發(fā)布時間：2018-05-10 15:33

  本文選題：TNF-α + IL-1β��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景:糖尿病(Diabetes Mellitus,DM)是一種由遺傳和環(huán)境因素共同作用而引起的以慢性高血糖為特征的代謝性疾病,其本質(zhì)是胰島素的分泌和(或)作用缺陷。當(dāng)前全世界范圍內(nèi)糖尿病的發(fā)病率和患病率都呈明顯上升趨勢,而我國作為一個人口龐大的發(fā)展中國家,已成為世界第一糖尿病大國。糖尿病主要分為1型和2型,但不論是哪種類型的糖尿病,胰島β細(xì)胞損傷和凋亡都是糖尿病發(fā)生發(fā)展的重要原因。TNF-α和IL-1β是糖尿病時存在的慢性系統(tǒng)性炎癥的重要介導(dǎo)因子,可以誘導(dǎo)胰島細(xì)胞凋亡,因而在糖尿病中發(fā)揮著重要作用。硫氧還蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)是目前發(fā)現(xiàn)的唯一內(nèi)源性硫氧還蛋白(thioredoxin,TRX)結(jié)合及抑制蛋白,可以削弱TRX的抗自由基和抗凋亡功能。糖尿病時各組織TXNIP表達(dá)明顯增加,研究發(fā)現(xiàn)高糖可以誘導(dǎo)INS-1細(xì)胞TXNIP m RNA和蛋白水平顯著升高且伴有明顯細(xì)胞凋亡。本實驗室前期研究證實過表達(dá)TXNIP可通過caspase-8、9及PERK參與的內(nèi)質(zhì)網(wǎng)應(yīng)激途徑誘導(dǎo)INS-1細(xì)胞凋亡,因此,TXNIP被認(rèn)為是連接糖尿病和β細(xì)胞死亡的關(guān)鍵環(huán)節(jié)。那么,炎性因子TNF-α和IL-1β是否可以影響INS-1細(xì)胞TXNIP的表達(dá)進(jìn)而影響凋亡,若可其機(jī)制又如何?本研究的目的就在于探討TNF-α和IL-1β誘導(dǎo)TXNIP變化情況及其可能機(jī)制,從而為糖尿病的預(yù)防和治療提供新的干預(yù)靶點。目的:在正常糖脂濃度下培養(yǎng)INS-1細(xì)胞,外源性給予TNF-α(5ng/ml)和IL-1β(10ng/ml)刺激細(xì)胞24h,觀察TNF-α和IL-1β是否引起INS-1細(xì)胞TXNIP表達(dá)變化并研究其可能機(jī)制。方法:1.實驗分組:將正常糖脂濃度下培養(yǎng)的INS-1細(xì)胞分四組,分別為正常對照組、TNF-α組(5ng/ml,24h)、IL-1β組(10ng/ml,24h)和TNF-α+IL-1β組(5ng/ml+10ng/ml,24h)。2.外源性給予炎性因子TNF-α和IL-1β干預(yù)INS-1細(xì)胞,用CCK-8檢測細(xì)胞存活率。3.流式細(xì)胞術(shù)和cleaved caspase-3兩種方法檢測TNF-α和IL-1β作用下INS-1細(xì)胞凋亡情況。4.Real-time PCR法檢測TNF-α和IL-1β作用下INS-1細(xì)胞中TXNIP、NFκB和Ch REBP m RNA的水平。5.Western blot法檢測TNF-α和IL-1β作用下INS-1細(xì)胞中TXNIP、NFκB、Ch REBP和FOXO1的蛋白表達(dá)量。6.Real-time PCR法和Western blot法檢測NFκB抑制劑PDTC、p38MAPK抑制劑SB203580預(yù)處理后,INS-1細(xì)胞TXNIP、NFκB和Ch REBP m RNA和蛋白表達(dá)量。結(jié)果:1.TNF-α和IL-1β降低INS-1細(xì)胞的存活率使用不同濃度炎性因子干預(yù)INS-1細(xì)胞后,與正常對照組相比,TNF-α和IL-1β都可以濃度依賴性地誘導(dǎo)INS-1細(xì)胞損傷,降低細(xì)胞的存活率;且TNF-α在(5ng/ml,24h)與IL-1β在(10ng/ml,24h)時引起較明顯的細(xì)胞活力下降,因此設(shè)定后期實驗的TNF-α和IL-1β作用濃度分別為5ng/ml和10ng/ml,作用時間為24h。2.TNF-α和IL-1β誘導(dǎo)INS-1細(xì)胞凋亡Annexin-V染色流式細(xì)胞儀分析的結(jié)果顯示,與對照組相比,TNF-α組、IL-1β組和TNF-α+IL-1β組細(xì)胞的凋亡率都顯著升高(P0.05),Western blot結(jié)果中代表凋亡的cleaved caspase-3表達(dá)量也升高明顯,提示炎性因子TNF-α和IL-1β可以引起INS-1細(xì)胞凋亡的增加,當(dāng)二者聯(lián)合作用時發(fā)揮協(xié)同作用,誘導(dǎo)更加顯著的細(xì)胞凋亡。3.TNF-α和IL-1β誘導(dǎo)TXNIP m RNA表達(dá)上調(diào)與對照組相比,TXNIP m RNA表達(dá)水平在5ng/ml的TNF-α和10ng/ml的IL-1β時達(dá)到高峰;而選用不同作用時間的實驗也表明,此濃度下作用24h時TXNIP m RNA表達(dá)水平也顯著升高。因此,后續(xù)實驗分組為正常對照組、TNF-α組(5ng/ml,24h)、IL-1β組(10ng/ml,24h)和TNF-α+IL-1β組(5ng/ml+10ng/ml,24h)。Real-time PCR結(jié)果顯示,各炎性因子干預(yù)組,包括TNF-α+IL-1β共同作用組,TXNIP m RNA較對照組有顯著升高(P0.05)。4.TNF-α和IL-1β誘導(dǎo)TXNIP蛋白表達(dá)量升高與對照組相比,TNF-α組、IL-1β組和TNF-α+IL-1β組細(xì)胞TXNIP蛋白水平升高明顯(P0.05)。5.p-NFκB、Ch REBP、FOXO1等轉(zhuǎn)錄因子的測定5.1炎性因子上調(diào)Ch REBP m RNA和蛋白水平與對照組相比,TNF-α、IL-1β和二者聯(lián)合作用,引起細(xì)胞Ch REBP m RNA和蛋白水平都顯著升高(P0.05)。5.2炎性因子下調(diào)FOXO1蛋白表達(dá)接受TNF-α和IL-1β刺激后,各組細(xì)胞FOXO1蛋白表達(dá)量與對照組相比都顯著下降(P0.05)。5.3炎性因子上調(diào)NFκB表達(dá)與對照組相比,TNF-α和IL-1β作用下細(xì)胞NFκB的m RNA水平顯著升高,同時蛋白檢測顯示p-NFκB水平也上升(P0.05)。5.4 NFκB抑制劑PDTC下調(diào)炎性因子誘導(dǎo)的TXNIP和Ch REBP的表達(dá)應(yīng)用20μM PDTC預(yù)處理INS-1細(xì)胞6h后,與炎性因子共同孵育細(xì)胞,PDTC顯著抑制炎性因子TNF-α、IL-1β誘導(dǎo)的TXNIP、Ch REBP的m RNA和蛋白表達(dá)(P0.05),提示炎性因子可能通過調(diào)節(jié)NFκB和Ch REBP來增加TXNIP表達(dá),且NFκB作為上游轉(zhuǎn)錄因子參與調(diào)控Ch REBP。5.5 p38MAPK抑制劑SB203580減弱炎性因子誘導(dǎo)的p-NFκB的激活和TXNIP的表達(dá)應(yīng)用20μM SB203580預(yù)處理INS-1細(xì)胞6h后,發(fā)現(xiàn)炎性因子作用下INS-1細(xì)胞磷酸化NFκB和TXNIP表達(dá)量都顯著降低(P0.05),提示p38MAPK作為炎癥反應(yīng)中的一種重要蛋白,可以通過激活核轉(zhuǎn)錄因子κB來誘導(dǎo)TXNIP改變。結(jié)論:1.炎性因子TNF-α、IL-1β可以誘導(dǎo)INS-1細(xì)胞凋亡。2.炎性因子TNF-α、IL-1β誘導(dǎo)TXNIP表達(dá)與INS-1細(xì)胞凋亡有相關(guān)性。3.炎性因子TNF-α、IL-1β可能通過p38MAPK-NFκB-Ch REBP通路上調(diào)TXNIP的表達(dá)。
[Abstract]:Research background: Diabetes Mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia caused by the combination of genetic and environmental factors. Its essence is insulin secretion and (or) deficiency. The prevalence and prevalence of diabetes in the world are obviously rising, and China is a one. The large population of developing countries has become the world's largest diabetes country. Diabetes is mainly divided into type 1 and type 2. However, no matter which type of diabetes, islet beta cell damage and apoptosis are important causes for the development of diabetes,.TNF- A and IL-1 beta are important mediating factors for chronic systemic inflammation in diabetes. In order to induce islet cell apoptosis, it plays an important role in diabetes. Thioredoxin interacting protein (TXNIP) is the only endogenous thioredoxin (thioredoxin, TRX) binding and inhibitory protein found at present. It can reduce the anti free radical and anti apoptosis function of TRX. All groups in diabetes mellitus The expression of TXNIP was significantly increased. The study found that high sugar could induce a significant increase in the level of TXNIP m RNA and protein in INS-1 cells with obvious apoptosis. This laboratory study confirmed that the expression of TXNIP could induce the apoptosis of INS-1 cells through the endoplasmic reticulum stress pathway involved in caspase-8,9 and PERK. Therefore, TXNIP is considered to be linked to diabetes. The key link of beta cell death. Then, whether the inflammatory factors TNF- alpha and IL-1 beta can affect the expression of TXNIP in INS-1 cells and then affect the apoptosis, and what is the mechanism? The aim of this study is to explore the possible mechanisms of TNF- and IL-1 beta induced TXNIP changes and the possible mechanisms to provide new intervention for the prevention and treatment of diabetes. Target. Objective: to cultivate INS-1 cells under normal sugar and fat concentration, and to give exogenous TNF- alpha (5ng/ml) and IL-1 beta (10ng/ml) to stimulate cell 24h, and observe whether TNF- A and IL-1 beta cause INS-1 cell TXNIP expression changes and study its possible mechanism. Methods: 1. experimental groups: four groups of INS-1 cells cultured under normal glycolipid concentration were divided into normal pairs, respectively. Group TNF- alpha (5ng/ml, 24h), IL-1 beta group (10ng/ml, 24h) and TNF- alpha +IL-1 beta group (5ng/ml+10ng/ml, 24h) were given inflammatory factors TNF- alpha and beta interfering cells. The cell survival rate was detected by flow cytometry and two methods for detecting apoptosis of the cells under the action of alpha and beta. CR method was used to detect TXNIP, NF kappa B and Ch REBP m under the action of TNF- alpha and IL-1 beta. TXNIP, NF kappa B and Ch REBP m RNA and protein expression. Results: the survival rate of 1.TNF- A and IL-1 beta INS-1 cells was reduced by the interference of different concentrations of inflammatory factors in INS-1 cells. 1 beta caused a significant decrease in cell viability at (10ng/ml, 24h). Therefore, the concentration of TNF- alpha and IL-1 beta in the later experiment was 5ng/ml and 10ng/ml respectively. The effect time was 24h.2.TNF- A and IL-1 beta induced INS-1 cell apoptosis by Annexin-V staining flow cytometry. The apoptosis rate of L-1 beta group increased significantly (P0.05), and the expression of cleaved caspase-3 representing apoptosis was also increased in Western blot, suggesting that the inflammatory factor TNF- alpha and IL-1 beta could induce the increase of INS-1 cell apoptosis. The synergistic effect was played when the two combined action, inducing more significant apoptosis.3.TNF- a and IL-1 beta induction. The expression level of TXNIP m RNA was higher than that of the control group, and the expression level of TXNIP m RNA reached the peak at 5ng/ml TNF- A and 10ng/ml IL-1 beta. The results of group (10ng/ml, 24h) and TNF- alpha +IL-1 beta group (5ng/ml+10ng/ml, 24h).Real-time PCR showed that every inflammatory factor intervention group, including TNF- alpha +IL-1 beta group, was significantly higher than the control group. The level of cell TXNIP protein increased significantly (P0.05).5.p-NF kappa B, Ch REBP, FOXO1 and other transcription factors. The 5.1 inflammatory factors up regulation of Ch REBP m RNA and protein levels compared with the control group. After F- alpha and IL-1 beta stimulation, the expression of FOXO1 protein in each group decreased significantly compared with the control group (P0.05).5.3 inflammatory factor up regulation of NF kappa B expression compared with the control group. The NF kappa B M levels increased significantly under the action of TNF- A and IL-1 beta. The expression of factor induced TXNIP and Ch REBP used 20 mu M PDTC to pretreat INS-1 cell 6h and incubate cells with inflammatory factors together. PDTC significantly inhibits the inflammatory factor TNF- alpha, IL-1 beta induced TXNIP. Transcriptional factors involved in the regulation of the activation of p-NF kappa B induced by Ch REBP.5.5 p38MAPK inhibitor SB203580 and the expression of TXNIP in 20 u M SB203580 preprocessing INS-1 cell 6h. An important protein can induce TXNIP change by activating nuclear factor kappa B. Conclusion: 1. inflammatory factor TNF- alpha, IL-1 beta can induce the.2. inflammatory factor TNF- alpha in INS-1 cells. IL-1 beta induced TXNIP expression and INS-1 cell apoptosis are related to.3. inflammatory factor TNF- alpha. Expression.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1

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