TAT-tCNTF对CD损伤细胞的作用机制研究

Studies of the mechanism of TAT-tCNTF on cell injury caused by cytochalasin D

目的:探讨TAT-tCNTF对细胞松弛素D(CD)导致的人脐静脉内皮细胞株(HUVECs)损伤的作用机制。方法:TATtCNTF处理经CD损伤的细胞,CCK-8法检测其对细胞活力的影响;荧光显微镜观察罗丹明-鬼笔环肽标记的F-actin的变化;荧光显微镜和流式细胞术(FCM)检测细胞内Fluo 4-AM标记的Ca2+浓度变化;Western blot检测F-actin蛋白水平变化。结果:10μmol·L-1 CD降低HUVECs的细胞活动度(P<0.000 1),而10μg·mL-1的TAT-tCNTF明显升高HUVECs的细胞活动度(P=0.003);荧光显微镜下观察到CD作用后的细胞F-actin断裂且分布减少,而TAT-tCNTF可改善这一现象;TAT-tCNTF可降低由CD引起的细胞内Ca2+超载;FCM显示CD组Ca2+荧光强度(MFI)较对照组增强,而(CD+TAT-tCNTF)组的MFI较CD组减弱。Western blot结果显示各组F-actin表达量无明显差异。结论:TAT-tCNTF对CD引起的细胞损伤有改善作用,其机制与维持细胞骨架结构和缓解细胞内Ca2+超载有关。

Objective：To investigate the mechanism of TAT-tCNTF on human umbilical vein endothelial cells （HUVECs） injured by cytochalasin D （CD）. Methods：After HUVECs were treated with CD and TAT-tCNTF, cell viability was determined by CCK-8. The structural changes of F-actin stained by rhodamine labeled ghost cyclic peptide were observed through the lfuorescence microscope. Concentrations of intracellular free calcium by Fluo 4-AM loading were detected through the lfuorescence microscope and lfow cytometry. Western blot was used to detect the expression of F-actin protein. Results：CD （10μmol·L-1） reduced the cell viability of HUVECs （P〈 0.000 1）, while TAT-tCNTF （10 μg·mL-1 ） significantly increased the cell viability of HUVECs （P=0.003）. F-actin with CD was fractured and reduced, while it was improved after subsequent treatment with TAT-tCNTF. TAT-tCNTF alleviated the calcium overload caused by CD. Flow cytometry showed mean lfuorescence intensity （MFI） of Ca2＋in CD group was stronger than that in control group, the MFI of Ca2＋in （CD＋TAT-tCNTF） group was lower than that in CD group. Western blot showed the expression of F-actin protein had almost no change among all groups. Conclusion：TAT-tCNTF ameliorated the cell injury caused by CD. The mechanism was associated with maintaining actin cytoskeleton and alleviating the calcium overload.