Cdk5活性抑制肽（TFP5）以CDK5为靶点治疗2型糖尿病的机制的研究

The pathophysiological basis of diabetes mellitus (DM) is islet beta cell damage and decrease in the number. In type 2 diabetes (T2DM), the islet beta cells will be under a chronic progressive processing, in which cells will be proliferation and apoptosis repeatedly, and finally, become irreversibly damaged and apoptosis in long-term high glucose stimulation. The mechanism of islet beta cell apoptosis includes reactive oxygen species (ROS) increased, mitochondrial damage, fat toxicity, toxicity of higher levels of inflammatory factors, glucose toxicity etc. In recent years, the studies found that the cell cycle dependent protein kinase 5 (Cdk5), and It's activator p35 have expressed in islet beta cells and play a role in regulating insulin secretion and in islet beta cell injury. Our previous studies found that chronic toxicity of high glucose may induce pancreatic islet cells produce p25 which causes abnormal Cdk5 activity, leads to increase islet βcell apoptosis, and reduces the secretion of insulin. But the mechanism keeps unknown. Our study also revealed that Cdk5 inhibitory peptides, CIP, can inhibit p25/Cdk5 abnormal activity induced by high glucose, reduce apoptosis of pancreatic islet beta cells, and restore the secretion of insulin. At the same time, CIP does not affect the activation of endogenous Cdk5/ p35 and other Cdks. Therefore, CIP may become a new way for the therapy of type 2 diabetes. However, CIP molecular weight is too big. It is difficult to achieve within the cell biology effect of concentration and also will be more side effects. In order to find smaller fragments, we further cloned a smaller fragment which is truncated from the CIP containing 24 amino acid peptide named as p5. We connected p5 with FITC - TAT tag and synthesis a short peptide, TFP5. Our previous studies have showed that both p5 and TFP5 has better Cdk5 inhibitory function than CIP and has the therapeutic effects in degenerative neurons and mouse brain. In this project, our purpose is focus on studying the mechanism of p25/Cdk5 inducing islet beta cell injury and the therapeutic effects of TFP5 in T2DM by both in vitro and in vivo studies, and in turn, to exploring the new therapeutic method for T2DM treatment targeting on Cdk5.

胰岛β细胞损伤功能障碍是糖尿病（DM）的病理生理基础。T2DM时在高糖刺激下，胰岛β细胞反复凋亡、增生形成慢性进行性不可逆的损伤。活性氧（ROS）增多、线粒体损伤及炎症因子是主要原因。我们的研究发现高糖诱发β细胞产生p25，p25/Cdk5异常活性导致细胞凋亡增加，胰岛素分泌减少。但p25/Cdk5在β细胞损伤中的机制如何？是否与上述的原因相关？尚鲜为人知。前期研究还揭示了Cdk5抑制肽，CIP可特异性抑制p25/Cdk5活性，减少β细胞凋亡，恢复胰岛素的分泌。可能成为治疗2型糖尿病的新途径。但CIP分子量过大，难以达到细胞内有效浓度、毒副作用大。我们发现一个分子量更小，更有效的Cdk5抑制短肽（TFP5）。本课题将进一步研究p25/Cdk5致胰岛β细胞损伤的机制；应用体内外实验观察TFP5在干预胰岛β细胞损伤中的作用和机制，旨在以Cdk5为靶点，寻找治疗2型糖尿病的新途径。

胰岛β细胞损伤功能障碍是糖尿病（DM）的病理生理基础。在高糖刺激下，胰岛β 细胞反复凋亡、增生形成慢性进行性不可逆的损伤。活性氧（ROS）增多、线粒体损伤及炎症因子是主要原因。我们的研究发现 Cdk5抑制短肽（TFP5）可以抑制由p25/Cdk5引起的胰岛β细胞损伤，故应用体内外实验观察TFP5在干预胰岛β 细胞损伤中存在的作用和机制，在以Cdk5为靶点，寻找治疗2型糖尿病的新途径。.具体结果如下：1.明确前期研究Cdk5抑制短肽-CIP在慢性葡萄糖毒性试验中抑制p25/Cdk5活性和防止胰岛β细胞凋亡的因果关系，进一步完成TFP5在慢性葡萄糖毒性体外实验，证实了慢性葡萄糖毒性可诱导细胞产生内源性p25, 而TFP5可抑制p25引起的Cdk5过度活性，抑制Cdk5过度活化引起的tau蛋白过度磷酸化，同时，TFP5从氧化应激中拯救了葡萄糖毒性细胞，减少了细胞培养物中由高葡萄糖诱导的炎性细胞因子的增加，改善高糖诱导的细胞凋亡，拯救了葡萄糖毒性细胞死亡（在胰岛β 细胞，足细胞及神经元中都得到以上结果）。2.应用db/db小鼠模型完成TFP5抑制的体内实验，P25在db/db 小鼠胰腺胰小岛细胞中的表达及CDK5的过度活性损伤小鼠胰岛细胞，TFP5保护db / db小鼠胰岛，增加胰岛素分泌和胰岛基因的表达，保护小鼠的胰岛细胞；同时，Cdk5过度活性在db/db模型肾小球引起炎症因子包括TGF-β，TNF-α及IL-1β增高，TFP5 可以抑制db / db小鼠胰岛细胞的炎症因子在db/db小鼠足细胞中Cdk5表达和微炎症因子，细胞因子如TGF-β、TNF-α、VEGF等呈正相关，并与蛋白尿形成相关，说明Cdk5及上述炎症因子与足细胞损害密切相关。