HDACi对炎症因子诱导INS-1细胞凋亡的保护作用及其分子机制研究

发布时间：2018-05-06 07:08

本文选题：Ⅰ型糖尿病 + 细胞因子　；参考：《东北师范大学》2017年硕士论文

【摘要】：Ⅰ型糖尿病以胰岛β细胞损伤及胰岛素绝对缺乏为特点,组蛋白去乙酰化酶抑制剂(HDACi)可以改善胰岛β细胞功能,促进胰岛素合成与释放,减轻炎症反应对胰岛β细胞的损伤。目前,HDACi对炎症因子损伤胰岛细胞研究尚缺乏完善的研究资料,本论文体外建立细胞因子(IL-1β,TNF-α和IFN-γ)所致胰岛细胞的损伤模型,借助分子对接软件筛选出五种异羟肟酸类HDACi,探究其对炎症因子诱导的胰岛细胞凋亡的保护作用,并进行分子机制研究。为进一步的糖尿病药物研发提供了新的候选化合物,为HDACi治疗糖尿病提供理论依据。为了研究细胞因子的促炎症反应中的作用,我们在体外用细胞因子刺激INS-1细胞构建炎症反应模型。首先,我们使用单个细胞因子(IL-1β,TNF-α,IFN-γ)以及细胞因子组合方式作用于INS-1细胞,用MTT实验检测了处理不同时间后的细胞活力的影响,以确定后续研究中细胞因子作用的浓度和时间。然后,通过DAPI染色观察发现细胞因子作用后核小体的数量显著增多,从细胞核形态上说明细胞因子促进了INS-1细胞凋亡。同时,我们也使用流式细胞术检测了细胞因子对INS-1细胞凋亡以及周期的影响,结果显示,细胞因子促进INS-1细胞凋亡并且阻滞周期为S期。接下来,我们根据文献选取17个HDAC抑制剂,通过GOLD5.2分子对接软件将17个HDACi小分子分别与HDAC2,HDAC3,HDAC5,HDAC6,HDAC7,HDAC8活性空腔对接,综合配体和蛋白之间的结合构象以及打分情况来评价分子对接效果,选择了五种HDACi(TSA、SAHA、PXD101、LBH589和PCI-24781)用于体外实验,筛选对细胞因子诱导的INS-1细胞凋亡有保护作用的HDACi。购买五种HDACi,使用一系列浓度梯度作用于INS-1细胞,通过MTT检测这五种HDACi对INS-1细胞活力的影响,并用SPSS软件分析出每种抑制剂的IC10值,确定后续研究中用于保护INS-1细胞的HDACi使用浓度。然后使用无毒剂量的HDACi预处理细胞不同时间后加入细胞因子处理,通过MTT对细胞活力的测定证明组蛋白去乙酰化酶抑制剂TSA、SAHA、PXD101和PCI-24781对炎症因子诱导的INS-1细胞凋亡均有抑制作用。最后我们用Western-blot实验对抑制剂PXD101的保护机制进行了探究。在细胞因子作用下,PXD101能够抑制IκB磷酸化以及p65入核,HDACi可能部分通过抑制细胞因子介导的NF-κB信号通路的激活来降低INS-1细胞的凋亡程度。综上可知,细胞因子能够促进INS-1细胞凋亡,异羟肟酸类组蛋白去乙酰化酶抑制剂TSA、SAHA、PXD101和PCI-24781能够保护INS-1细胞抑制细胞凋亡的发生,其中PXD101的保护作用部分是通过调节NF-κB信号通路来实现的。因此,HDACi有希望成为以HDACs为治疗靶点的潜在的Ⅰ型糖尿病治疗药物。
[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- 伪 and IFN- 纬 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- 伪 IFN- 纬) 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.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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