【摘要】：目的:本研究旨在研究細胞水平中高糖對細胞的脂代謝作用及poldip2的表達水平;并研究2型糖尿病小鼠注入Poldip2腺病毒后對胰島素信號通路中的作用及對KKAy小鼠脂代謝的調節(jié)作用。方法:(1)將Mc Ardle 7777細胞分別培養(yǎng)于高糖培養(yǎng)基和正常葡萄糖培養(yǎng)基中,檢測在高糖和正常葡萄糖培養(yǎng)下TG,TCH水平及Poldip2的表達情況。(2)利用不同胰島素劑量建立不同水平的胰島素抵抗,檢測Poldip2在胰島素作用下的劑量反應關系、時間效應關系;以及在胰島素最優(yōu)劑量和時間下,Poldip2對Mc Ardle7777細胞的脂代謝作用。(3)體內實驗中,KKAy小鼠經(jīng)過4周高脂飲食后,將KKAy小鼠隨機分為Poldip2病毒組;GFP組;Saline組。此外,將C57BL/6J小鼠作為空白對照組測定小鼠中血脂四項,餐后脂耐受水平,血清游離脂肪酸,胰島素,瘦素,肝臟脂水平,肝臟中PTP1B酶活性、H_2O_2水平,并檢測胰島素信號通路中相關蛋白的表達水平。結果:細胞實驗中,高糖情況下Poldip2的表達水平顯著下降,同時高糖刺激能夠降低脂代謝水平。在胰島素刺激實驗中,正常葡萄糖下,胰島素劑量為20μmol,24h內Poldip2蛋白的表達最為豐富;高糖下,胰島素劑量5μmol,培養(yǎng)6h后Poldip2的表達最為豐富,其細胞TCH水平顯著高于未加胰島素刺激下的TCH水平。同時發(fā)現(xiàn)在高糖培養(yǎng)下,較低劑量的胰島素,就會使Poldip2的表達抑制。體內動物試驗中,Poldip2腺病毒能夠顯著改善KKAy小鼠的血糖水平,并能降低血清中TG,TCH,LDL-C及肝臟中TCH的水平;降低血清中游離脂肪酸及胰島素水平;對小鼠新鮮肝臟中PTP1B酶活性和H_2O_2的檢測發(fā)現(xiàn),Poldip2能提高肝臟中H_2O_2水平,并顯著降低PTP1B酶的活性;通過餐后脂耐受水平檢測發(fā)現(xiàn)Poldip2可加速小鼠外源性脂肪代謝。通過對胰島素信號通路中相關蛋白的檢測,Poldip2能夠降低KKAy小鼠中PTEN,PTP1B蛋白的表達,激活Akt的活性進而促進胰島素信號通路的傳遞;另外Poldip2可能通過降低PEPCK蛋白的表達,進而抑制糖異生的合成,使肝糖葡萄糖輸出減少。結論:細胞在高糖培養(yǎng)下發(fā)現(xiàn)高糖對細胞具有高毒作用,產生了脂代謝紊亂,這可能與細胞在高糖培養(yǎng)下Poldip2的蛋白水平下降相關。Poldip2能夠扭轉外源性脂代謝的異常,改善小鼠的脂代謝情況。Poldip2改善糖尿病小鼠糖脂代謝的機制可能是通過Poldip2的表達升高,激活Nox4產生H_2O_2,過度氧化抑制了PTP1B,PTEN的活性,進而激活Akt蛋白的活性,改善了胰島素信號通路及糖脂代謝紊亂。
[Abstract]:Aim: to study the effect of high glucose on lipid metabolism and the expression of poldip2 in cells, and to study the role of Poldip2 adenovirus in insulin signaling pathway and the regulation of lipid metabolism in KKAy mice in type 2 diabetic mice. Methods: (1) Mc Ardle 7777 cells were cultured in high glucose medium and normal glucose medium respectively. The levels of TG,TCH and the expression of Poldip2 were detected under high glucose and normal glucose culture. (2) different levels of insulin resistance were established with different insulin doses. The dose-response relationship, time-effect relationship and lipid metabolism of Poldip2 on Mc Ardle7777 cells under the optimal insulin dose and time were measured. (3) in vivo, the KKAy mice were fed a high-fat diet for 4 weeks. KKAy mice were randomly divided into Poldip2 virus group, GFP group and Saline group. In addition, C57BL/6J mice were used as blank control group to determine four items of blood lipids, postprandial lipid tolerance, serum free fatty acids, insulin, leptin, liver lipid, liver PTP1B enzyme activity and H_2O_2 level. The expression of related proteins in insulin signaling pathway was detected. Results: in the cell experiment, the expression of Poldip2 was significantly decreased under high glucose condition, and the lipid metabolism was decreased by high glucose stimulation. In insulin stimulation experiment, the expression of Poldip2 protein was the most abundant in normal glucose at 20 渭 mol,24h, and in high glucose at 5 渭 mol, for 6 h. The level of TCH was significantly higher than that of TCH without insulin stimulation. At the same time, it was found that the low dose of insulin inhibited the expression of Poldip2 in high glucose culture. In animal experiment in vivo, Poldip2 adenovirus could significantly improve the blood glucose level of KKAy mice, and decrease the levels of TG,TCH,LDL-C in serum and TCH in liver, free fatty acid and insulin in serum. The detection of PTP1B enzyme activity and H_2O_2 in fresh liver of mice showed that Poldip2 could increase the level of H_2O_2 in liver and decrease the activity of PTP1B enzyme significantly, and Poldip2 could accelerate the exogenous fat metabolism of mice by detecting the level of postprandial lipid tolerance. By detecting the related proteins in insulin signaling pathway, Poldip2 can reduce the expression of PTEN,PTP1B protein in KKAy mice, activate the activity of Akt and promote the transmission of insulin signal pathway. In addition, Poldip2 may decrease the expression of PEPCK protein. Furthermore, the synthesis of glucose allogeneic was inhibited, and the glucose output of liver was decreased. Conclusion: high glucose has high toxicity to cells in high glucose culture and leads to lipid metabolism disorder, which may be related to the decrease of protein level of Poldip2 in high glucose culture. Poldip2 can reverse the abnormality of exogenous lipid metabolism. Improving lipid metabolism in mice. The mechanism of Poldip2 in improving glucose and lipid metabolism in diabetic mice may be through the increase of Poldip2 expression and activation of Nox4 to produce H _ 2O _ 2. Excessive oxidation inhibits the activity of PTP1B,PTEN and then activates the activity of Akt protein. Improved insulin signaling pathway and glycolipid metabolism disorder.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.1

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