孤儿核受体NR2E1在2型糖尿病发病机制中的研究

发布时间：2018-05-02 01:04

本文选题：2型糖尿病 + 炎症　；参考：《武汉大学》2015年博士论文

【摘要】：目的：探讨孤儿核受体NR2E1在新诊断2型糖尿病患者的外周血单个核细胞中的表达。研究NR2E1对胰岛β细胞增殖凋亡的影响及高糖条件下NR2E1对胰岛p细胞损伤的调控机制。方法：研究对象分为糖尿病组(54名新诊断2型糖尿病患者)和对照组(88名健康志愿者)。测定研究对象身高、体重,抽取空腹静脉血测定血浆游离脂肪酸(FFAs)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-c)、低密度脂蛋白(LDL-c)、空腹血浆胰岛素(FIN)、空腹血糖(FBG)等临床指标,采用稳态模型评估胰岛素抵抗指数(HOMA-IR),分离外周血单个核细胞(PBMCs)实时荧光定量RT-PCR检测NR2E1mRNA 水平,V Western blotting检测NR2E1蛋白水平,ELISA检测肿瘤坏死因子α(TNF-α)、白介素6(IL-6)表达水平；健康志愿者外周血PBMCs分别给与0、16.7mmol/L葡萄糖、250μmol/L棕榈酸刺激后培养24h, Western blot检测NR2E1蛋白水平,ELISA检测上清中TNF-α、IL-6表达水平。分析糖尿病患者NR2E1表达水平和其他变量的相关性。建立慢病毒包装的过表达NR2E1载体,转染小鼠胰岛系MIN6细胞,利用MTT法、RT-PCR、Western-blot、流式细胞仪等检测NR2E1对胰岛β细胞增殖活力、细胞周期、细胞凋亡等的调控机制。在高糖(25mmol/L葡萄糖)条件下,通过慢病毒包装的过表达载体和RNAi (RNA interference)方法来调节NR2E1的表达,并利用MTT、Western-blot、流式细胞仪、实时定量PCR等方法来检测改变NR2E1表达水平对细胞增殖活力、细胞周期、凋亡、胰岛素分泌相关基因的影响。结果：54例糖尿病组与88例对照组间年龄、性别、身高、体重、TC、LDL-c、ALT无统计学差异(均P0.05),糖尿病组HDL-c较对照组明显降低(P0.01),FIN、FBG、TG、HOMA-IR、FFAs、TNF-α、IL-6表达较对照组明显升高(均P0.01)；糖尿病组NR2E1表达较对照组明显增加(P0.01),且糖尿病组NR2E1mRNA与FIN、FBG以及TNF-α、IL-6呈明显正相关(P0.01)。体外培养PBMCs经16.7mmol/L葡萄糖、250μmol/L棕榈酸刺激后NR2E1、TNF-α、IL-6表达较空白对照组明显升高(P0.01)。通过在MIN6细胞中过表达NR2E1,发现NR2E1能够通过增加细胞周期S期的比例及增加PCNA(Proliferating cell nuclear antigen)基因表达来提高MIN6细胞增殖活力,同时过表达NR2E1可以增加凋亡相关Bcl-2蛋白的表达以及减少ERK的磷酸化水平,但是过表达NR2E1的MIN6细胞的凋亡率无明显改变,与胰岛素分泌相关基因PDX-1表达无明显改变。在高糖条件下,过表达NR2E1能够减少高糖诱导的胰岛p细胞凋亡率,提高胰岛p细胞的增殖活力,同时减少细胞增殖凋亡相关的信号通路JNK(c-Jun N-terminal kinases)以及ERK磷酸化水平,增加Bcl-2表达,抑制Bax表达；过表达NR2E1能够增加胰岛素分泌相关基因PDX-1表达。相反当利用RNAi下调NR2E1表达时可增加高糖对胰岛p细胞MIN6损伤。结论：在新诊断2型糖尿病中,NR2E1的升高可能与糖脂毒性及2型糖尿病患者体内炎症状态有关。NR2E1参与了胰岛β细胞增殖凋亡。NR2E1参与了胰岛p细胞糖毒性损伤的调控,过表达NR2E1能够改善胰岛p细胞糖毒性损伤。
[Abstract]:Objective: To investigate the expression of orphan nuclear receptor NR2E1 in peripheral blood mononuclear cells of newly diagnosed type 2 diabetic patients. The effect of NR2E1 on the proliferation and apoptosis of islet beta cells and the regulation mechanism of NR2E1 on islet P cell damage under high glucose conditions. Methods: the subjects were divided into diabetes group (54 newly diagnosed type 2 diabetes patients) and control. Group (88 healthy volunteers). Determine the height and body weight of the subjects, determine the plasma free fatty acid (FFAs), triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL-c), low density lipoprotein (LDL-c), fasting plasma islet (FIN) and fasting blood glucose (FBG), and evaluate insulin resistance by steady state model. Index (HOMA-IR), separate peripheral blood mononuclear cells (PBMCs) real-time fluorescence quantitative RT-PCR detection NR2E1mRNA level, V Western blotting detection NR2E1 protein level, ELISA detection of tumor necrosis factor alpha (TNF- alpha), interleukins 6 (IL-6) expression level; healthy volunteers peripheral blood PBMCs, respectively, glucose, 250 Mu palmitate acid thorn After stimulated 24h, Western blot detection of NR2E1 protein level, ELISA detection of TNF- alpha and IL-6 expression level in the supernatant, the correlation between the level of NR2E1 expression and other variables in diabetic patients was analyzed. The overexpressed NR2E1 carrier in the lentivirus package was established, and the mouse islet MIN6 cells were transfected, and the MTT method, RT-PCR, flow cytometry and so on were detected. The regulatory mechanism of 2E1 on the proliferation activity, cell cycle and apoptosis of islet beta cells. Under the condition of high glucose (25mmol/L glucose), the expression of NR2E1 was regulated by the overexpressed vector packed with lentivirus and RNAi (RNA interference) method, and the MTT, Western-blot, flow cytometry and real-time quantitative PCR were used to detect the change of the NR2E1 table. Results: there was no significant difference in age, sex, height, weight, TC, LDL-c, ALT (all P0.05) between 54 diabetic and 88 control groups, and HDL-c in diabetic group was significantly lower than that of the control group (P0.01), FIN, FBG, TG, HOMA-IR, FFAs, TNF- a The NR2E1 expression in the diabetic group was significantly higher than that in the control group (P0.01), and the NR2E1mRNA in the diabetic group was significantly positively correlated with FIN, FBG and TNF- alpha (P0.01). In vitro culture, PBMCs was stimulated by 16.7mmol/L glucose and 250 micron mol/L palmitic acid was stimulated, and the expression was significantly higher than that in the blank control group. Over expression of NR2E1 in MIN6 cells, it is found that NR2E1 can increase the proliferation activity of MIN6 cells by increasing the proportion of cell cycle S and increasing the expression of PCNA (Proliferating cell nuclear antigen) gene, while overexpression NR2E1 can increase the expression of apoptotic related Bcl-2 proteins and reduce the phosphorylation level of the MIN6, but overexpressed There was no obvious change in the apoptosis rate of MIN6 cells and the expression of PDX-1 expression related to insulin secretion. Under high glucose conditions, overexpression of NR2E1 could reduce the apoptosis rate of high glucose induced islet P cells, increase the proliferation activity of islet P cells, and reduce the signal pathway JNK (c-Jun N-terminal kinases) of cell proliferation and apoptosis phase (c-Jun N-terminal kinases). ERK phosphorylation level, increase the expression of Bcl-2, inhibit Bax expression, overexpression of NR2E1 can increase the expression of insulin secreting related gene PDX-1. Conversely, RNAi can increase the MIN6 damage to islet P cells when RNAi downregulation NR2E1 expression. Conclusion: in the newly diagnosed type 2 diabetes, the increase of NR2E1 may be associated with the toxicity of glycolipid and the body of type 2 diabetic patients. .NR2E1 participates in the proliferation and apoptosis of islet beta cells involved in the regulation of pancreatic islet P cell glucose toxicity, and the overexpression of NR2E1 can improve the glucose toxicity of pancreatic islet P cells.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.1

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