细胞內腺苷通过表观遗传学修饰调控胰岛β细胞增殖凋亡的机制研究

Islet β cell dysfunction is the core of the development of diabetes, but there still lacks effective means to reverse its progressive failure. Recently, studies show that inhibition of adenosine kinase(ADK) can significantly promote β cell proliferation by increasing intracellular adenosine, however the mechanism is unknown. Our previous studies revealed that ADK inhibitors can promote β cell proliferation and inhibit its apoptosis, the specific mechanism may be related to DNA methylation of PDX-1 promoter. Accordingly, we hypothesize that intracellular adenosine may regulate the expression of key genes in β cell proliferation and apoptosis through epigenetic modification, thereby regulating the β cell mass. The present study intends to verify that intracellular adenosine regulates islet β cell proliferation and apoptosis through methylation modification. Specific contents include: to observe the changes of adenosine pathway in diabetic mice; To regulate the endogenous adenosine by means of gene expression regulation, intending to observe the expression and activity of key enzymes in methylation as well as the changes of methylation and expression of key genes in proliferation and apoptosis in vivo and in vitro; Through up-regulation / down-regulation of the key enzymes of methylation to confirm the regulation of adenosine on the methylation level is the key to control the proliferation of β cells; Based on this, adenosine receptor knockout and nucleoside transporter inhibitors will be used to study whether the promoted effect of intracellular adenosine is receptor dependent. The results are expected to provide new ideas for the prevention and treatment of diabetes.

胰岛β细胞功能障碍是糖尿病发生发展的核心环节，但目前仍缺乏有效手段逆转其进行性衰竭。研究显示抑制腺苷激酶（ADK）以增加内源性腺苷可显著促进β细胞增殖，但机制不明。我们前期发现：ADK抑制剂可促进β细胞增殖并抑制其凋亡，其机制可能与调节PDX-1启动子区DNA甲基化有关。故此，我们推测细胞內腺苷通过甲基化修饰调控PDX-1等关键基因表达，从而调节β细胞增殖凋亡。为证明推测，本研究拟观察糖尿病模型鼠病程中腺苷通路的变化；通过基因表达调控等手段调节内源性腺苷含量，在大体及细胞水平观察甲基化关键酶的表达及活性、增殖凋亡关键基因甲基化修饰及表达水平的改变；针对性上调／下调甲基化关键酶，以证实腺苷对甲基化水平的调节是调控β细胞增殖凋亡的关键；在此基础上运用腺苷受体敲除和核苷转运子抑制剂，研究细胞內腺苷的促增殖效应是否为受体依赖性。研究结果有望为糖尿病防治提供新的思路。

胰岛β细胞功能障碍是糖尿病发生发展的核心环节，但目前仍缺乏有效手段逆转其进行性衰竭。腺苷通路从多种途径参与细胞代谢调节，细胞内腺苷水平主要受腺苷激酶（ADK）调控，研究显示ADK抑制剂可显著促进β细胞增殖。ADK与基因和蛋白的甲基化修饰的密切关系已被多个研究证实。有研究观察到糖尿病患者胰岛整体甲基化水平上升，PDX-1启动子区域甲基化水平上调，mRNA水平下调。结合以上，我们认为细胞內腺苷通过甲基化修饰调控PDX-1等关键基因表达，从而调节β细胞增殖凋亡。经一系列实验，我们发现： （1）胰腺组织ADK分布存在种属差异，人类胰腺组织ADK主要表达于胰岛，α和β细胞中均有表达，胰岛区域高于胰腺外分泌组织；小鼠胰腺ADK表达量近似肝脏，高于脂肪组织、脾、肺、肾，胰腺外分泌腺ADK表达量显著高于胰岛。；（2）ADK在人类胰岛素瘤患者过度增殖的瘤体部位低表达，而在瘤周胰岛β细胞胞质中表达高于瘤体，在经典增殖模型胰腺部分切除小鼠中，也观察到Px（partial pancreatectomy，胰腺部分切除）组小鼠胰岛ADK表达较Sham（假手术）组下降。（3）ADKβ细胞选择性过表达，导致胰岛分泌功能受损，糖耐量异常，β细胞增殖受抑制（4）构建ADK过表达小鼠胰岛素瘤细胞系beta-TC-6，蛋白组学检测显示相较于对照组，ADK过表达的β细胞增殖、胰岛素分泌、葡萄糖代谢相关分子表达有明显差异；部分胰腺β增殖凋亡相关的蛋白水平发生变化；（5）机制探索：ADK过表达β细胞系中观察到PDX-1表达下调，其机制可能与转甲基酶Dnmt3a水平明显上调引起的PDX-1启动子区甲基化修饰水平变化相关，AKT磷酸化水平的改变也被观察到。结论：ADK在多个β细胞增殖模型中表达降低，ADK过表达β细胞系和ADKβ细胞特异性过表达转基因小鼠中均观察到β细胞增殖受抑制，胰岛素分泌功能受损和糖耐量异常 ，提示ADK在β细胞增殖和胰岛素分泌过程中起负向调节作用。其机制与DNA转甲基酶Dnmt3a介导的PDX-1基因启动子区甲基化水平改变相关。