase-3 fragments and PARP-1 fragments in HL-H group, and the number of adherent cells in HL group was higher than that in group C. The decrease of SOD activity in suspension cells increased the content of MDA and the rate of apoptosis, and the expression of activated Caspase-3 and PARP-1 fragments decreased significantly. Conclusion: high glucose can induce the damage of Schwann cells, and it is not related to the high osmotic pressure induced by high glucose. L-carnitine has protective effect on the injury of Schwann cells induced by high glucose. The mechanism may be related to the decrease of oxidative stress level and the down-regulation of the expression of PARP-1 and 89kDaCaspase-3Caspase-317kDa. It is related to reducing apoptosis.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614

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