本文選題：硒蛋白 + mSelK　； 參考：《遼寧大學》2017年碩士論文

【摘要】：微量元素硒在機體內具有胰島素樣的作用,但其機理一直不清楚。前人研究發(fā)現細胞內游離鈣離子濃度的升高是促進胰島β細胞MIN6分泌胰島素的重要條件之一;硒蛋白mSelK能夠促進內質網上IP3受體IP3R的表達,進而促進內質網鈣庫中鈣離子的釋放,從而提高細胞漿中的游離鈣離子水平。但是硒蛋白mSelK對胰島細胞內胰島素釋放的影響一直沒有人研究。本文通過對小鼠胰島β細胞MIN6進行mSelK的基因敲減和過表達,對硒蛋白mSelK在促進胰島素釋放中的作用進行了研究,并通過研究mSelK的基因敲減和過表達對Sel KMIN6細胞胞漿中游離鈣離子水平的作用,對內質網上三種IP3受體表達的影響,對硒蛋白mSelK影響胰島素釋放的機制進行了探討。首先探究微量元素硒與胰島素釋放的關系。用最適濃度的Na2Se O3處理小鼠胰島β細胞MIN 6,用小鼠胰島素ELISA試劑盒檢測藥物處理后MIN6細胞胰島素釋放水平的變化。同時利用實時定量PCR和western blot檢驗微量元素硒處理后細胞內硒蛋白mSelK的變化。實驗結果表明,亞硒酸鈉的處理會導致MIN6細胞中胰島素釋放量的增加,同時明顯上調細胞內硒蛋白mSelK的表達量。葡萄糖的攝入是胰島細胞分泌胰島素的開端,高濃度的葡萄糖會促使胰島素分泌量的增加。用由低到高不同濃度的葡萄糖溶液(2.8 m M,5.6 m M,16.7mM,25 mM)處理小鼠胰島β細胞Min 6,24h后提取蛋白,Western blot驗證葡萄糖濃度與硒蛋白mSelK表達量的的關系。結果顯示細胞內葡萄糖的增加也會引起細胞內硒蛋白mSelK表達量的增加。然后利用慢病毒載體對MIN6細胞進行mSelK基因敲減,隨后利用腺病毒載體對MIN6細胞進行mSelK基因過表達,用小鼠胰島素ELISA試劑盒檢測mSelK基因敲減和過表達后胰島素釋放水平的變化。結果表明硒蛋白mSelK的敲減會引起細胞內胰島素釋放量的減少,而硒蛋白mSelK的過表達會引起細胞內胰島素釋放量的增加,并且呈劑量依賴關系。為探討硒蛋白mSelK影響胰島素釋放的機制,采用流式細胞儀法對mSelK基因敲減和過表達后MIN6細胞中游離鈣離子水平的變化進行了研究。研究發(fā)現硒蛋白mSelK的基因敲減引起細胞內的游離鈣離子水平的顯著下降,而硒蛋白mSelK的過表達引起細胞內的游離鈣離子水平的顯著上升。最后,采用實時定量PCR和western blot對mSelK基因敲減和過表達后IP3R的表達情況進行了研究。實時定量PCR結果顯示,IP3的Ⅰ、Ⅱ型受體不隨細胞內硒蛋白mSelK表達量的變化而改變,但是IP3 Ⅲ型受體在細胞中的表達量會隨著細胞內硒蛋白mSelK表達量的降低而降低,隨著細胞內硒蛋白mSelK表達量的升高而升高,western blot檢驗證實了上述結果。綜上所述:微量元素硒和葡萄糖均能引起小鼠胰島β細胞內胰島素釋放水平的升高,并且能夠促進細胞內硒蛋白mSelK的表達量的升高。硒蛋白mSelK的表達量的降低及升高可以引起小鼠胰島β細胞內胰島素釋放水平的降低及升高。這種胰島素釋放水平的降低及升高是由于硒蛋白mSelK的敲減和過表達能夠直接調控IP3 Ⅲ型受體的表達水平,進而影響細胞內游離鈣離子的水平而實現的。此結果進一步闡明了小鼠硒蛋白mSelK的生物學功能,也為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.
【學位授予單位】：遼寧大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.1

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本文編號：2008514