TFP5对高糖诱发的细胞周期素依赖性激酶过度激活及胰岛β细胞凋亡的影响

Effects of TFP5 on high glucose-induced hyperactivity of cyclin dependent kinase 5 and pancreatic beta cells apoptosis

目的：探讨TFP5对高糖诱发的细胞周期素依赖性激酶5（Cdk5）活性的抑制作用及其对胰岛β细胞的保护作用。方法体外培养小鼠胰岛细胞株Min6。将TFP5转染Min6细胞，分别用免疫印迹和免疫荧光观察其表达。实验分3组：低糖（5mmol/L）组，高糖（25mmol/L）＋空病毒载体（EV）组和高糖（25mmol/L）＋TFP5组，分别用同位素标记法测定3组细胞内Cdk5激酶活性；用酶联免疫吸附法测定3组细胞胰岛素分泌水平；用免疫印迹法测定胰岛β细胞凋亡。结果（1）表明TFP5来源和它的分子序列；（2）TFP5在Min6细胞内表达良好；（3）在高糖＋TFP5组Cdk5的活性明显低于高糖＋EV组（ P＜0.01），而且在高糖＋TFP5组胰岛素分泌明显高于高糖＋EV组（P＜0.01）；（4）在高糖组Min6细胞的Bax表达增加，Bcl-2表达减少，Bax/Bcl-2比值升高，细胞凋亡增加（P＜0.01，vs 低糖组），而加入TFP5后，Bax表达减少，Bcl-2表达增加，Bax/Bcl-2的比值减低（P＜0.01，vs 高糖组），细胞的凋亡减少。结论 TFP5可抑制高糖诱发的Cdk5激酶的过度活性、减少高糖刺激下胰岛细胞凋亡，从而恢复胰岛素分泌，具有潜在的以Cdk5为靶点治疗2型糖尿病的前景。

Objective To determine the inhibitory effect of TFP5 on the hyperactivity of cyclin dependent kinase 5 （Cdk5） induced by high glucose and its protection for the pancreatic beta cells from apoptosis. Methods After TFP5 was delivered into mouse insulinoma line 6 （Min 6）, the expression of TFP5 was detected in the cells by Western blotting and immunofluorescence staining. Then, the cells were treated by low （5mmol/L）, high glucose （25mmol/L）＋empty vector （EV）, and high glucose ＋TFP5, respectively. Cdk5 activities were measured by immune precipitation and isotope labeling. The level of insulin secretion were detected with ELISA;and the apoptosis of Min 6 cells were analyzed by Western blotting. Results （1） Our results indicated the derivation of TFP5 peptide from p35 and its sequence. （2） TFP5 was well expressed in the Min 6 cells. （3） Cdk5 activity was significantly lower in the high glucose＋TFP5 cells than in the high glucose＋EV cells （P〈0.01）; the insulin secretion level in the high glucose＋TFP5 cells was significantly higher than that in the high glucose cells （P〈0.01）. （4） In the high glucose cells, the expression of Bax was increased while that of Bcl-2 was decreased, and the ratio of Bax/Bcl-2 was increased （P〈0.01, vs the low glucose cells）, indicating the increase of cell apoptosis. However, the expression of the 2 proteins was opposite, and the ratio was decreased in the high glucose＋TFP5 cells, showing lesser apoptotic cells （P〈0.01, vs the high glucose cells）. Conclusion TFP5 inhibits the hyperactivity of Cdk5 induced by high glucose, protects pancreatic cells from apoptosis, and recovers the insulin secretion. Therefore, it may have a potential therapeutic agent for type 2 diabetes targeting on Cdk5.