本文選題：游離脂肪酸 + 軟脂酸�。� 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：研究背景:糖尿病的患病率逐年上升,目前已成為嚴重威脅人類健康的疾病之一,胰島β細胞凋亡增加是糖尿病發(fā)生、發(fā)展的關(guān)鍵因素[1]。近年來有研究表明,2型糖尿病患者體內(nèi)長期高水平的游離脂肪酸(free fatty acids,FFAs),特別是飽和脂肪酸可以引起胰島素抵抗、胰島β細胞功能紊亂,以及β細胞凋亡的增加,進而加重糖尿病[2-4]。硫氧還蛋白相互作用蛋白(Thioredoxin interacting protein,TXNIP)又名VDUP1(Vitamin D(3)up-regulated protein 1)和TBP2(Thioredoxin-binding protein-2),屬于α抑制蛋白家族,是目前發(fā)現(xiàn)的唯一內(nèi)源性的硫氧還蛋白(Thioredoxin,Trx)結(jié)合抑制蛋白,在糖尿病各組織中其表達均顯著升高,已有大量研究證明高糖可引起TXNIP表達明顯升高,并介導β細胞功能損傷和凋亡[5-7]。但是,作為糖尿病主要損傷因素之一的游離脂肪酸,其對TXNIP表達的影響,以及不同飽和程度的游離脂肪酸對TXNIP表達的可能差別目前尚不清楚。因此本課題旨在研究不同飽和程度的脂肪酸對INS-1胰島細胞TXNIP表達的影響,并分析其可能機制。目的:1.使用不同飽和程度的游離脂肪酸孵育INS-1細胞,觀察不同類型脂肪酸對INS-1胰島細胞凋亡及TXNIP表達的影響。2.分析脂肪酸調(diào)節(jié)TXNIP表達的可能機制。方法:1.實驗分組:將正常培養(yǎng)的INS-1細胞分為四組:分別是正常對照組(含0.55%fatty acid free-BSA的RPMI1640培養(yǎng)基)、飽和脂肪酸-軟脂酸組(0.5 mmol/L軟脂酸+0.55%fatty acid free-BSA)、單不飽和脂肪酸-棕櫚油酸組(0.5 mmol/L棕櫚油酸+0.55%fatty acid free-BSA)、多不飽和脂肪酸-DHA 組(0.5 mmol/L DHA+0.55%fatty acid free-BSA),均于培養(yǎng)24 h后收集細胞進行指標測定。2.采用Real-time PCR方法測定FFAs處理后INS-1細胞TXNIP mRNA表達量。3.采用 Western blot方法檢測FFAs處理后 INS-1 細胞 TXNIP、Cleaved caspase-3、Caspase-3、ChREBP、FOXO1、p-NF-KB 蛋白表達。4.采用免疫熒光技術(shù)檢測FFAs處理后INS-1細胞中ChREBP蛋白表達。5.使用Annexin V-FITC/PI染色流式細胞術(shù)檢測INS-1細胞凋亡情況。結(jié)果:1.不同飽和程度的FFAs對INS-1細胞TXNIP表達的影響不同。與對照組相比,飽和脂肪酸軟脂酸組TXNIP mRNA和蛋白表達均顯著上調(diào),而不飽和脂肪酸棕櫚油酸組和DHA組INS-1細胞TXNIP表達均沒有明顯的改變(見圖1)。如圖2所示,軟脂酸引起的TXNIP的蛋白表達從18 h開始增加,并于24 h達到最高。36 h時,軟脂酸誘導的TXNIP表達降低到原蛋白水平。而棕櫚油酸和DHA在12 h、18 h、24 h、36 h不同的時間點對TXNIP表達與對照組相比均無統(tǒng)計學差異。2.FFAs由于其飽和程度的不同對INS-1細胞凋亡也具有不同的影響。如圖3所示,軟脂酸組Cleaved caspase-3/caspase-3的比值與對照組相比明顯增加。棕櫚油酸組和DHA組Cleaved caspase-3/caspase-3比值與對照組相比無顯著性差異。流式細胞術(shù)結(jié)果顯示,軟脂酸組INS-1細胞的凋亡指數(shù)明顯增加,而棕櫚油酸組和DHA組對INS-1細胞凋亡的影響與對照組相比無統(tǒng)計學差異(見圖4)。3.軟脂酸對轉(zhuǎn)錄因子ChREBP和FOXO1蛋白表達的影響。使用Western blot和免疫熒光方法研究發(fā)現(xiàn),與對照組相比軟脂酸組ChREBP蛋白表達顯著上調(diào),轉(zhuǎn)錄因子FOXO1蛋白表達降低。(見圖5-6)4.軟脂酸調(diào)節(jié)INS-1細胞TXNIP蛋白表達與NF-κB磷酸化相關(guān)。與對照組相比,軟脂酸組p-NF-κB p65蛋白表達量明顯增加(見圖7)。加入NF-κB抑制劑PDTC后,與不加抑制劑組相比,軟脂酸孵育后引起的TXNIP mRNA和蛋白水平的上調(diào)受到明顯的抑制(見圖8)。同樣,使用一種多肽類的NF-κB抑制劑SN50也可降低軟脂酸誘導的TXNIP表達上調(diào)(見圖9)。同時發(fā)現(xiàn),PDTC能夠顯著降低軟脂酸誘導的ChREBP表達上調(diào)(見圖10),而對軟脂酸下調(diào)FOXO1的表達沒有影響(見圖11)。5.p38 MAPK參與軟脂酸誘導的TXNIP表達上調(diào)。與不加抑制劑組相比,使用p38 MAPK抑制劑SB203580可顯著抑制軟脂酸處理后引起的INS-1細胞TXNIP mRNA和蛋白水平的增加(見圖12)。同時,SB203580也可阻斷了軟脂酸誘導的p-NF-κB p65蛋白表達上調(diào)(見圖13),以及軟脂酸對轉(zhuǎn)錄因子ChREBP和FOXO1蛋白表達的調(diào)節(jié)作用(見圖14,15)。結(jié)論:1.飽和脂肪酸軟脂酸可能通過誘導TXNIP表達上調(diào),進而促進了 INS-1胰島細胞凋亡。而不飽和脂肪酸棕櫚油酸和DHA對TXNIP表達以及INS-1細胞凋亡均無顯著作用。2.軟脂酸上調(diào)TXNIP表達的機制與p38 MAPK/NF-κB/ChREBP信號通路有關(guān),FOXO1可能也參與了 TXNIP的調(diào)節(jié)。
[Abstract]:Background: the prevalence of diabetes is increasing year by year, and now it has become one of the serious threats to human health. The increase of islet beta cell apoptosis is the occurrence of diabetes. The key factor in the development of diabetes [1]. in recent years has shown that the long-term high level of free fatty acids (FFAs), especially saturated fat, in patients with type 2 diabetes mellitus Fatty acids can cause insulin resistance, islet beta cell dysfunction, and the increase of beta cell apoptosis, and further aggravate the diabetes [2-4]. thioredoxin interaction protein (Thioredoxin interacting protein, TXNIP), also known as VDUP1 (Vitamin D (3) up-regulated protein 1) and TBP2 (Thioredoxin-binding), belonging to the alpha suppressor family. The only endogenous thioredoxin (Thioredoxin, Trx) binding inhibitor is found at present, and its expression is significantly increased in all diabetic tissues. A large number of studies have shown that high glucose can cause a significant increase in the expression of TXNIP and mediate the function damage and apoptosis of beta cells, but it is one of the major damage factors of diabetes. The effects of fatty acids on the expression of TXNIP and the possible difference in the expression of TXNIP with different saturation degrees of free fatty acids are still unclear. Therefore, the purpose of this study is to investigate the effect of fatty acids on the expression of TXNIP in INS-1 islet cells at different saturation levels and to analyze its possible mechanism. Objective: 1. the use of free fat with different saturation degrees is used. INS-1 cells were incubated with fatty acids to observe the effects of different types of fatty acids on the apoptosis of INS-1 islet cells and the expression of TXNIP..2. analysis of the possible mechanism of TXNIP expression by fatty acids. Methods: 1. experimental groups: the normal cultured INS-1 cells were divided into four groups: the normal control group (RPMI1640 culture containing 0.55%fatty acid free-BSA), saturated fat, respectively. Fatty acid and palmitic acid group (0.5 mmol/L +0.55%fatty acid free-BSA), monounsaturated fatty acid palmitic acid group (0.5 mmol/L palmitic oleic acid +0.55%fatty acid free-BSA), polyunsaturated fatty acid -DHA group (0.5 mmol/L DHA+0.55%fatty acid) The expression of TXNIP mRNA in INS-1 cells after FFAs treatment was measured by Western blot method to detect INS-1 cell TXNIP, Cleaved caspase-3. Cell apoptosis was used to detect the apoptosis of INS-1 cells. Results: 1. the effects of FFAs on the expression of TXNIP in INS-1 cells were different. Compared with the control group, the expression of TXNIP mRNA and protein in the saturated fatty acid palmitic acid group were significantly up, while the TXNIP expression of the unsaturated fatty acid palmitoleic acid group and the DHA group had no significant changes (see Figure 1). As shown in Figure 2, the protein expression of TXNIP caused by palmitic acid increased from 18 h, and the TXNIP expression induced by palmitic acid decreased to the original protein level when 24 h reached the highest.36 H. While palmitic acid and DHA were at 12 h, 18 h, 24 h, 36 h, there was no significant difference between the expression of TXNIP and the control group because of its saturation degree The difference in the apoptosis of INS-1 cells was also different. As shown in Figure 3, the ratio of Cleaved caspase-3/caspase-3 in the palmitic acid group was significantly increased compared with the control group. The ratio of Cleaved caspase-3/caspase-3 in the palmitic and DHA groups was not significantly different from that of the control group. The flow cytometry results showed that the INS-1 cell in the palmitic acid group was withered. The death index increased significantly, while the effect of palmitic acid group and DHA group on INS-1 cell apoptosis was not significantly different from that of the control group (see Figure 4) the effect of.3. palmitic acid on the expression of ChREBP and FOXO1 protein. The use of Western blot and immunofluorescence methods found that the expression of ChREBP protein in the palmitic acid group was significantly up-regulated compared with the control group. The expression of transcription factor FOXO1 protein decreased. (see Figure 5-6) 4. palmitic acid regulated INS-1 cell TXNIP protein expression was associated with NF- kappa B phosphorylation. Compared with the control group, the expression of p-NF- kappa B p65 protein in the palmitic acid group increased significantly (see Figure 7). After adding NF- kappa B inhibitor PDTC, the TXNIP and egg caused by the incubation of palmitic acid compared with the non inhibitor group. The upregulation of white level was significantly inhibited (see Figure 8). Similarly, the use of a peptide NF- kappa B inhibitor SN50 could also reduce the up-regulated TXNIP expression induced by palmitic acid (see Figure 9). At the same time, it was found that PDTC could significantly reduce the up-regulated ChREBP expression induced by palmitic acid (see Figure 10), but no effect on the expression of palmitic acid down FOXO1 (see Figure 11).5.p 38 MAPK was involved in the up-regulated TXNIP expression induced by palmitic acid. Compared with the non inhibitor group, the use of p38 MAPK inhibitor SB203580 significantly inhibited the increase of TXNIP mRNA and protein levels in INS-1 cells induced by palmitic acid (see Figure 12). At the same time, SB203580 also blocked the up regulation of p-NF- kappa B p65 protein expression induced by palmitic acid (see Figure 13). And the regulation of palmitic acid on the expression of transcription factor ChREBP and FOXO1 protein (see Figure 14,15). Conclusion: 1. saturated fatty acid palmitic acid may increase the expression of TXNIP by inducing TXNIP expression, and thus promote the apoptosis of INS-1 islet cells, while unsaturated fatty acid palmitoleic acid and DHA have no significant effect on TXNIP expression and INS-1 cell apoptosis. The mechanism of upregulation of TXNIP expression is related to p38 MAPK/NF- kappa B/ChREBP signaling pathway. FOXO1 may also play a role in TXNIP regulation.
【學位授予單位】：山西醫(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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