小鼠硒蛋白mSelK在调控胰岛素分泌过程中的作用

发布时间：2018-06-12 05:16

  本文选题：硒蛋白 + mSelK　； 参考：《辽宁大学》2017年硕士论文

【摘要】：微量元素硒在机体内具有胰岛素样的作用,但其机理一直不清楚。前人研究发现细胞内游离钙离子浓度的升高是促进胰岛β细胞MIN6分泌胰岛素的重要条件之一;硒蛋白mSelK能够促进内质网上IP3受体IP3R的表达,进而促进内质网钙库中钙离子的释放,从而提高细胞浆中的游离钙离子水平。但是硒蛋白mSelK对胰岛细胞内胰岛素释放的影响一直没有人研究。本文通过对小鼠胰岛β细胞MIN6进行mSelK的基因敲减和过表达,对硒蛋白mSelK在促进胰岛素释放中的作用进行了研究,并通过研究mSelK的基因敲减和过表达对Sel KMIN6细胞胞浆中游离钙离子水平的作用,对内质网上三种IP3受体表达的影响,对硒蛋白mSelK影响胰岛素释放的机制进行了探讨。首先探究微量元素硒与胰岛素释放的关系。用最适浓度的Na2Se O3处理小鼠胰岛β细胞MIN 6,用小鼠胰岛素ELISA试剂盒检测药物处理后MIN6细胞胰岛素释放水平的变化。同时利用实时定量PCR和western blot检验微量元素硒处理后细胞内硒蛋白mSelK的变化。实验结果表明,亚硒酸钠的处理会导致MIN6细胞中胰岛素释放量的增加,同时明显上调细胞内硒蛋白mSelK的表达量。葡萄糖的摄入是胰岛细胞分泌胰岛素的开端,高浓度的葡萄糖会促使胰岛素分泌量的增加。用由低到高不同浓度的葡萄糖溶液(2.8 m M,5.6 m M,16.7mM,25 mM)处理小鼠胰岛β细胞Min 6,24h后提取蛋白,Western blot验证葡萄糖浓度与硒蛋白mSelK表达量的的关系。结果显示细胞内葡萄糖的增加也会引起细胞内硒蛋白mSelK表达量的增加。然后利用慢病毒载体对MIN6细胞进行mSelK基因敲减,随后利用腺病毒载体对MIN6细胞进行mSelK基因过表达,用小鼠胰岛素ELISA试剂盒检测mSelK基因敲减和过表达后胰岛素释放水平的变化。结果表明硒蛋白mSelK的敲减会引起细胞内胰岛素释放量的减少,而硒蛋白mSelK的过表达会引起细胞内胰岛素释放量的增加,并且呈剂量依赖关系。为探讨硒蛋白mSelK影响胰岛素释放的机制,采用流式细胞仪法对mSelK基因敲减和过表达后MIN6细胞中游离钙离子水平的变化进行了研究。研究发现硒蛋白mSelK的基因敲减引起细胞内的游离钙离子水平的显著下降,而硒蛋白mSelK的过表达引起细胞内的游离钙离子水平的显著上升。最后,采用实时定量PCR和western blot对mSelK基因敲减和过表达后IP3R的表达情况进行了研究。实时定量PCR结果显示,IP3的Ⅰ、Ⅱ型受体不随细胞内硒蛋白mSelK表达量的变化而改变,但是IP3 Ⅲ型受体在细胞中的表达量会随着细胞内硒蛋白mSelK表达量的降低而降低,随着细胞内硒蛋白mSelK表达量的升高而升高,western blot检验证实了上述结果。综上所述:微量元素硒和葡萄糖均能引起小鼠胰岛β细胞内胰岛素释放水平的升高,并且能够促进细胞内硒蛋白mSelK的表达量的升高。硒蛋白mSelK的表达量的降低及升高可以引起小鼠胰岛β细胞内胰岛素释放水平的降低及升高。这种胰岛素释放水平的降低及升高是由于硒蛋白mSelK的敲减和过表达能够直接调控IP3 Ⅲ型受体的表达水平,进而影响细胞内游离钙离子的水平而实现的。此结果进一步阐明了小鼠硒蛋白mSelK的生物学功能,也为糖尿病的防治提供了新的思路。
[Abstract]:Trace element selenium has the role of insulin like in the body, but its mechanism is not clear. Previous studies have found that the increase of intracellular free calcium concentration is one of the important conditions to promote insulin secretion in islet beta cell MIN6; selenoprotein mSelK can promote the expression of IP3 receptor IP3R in the endoplasmic reticulum, and then promote calcium in the endoplasmic reticulum calcium pool. The release of ions increases the level of free calcium ions in the cytoplasm. However, the effect of selenoprotein mSelK on the release of insulin in the islet cells has not been studied. In this paper, the gene knockout and overexpression of mSelK in the mouse islet beta cell MIN6 were knocked down and overexpressed, and the role of selenoprotein mSelK in the insulin release was studied. The effect of mSelK gene knockout and overexpression on the level of free calcium ion in the cytoplasm of Sel KMIN6 cells, the effect on the expression of three IP3 receptors in the endoplasmic reticulum, the mechanism of the effect of selenoprotein mSelK on the release of insulin was discussed. First, the relationship between selenium and insulin release was explored. The optimum concentration of Na2Se O3 was used to treat the release of insulin. The mouse islet beta cell MIN 6 was used to detect the changes in the insulin release level of MIN6 cells after treatment with the mouse insulin ELISA kit. Meanwhile, the changes in the intracellular selenoprotein mSelK were detected by real-time quantitative PCR and Western blot. The experimental results showed that the treatment of sodium selenite could lead to insulin release in MIN6 cells. The increase in volume increased the expression of intracellular selenoprotein mSelK. Glucose intake was the beginning of insulin secretion in islet cells, and high concentration of glucose could increase the insulin secretion. The glucose solution from low to high concentrations (2.8 m M, 5.6 m M, 16.7mM, 25 mM) treated the mouse pancreatic beta cells after Min 6,24h. The relationship between the concentration of glucose and the mSelK expression of selenoprotein was verified by Western blot. The results showed that the increase of glucose in the cells also resulted in the increase in the expression of mSelK in the cells. Then the mSelK gene was knocked down by the lentivirus vector and the mSelK gene of MIN6 cells was followed by the adenovirus vector. Over expression, a mouse insulin ELISA kit was used to detect the changes in insulin release and insulin release after mSelK gene knockout and overexpressed. The results showed that the reduction of selenoprotein mSelK caused the decrease of intracellular insulin release, and the overexpression of selenoprotein mSelK could cause an increase in intracellular insulin release and a dose-dependent relationship. The mechanism of the effect of selenoprotein mSelK on the release of insulin was investigated. Flow cytometry was used to study the changes of free calcium ions in MIN6 cells after mSelK knockout and overexpressed. The study found that the gene knockout of selenoprotein mSelK significantly decreased the level of free calcium ions in the cells, and the overexpression of selenoprotein mSelK was induced. The level of free calcium ions in the cells increased significantly. Finally, the expression of IP3R was studied by real-time quantitative PCR and Western blot after the mSelK gene knockout and overexpressed. Real-time quantitative PCR results showed that IP3's I, type II receptor did not change with the changes in the amount of selenoprotein mSelK, but the IP3 type III receptor was in the cell The expression in the cells decreased with the decrease of the expression of selenoprotein mSelK, and increased with the increase of the expression of selenoprotein mSelK. The Western blot test confirmed the above results. The increase of the expression of selenoprotein mSelK in the incoming cells. The decrease and increase of the expression of selenoprotein mSelK can cause the decrease and increase of insulin release level in the islet beta cells of mice. The decrease and increase of the level of insulin release is due to the direct regulation of the expression of IP3 III receptor by the knockout of selenoprotein mSelK and the overexpression of selenoprotein. The results further elucidate the biological function of the mouse selenoprotein mSelK and provide a new idea for the prevention and treatment of diabetes.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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