TAT-tCNTF对CD损伤细胞的作用机制研究

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：TAT-tCNTF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＜0.0001），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。

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

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.