糖尿病溃疡患者血清诱导组蛋白H3K27甲基化修饰失衡损伤血管内皮的表观调控机制

The microenvironment of serum from diabetic ulcer patients (diabetic ulcer serum) , consisted of high level of blood glucose, advanced glycation end products (AGEs) and inflammatory mediators, induced endothelial injury. We found that diabetic ulcer serum caused aging and cell cycle block in human umbilical vein endothelial Cells (HUVECs). In addition, endothelial cells were found distributing disorderly in the diabetic ulcer tissues without forming lumen and the underlying mechanisms remain unknown. After we reported that in LPS-induced H3K27me3 demethylation in HUVECs, and this reaction promoted the release of pro-inflammatory factors which resulted in the endotheliocytes injury. We observed that H3K27me3 demethylated significantly in HUVECs induced by diabetic serum, promoting the release of inflammatory mediators , lead to the injury of the endotheliocytes. Based on the advantage of epigenetic regulation which possesses the feature of both heritability and reversibility, and by using the ex vivo skin tissue culture which mimics the in vivo condition as well as routine in vitro endothelia cell culture, we plan to explore the unbalance of H3K27 demethylation and the expression and activation of molecules involved in the survive, differentiation and angiopoiesis of endothelial cells induced by diabetic ulcer. By applying the small molecular inhibitors, regulating the methylate status reversibly to relieve the endothelial cell injury caused by the diabetic ulcer serum. And all these would provide new strategy to prevent and cure the complication of diabetes mellitus.

糖尿病溃疡患者血清中存在的高血糖、终末糖基化产物及炎性介质等构成损害血管内皮细胞功能的微环境。课题组发现糖尿病溃疡患者血清可显著诱导人脐静脉内皮细胞衰老和周期阻滞，且与正常组织相比糖尿病溃疡组织中大量血管内皮细胞无序存在，不能形成血管管腔，其机制尚不明确。继报道内毒素诱导血管内皮细胞中组蛋白H3K27me3去甲基化而释放促炎细胞因子损伤内皮细胞后，课题组发现糖尿病溃疡血清显著促进血管内皮细胞H3K27me3去甲基化，释放促炎细胞因子损伤内皮细胞。基于表观遗传调控兼有可遗传性和可逆性优势，本项目拟运用ex vivo组织培养模拟体内环境结合常规体外细胞培养,研究糖尿病溃疡血清引起内皮细胞H3K27甲基化修饰失衡、干扰内皮细胞存活、分化和血管形成相关基因和信号分子序贯表达及活化的分子途径，运用小分子抑制剂可逆性调控H3K27甲基化修饰水平以减轻血清微环境对血管的损伤，为糖尿病并发症防治提供策略。

糖尿病创面迁延难愈，血管内皮损伤在其中起重要作用。我们发现糖尿病溃疡患者血清可显著诱导人脐静脉内皮细胞衰老和周期阻滞，且与正常组织相比糖尿病创面组织中大量血管内皮细胞无序存在，不能形成血管管腔，同时也发现患者血清显著促进血管内皮细胞H3K27me3去甲基化。我们应用ChIP-seq技术联合转录组测序技术，发现有201个差异基因的基因启动子区域H3K27me3富集水平发生变化，将这201差异基因与凋亡/血管形成相关基因进行匹对，发现有31个基因与血管形成的调控有关，有49个基因与细胞凋亡的调控有关，其中有19个共有基因。全基因组功能区域差异 peak相对应基因富集分析表明H3K27ac全基因组功能区域差异peak相对应基因主要参与Metabolic pathways, Ribosome 和Viral carcinogenesis上。通过ex vivo组织培养模拟体内环境结合常规体外细胞培养,证实患者血清引起内皮细胞H3K27甲基化修饰失衡、促进内皮细胞促炎基因和信号分子的表达，运用小分子抑制剂可逆性调控H3K27甲基化修饰水平抑制促炎细胞因子, ET-1及RAGE基因的表达以减轻血清微环境对血管的损伤。除完成原实验计划外，我们还证实糖尿病创面患者血清中存在参与ECM受体相互作用 、补体及凝血级联途径的差异蛋白质，这些分子可能是造成血管内皮损伤的始动因子，亦可能是内皮损伤的产物，仍有待进一步研究。本项目成果为研究糖尿病并发症防治提供新策略。