NR4A1抵抗胰岛β细胞内质网应激所致细胞凋亡及相关分子机制研究

The mechanism of pancreatic β-cell apoptosis in type 2 diabetes is far from being clarified. The most accepted mechanism for it is that continuous hyperglycemia and/or free fatty acid is able to induce β-cell ER-stress, which will results in β-cell apoptosis if it is not reduced effectively. Research on the mechanism of β- cell apoptosis is to find ways to protect β cell. Our preliminary data showed that β-cell line (MIN6) over-expressing NR4A1 is resistant to apoptosis induced by ER-stress reagents, such as TG or FFA. We propose a hypothesis that NR4A1 up-regulates the expression of one or more anti-apoptosis genes or anti-ER-stress genes, therefore, β-cell apoptosis induced by ER stress is reduced or inhibited. In order to verify the hypothesis, we design these experiments: 1）observe the changes of the UPR related molecules and apoptotic molecules in MIN6 over-expressing NR4A1; 2) observe the proteomics change of β-cell over-expressing NR4A1; 3) Separate and purify β-cells from mouse pancreas, infect these cells with adenovirus encoding NR4A1, and observe these infected cell under drug pressure of TG/TM or FFA; 4) Generate NR4A1 over-expressed and down-expressed mice, observe these two different mice on their endurance on hyperglycemia, high fat diet and ER-stress inducing drug ( TG/TM or FFA).

2型糖尿病中引起胰岛β细胞凋亡的机制尚未揭示清楚。人们普遍接受：持续高糖或游离脂肪酸(FFA)可致β细胞内质网应激(ER-stress)，若不及时消除则可引发细胞凋亡。研究β细胞凋亡机制是为了防止β细胞凋亡。我们前期工作发现：β细胞株(MIN6转染NR4A1分子后，由内质网应激诱导剂(TG，FFA)所致的β细胞凋亡被有效抑制。我们提出假说：NR4A1可上调某个或某些抗凋亡基因及抗内质网应激分子的表达，进而抵抗或抑制内质网应激引起的β细胞凋亡。为求证假说，特设如下研究：1）观察β细胞转基因表达NR4A1后，UPR相关分子及凋亡相关分子的改变；2）观察NR4A1引起的β细胞内蛋白组学的改变；3）分离小鼠β细胞，用腺病毒载体使之表达NR4A1分子，观察其抵抗TG及FFA引发细胞凋亡的情况；4）构建高\低表达NR4A1的转基因动物，观察NR4A1对高糖、高脂饮食及药物(TG\TM，FFA)的抵抗。

研究课题：NR4A1抵抗β细胞内质网应激所致细胞凋亡及 相关机制研究（编号81370881）在实施过程中，取得了很大进展。我们证实，用内质网应激诱导剂毒胡萝卜素（TG）或棕榈酸（PA）处理MIN6细胞和小鼠胰岛可导致NR4A1的mRNA和蛋白质水平增高。MTT结果表明在MIN6细胞中过表达NR4A1能够抵抗由TG或PA诱导的细胞损失；TUNEL实验结果表明，NR4A1过表达也能防止内质网应激诱导的细胞凋亡。利用siRNA敲出MIN6细胞中NR4A1的表达以及利用NR4A1敲除小鼠进一步证实了上述结论。在MIN6细胞中过表达NR4A1可降低TG或PA引起的C/ EBP同源蛋白（CHOP）的表达和Caspase3的活化。在MIN6细胞和小鼠胰岛中过表达NR4A1可导致SURVIVIN表达的上调。我们证实在SURVIVIN的启动子上（-1872bp至-1866bp）有一个NR4A1的结合位点，免疫共沉淀实验表明，NR4A1与SURVIVIN启动子有物理结合。综上所述，NR4A1通过上调SURVIVIN的表达和下调CHOP的表达来保护胰岛β细胞免受内质网应激介导的细胞凋亡.本课题组经4年的努力，顺利完成了课题的各项任务，取得了一些成果。课题组使用本课题的资源完成了7篇SCI文章，而且还会有文章在后续发表。本课题帮助我们培养了5位博士研究生，2位硕士毕业生。总之，本课题在实施过程中，我们收获丰富，既有科研成果，又有培养人才的成绩。