本文選題：Ⅰ型糖尿病 + 細(xì)胞因子��； 參考：《東北師范大學(xué)》2017年碩士論文

【摘要】：Ⅰ型糖尿病以胰島β細(xì)胞損傷及胰島素絕對缺乏為特點(diǎn),組蛋白去乙�；敢种苿�(HDACi)可以改善胰島β細(xì)胞功能,促進(jìn)胰島素合成與釋放,減輕炎癥反應(yīng)對胰島β細(xì)胞的損傷。目前,HDACi對炎癥因子損傷胰島細(xì)胞研究尚缺乏完善的研究資料,本論文體外建立細(xì)胞因子(IL-1β,TNF-α和IFN-γ)所致胰島細(xì)胞的損傷模型,借助分子對接軟件篩選出五種異羥肟酸類HDACi,探究其對炎癥因子誘導(dǎo)的胰島細(xì)胞凋亡的保護(hù)作用,并進(jìn)行分子機(jī)制研究。為進(jìn)一步的糖尿病藥物研發(fā)提供了新的候選化合物,為HDACi治療糖尿病提供理論依據(jù)。為了研究細(xì)胞因子的促炎癥反應(yīng)中的作用,我們在體外用細(xì)胞因子刺激INS-1細(xì)胞構(gòu)建炎癥反應(yīng)模型。首先,我們使用單個細(xì)胞因子(IL-1β,TNF-α,IFN-γ)以及細(xì)胞因子組合方式作用于INS-1細(xì)胞,用MTT實(shí)驗(yàn)檢測了處理不同時間后的細(xì)胞活力的影響,以確定后續(xù)研究中細(xì)胞因子作用的濃度和時間。然后,通過DAPI染色觀察發(fā)現(xiàn)細(xì)胞因子作用后核小體的數(shù)量顯著增多,從細(xì)胞核形態(tài)上說明細(xì)胞因子促進(jìn)了INS-1細(xì)胞凋亡。同時,我們也使用流式細(xì)胞術(shù)檢測了細(xì)胞因子對INS-1細(xì)胞凋亡以及周期的影響,結(jié)果顯示,細(xì)胞因子促進(jìn)INS-1細(xì)胞凋亡并且阻滯周期為S期。接下來,我們根據(jù)文獻(xiàn)選取17個HDAC抑制劑,通過GOLD5.2分子對接軟件將17個HDACi小分子分別與HDAC2,HDAC3,HDAC5,HDAC6,HDAC7,HDAC8活性空腔對接,綜合配體和蛋白之間的結(jié)合構(gòu)象以及打分情況來評價分子對接效果,選擇了五種HDACi(TSA、SAHA、PXD101、LBH589和PCI-24781)用于體外實(shí)驗(yàn),篩選對細(xì)胞因子誘導(dǎo)的INS-1細(xì)胞凋亡有保護(hù)作用的HDACi。購買五種HDACi,使用一系列濃度梯度作用于INS-1細(xì)胞,通過MTT檢測這五種HDACi對INS-1細(xì)胞活力的影響,并用SPSS軟件分析出每種抑制劑的IC10值,確定后續(xù)研究中用于保護(hù)INS-1細(xì)胞的HDACi使用濃度。然后使用無毒劑量的HDACi預(yù)處理細(xì)胞不同時間后加入細(xì)胞因子處理,通過MTT對細(xì)胞活力的測定證明組蛋白去乙酰化酶抑制劑TSA、SAHA、PXD101和PCI-24781對炎癥因子誘導(dǎo)的INS-1細(xì)胞凋亡均有抑制作用。最后我們用Western-blot實(shí)驗(yàn)對抑制劑PXD101的保護(hù)機(jī)制進(jìn)行了探究。在細(xì)胞因子作用下,PXD101能夠抑制IκB磷酸化以及p65入核,HDACi可能部分通過抑制細(xì)胞因子介導(dǎo)的NF-κB信號通路的激活來降低INS-1細(xì)胞的凋亡程度。綜上可知,細(xì)胞因子能夠促進(jìn)INS-1細(xì)胞凋亡,異羥肟酸類組蛋白去乙酰化酶抑制劑TSA、SAHA、PXD101和PCI-24781能夠保護(hù)INS-1細(xì)胞抑制細(xì)胞凋亡的發(fā)生,其中PXD101的保護(hù)作用部分是通過調(diào)節(jié)NF-κB信號通路來實(shí)現(xiàn)的。因此,HDACi有希望成為以HDACs為治療靶點(diǎn)的潛在的Ⅰ型糖尿病治療藥物。
[Abstract]:Type I diabetes mellitus is characterized by islet 尾 cell injury and absolute insulin deficiency. Histone deacetylase inhibitor (HDACii) can improve the function of islet 尾 cells, promote insulin synthesis and release, and alleviate the damage of inflammatory reaction to islet 尾 cells. At present, HDACi is still lack of perfect research data for the study of inflammatory cytokine injury of islet cells. In this paper, the model of islet cell injury induced by cytokine IL-1 尾 -TNF- 偽 and IFN- 緯 was established in vitro. Five kinds of hydroxamic acids HDACii were screened by molecular docking software to investigate the protective effect of HDACie on the apoptosis of islet cells induced by inflammatory cytokines and to study the molecular mechanism. It provides a new candidate compound for the further development of diabetes drugs and provides theoretical basis for the treatment of diabetes with HDACi. In order to study the role of cytokines in promoting inflammation, we used cytokines to stimulate INS-1 cells in vitro to construct inflammatory response models. First, we used single cytokine IL-1 尾 TNF- 偽 IFN- 緯) and cytokine combination to act on INS-1 cells. The effects of different treatment time on cell viability were detected by MTT experiment to determine the concentration and time of cytokine action in subsequent studies. Then, DAPI staining showed that the number of nucleosomes increased significantly after the action of cytokines, which indicated that cytokines promoted the apoptosis of INS-1 cells. The effects of cytokines on apoptosis and cell cycle of INS-1 cells were also detected by flow cytometry. The results showed that cytokines promoted apoptosis of INS-1 cells and blocked the cell cycle in S phase. Next, we selected 17 HDAC inhibitors according to the literature, and docked 17 HDACi small molecules with HDAC2HDAC3, HDAC5, HDAC7, HDAC8 active cavity by GOLD5.2 molecular docking software, and evaluated the molecular docking effect by combining the conformation and scoring between ligand and protein. Five kinds of HDACiTSAHAA PXD101 LBH589 and PCI-24781) were selected to screen HDACie which have protective effect on cytokine induced apoptosis of INS-1 cells. Five HDACis were purchased and treated with a series of concentration gradients on INS-1 cells. The effects of the five HDACi on the viability of INS-1 cells were detected by MTT, and the IC10 values of each inhibitor were analyzed by SPSS software. Determine the concentration of HDACi used to protect INS-1 cells in subsequent studies. Then the cells were pretreated with nontoxic dose of HDACi for different time and then treated with cytokines. The results showed that the histone deacetylase inhibitor TSASAHAHAPXD101 and PCI-24781 could inhibit the apoptosis of INS-1 cells induced by inflammatory factors. Finally, we use Western-blot experiment to explore the protective mechanism of inhibitor PXD101. PXD101 can inhibit the phosphorylation of I 魏 B and the activation of NF- 魏 B signaling pathway mediated by p65, which may reduce the degree of apoptosis of INS-1 cells by inhibiting the activation of NF- 魏 B signaling pathway mediated by cytokines. In conclusion, cytokines can promote apoptosis of INS-1 cells, and hydroxamic acid histone deacetylase inhibitor TSASAHAA PXD101 and PCI-24781 can protect INS-1 cells from apoptosis. The protective effect of PXD101 is partly achieved by regulating NF- 魏 B signaling pathway. Therefore, HDACi may become a potential type 1 diabetes drug targeting HDACs.
【學(xué)位授予單位】：東北師范大學(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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