DNMT1介导高血糖“代谢记忆”对糖尿病难愈创面的作用机理研究

我们前期研究显示，糖尿病微血管病变及创面愈合存在“代谢记忆”现象，即糖尿病患者血糖即使控制正常，微血管损伤仍持续发展、创面难愈。我们后续基因表达谱及甲基化芯片分析示，高血糖可激活DNMT1表达，导致ANG1启动子甲基化程度升高、表达降低，继而激活NF-κB通路而造成微血管损伤。由此我们推测：DNMT1/Ang-1/NF-κB信号网络可能是调控高血糖“代谢记忆”及创面难愈的重要机制。本项目拟：（1）采用硫化测序PCR、启动子甲基化荧光素酶报告、慢病毒双向调控及转基因小鼠等技术，在体内、外逐级纵向阶梯式验证DNMT1/Ang-1/NF-κB调控高血糖“代谢记忆”及创面愈合的作用；（2）利用“代谢记忆”动物模型验证单独/联合应用去甲基化药物、DNMT1-DsiRNA、cAng-1及NF-κB抑制剂对治疗糖尿病难愈创面的临床价值。本项目的完成，为糖尿病难愈创面的防治提供理论基础和新的治疗靶点。

Our previous studies have shown that there is a specially phenomenon called hyperglycemia “metabolic memory” that exists in the diabetic cardiovascular complications and wound healing, that is: even though the blood glucose of diabetic is controlled in normal levels, the cardiovascular complications still keep on deteriorate, which eventually lead to delayed wound healing. Our following relational analysis of whole genome microarrays combined with methylation chip showed that transient hyperglycemia activated the expression of DNMT1, which increased the methylation level of promoters of ANG1, thus decreased the expression of Ang-1, leading to the activation of NF-κB and the following microvascular injury and delayed wound healing. Based on the above results, we speculated that: DNMT1/Ang-1/NF-κB signaling pathway is the potential mechanism in regulating delayed wound healing that induced by hyperglycemia “metabolic memory”. In this project, our plan for the further research is: (1) By using molecular biological technologies, including bisulfite sequencing PCR (BSP), promoter methylation luciferase reporter gene assay system, lentiviral vector-inducd dual regulation assay, and transgenic mice. et al, we plan to further demonstrate step by step the function of DNMT1/Ang-1/NF-κB signaling pathway on regulating diabetic "metabolic memory" and the following microangiopathy in vitro and in vivo; (2) By establishing the animal model that mimic diabetic "metabolic memory" situation, we will determine the clinical therapeutic value of separated/combined applications of demethylation agents, DNMT1-DsiRNA, cAng-1, and NF-κB pathway inhibitor in the treatment of diabetic microangiopathy and the diabetic delayed wound healing. The meaningful results of this study give us new ideas for the prevention and comprehensive therapy of diabetic microangiopathy and delayed would healing, providing novel strategies for achieving a better recover of diabetes.