核受体NR2E1及其相关基因调控胰岛β细胞衰老和损伤修复的机制研究

Regeneration and restore differentiation is fundamental treatment for diabetes to rescue the number of islet β cells. Nuclear receptor NR2E1 can regulate the self-repair and proliferation of neural stem cells and retinal precursor cells. The development of nervous system is similar to that of endocrine system, thus it is possible that NR2E1 is also involved the proliferation and repair of islet β cells and its precursor cells. The preliminary results suggest that the expression of NR2E1 was detected in islet β cells and increased under the condition of high palmitate for compensation. In addition, the body weight and fat pad of NR2E1 knockout mice were reduced compared to control. Base on these results, it could be concluded that NR2E1 may regulate energy metabolism and restore function of islet β cells. However, the mechanism is not clear. we not only plan to use NR2E1 knockout mice, wild type mice,primary cells and islet cell line MIN6 as models,but adopt molecular and cell biological methods and rescue experiment to elaborate the role of NR2E1 in mouse pancreatic beta cell aging and glycolipotoxic damage, insulin secretion, proliferation and apoptosis, glucose and lipid metabolism in vivo and in vitro through upregulation or downregulation of NR2E1. The project may provide target for protecting the islet β cells against aging and glucolipotoxicity mediated dysfunction. Morerver,it is possible to find a new therapy target for type 2 diabetes.

再生和恢复去分化胰岛β细胞的分化能力是2型糖尿病治疗的根本。前期结果证实胰岛β细胞存在NR2E1表达，同时高脂条件下其代偿性表达增加。敲除NR2E1的小鼠体重和脂肪垫的含量较对照明显减少，而且能够调控干细胞和前体细胞的增殖和自我修复，提示NR2E1非常可能参与调控能量代谢和胰岛β细胞损伤后再生，但是其具体机制尚不明确。我们拟采用敲除NR2E1小鼠和野生型小鼠作为模型，借助原代细胞、胰岛细胞系培养和EMSA、染色质免疫共沉淀等细胞分子生物学技术及rescue实验等，从体内体外两个方面通过上调或下调NR2E1表达来阐述NR2E1在小鼠胰岛β细胞衰老和糖脂毒性损伤后的修复、去分化、再生、分泌、增殖凋亡及糖脂代谢中的调控机制，从而为衰老及糖脂毒性损伤后胰岛β细胞修复再生提供靶目标。

根据我们的研究结果显示，NR2E1在胰岛β细胞功能再生修复中起到重要作用。因此，我们采用敲除NR2E1小鼠和野生型小鼠作为模型，借助原代细胞和胰岛细胞系培养以及分子生物学、rescue实验、细胞生物学技术，通过上调或下调NR2E1表达从体内体外两个方面来阐述NR2E1在小鼠胰岛β细胞衰老和糖脂毒性损伤后的修复再生、分泌、增殖凋亡、糖脂代谢中的调控机制。我们的研究结果发现，与野生型小鼠相比，NR2E1-/-小鼠的血糖水平升高，葡萄糖负荷后的胰岛素分泌水平降低。血液及胰腺组织的氧化应激标志物包括谷胱甘肽（GSH）以及超氧化物歧化酶（SOD）、丙二醛（MDA）等水平升高。胰腺干细胞标记物nestin、 Pdx-1、Pax6等表达水平下降。免疫荧光分析和共聚焦显微镜显示NR2E1-/-小鼠β细胞衰老相关基因p16INK4a、Ezh2、PDGFR-α、p38、H3K4me3、SIRT1等表达升高，胰岛阳性细胞的增殖减少，凋亡增加。分离NR2E1-/-小鼠β细胞进行NR2E1过表达后，β细胞衰老相关基因p16INK4a、Ezh2、PDGFR-α、p38、H3K4me3、SIRT1等表达降低，凋亡减少，增殖增加。在糖尿病发展过程中，NR2E1-/-小鼠会出现更快进程的血糖升高和更多的胰岛β细胞凋亡。过表达NR2E1的MIN6细胞，在高糖，高脂的刺激下，胰岛素分泌相关的基因表达上调，细胞的增殖较空白对照组增加，凋亡减少。氧化应激标志物表达水平降低。荧光素酶活性分析提示，NR2E1可以结合PDGFRα启动区，提高PDGFRα转录活性，并通过这种机制调控β细胞的增殖活性。我们的研究结果提示，NR2E1是一种重要的胰岛β细胞转化和增殖调控因子，在2型糖尿病的发生过程中起到重要的保护性作用。本研究结果为进一步开发新的糖尿病治疗靶位提供了重要依据。