TAT-tCNTF对CD诱导HUVEC细胞损伤的作用研究

Effect of TAT-tCNTF to HUVECs' Injury Caused by Cytochalasin D

目的探讨TAT—tCNTF对细胞松弛素D（cD）诱导人脐静脉内皮细胞（HuvEc）损伤作用的影响。方法CCK-8法检测TAT—tCNTF对CD致HUVEC生长抑制的作用；荧光显微镜检测CD诱导HUVEC的F—actin的变化，以及在TAT—tCNTF作用下对细胞微丝的影响；荧光显微镜和流式细胞术检测CD和TAT—tCNTF作用后细胞内Ca^2＋浓度；Westernblot检测CD和TAT—tCNTF作用后F—actin的蛋白水平的变化。结果在1μg·ml^-1。的CD作用HUVEC后，细胞表现出明显的生长抑制，而在0．05～10μg·ml^-1。浓度范围内，TAT—tCNTF促进HUVEC的生长，并可改善CD对HUVEC的损伤，TAT—tCNTF组与CD＋TAT—tCNTF组细胞活动度具有明显差异；荧光显微镜下检测到经CD作用后细胞F-actin分布减少，而经TAT—tCNTF作用后细胞F-actin分布密集；采用Fluo4-AM荧光探针标记细胞内Ca^2＋浓度，荧光显微镜和流式细胞术检测到TAT—tCNTF能够降低经CD损伤HUVEC后的细胞内Ca^2＋浓度升高；FCM检测显示，CD组的MFI相对于对照组明显增加，而CD＋TAT—tCNTF组MFI较CD组明显降低。Westernblot显示各组之间的F—actin表达量没有明显改变。结论TAT—tCNTF对CD致内皮细胞的损伤有改善和逆转作用，其改善机制可能与增加细胞活性和维持细胞骨架有关。

Objective To investigate the effect and mechanism of TAT-tCNTF to injury of human umbilical vein endothelial cells led by cyto- chalasin D. Methods TAT-tCNTF and CD affected cell viability by CCK-8. Using fluorescence nficroscope observed changes of F-actin resulted by CD and TAT-tCNTF. Level of intracellular free calcium concentration was detected by Fluo 4-AM loading through the fluorescence microscope and flow cytometry. Western blot detect F-actin protein expression. Results HUVECs treated with 1μg·ml^-1 CD showed obvious growth inhibition while HUVECs with CD and 0.05-10 μg·ml^-1 TAT-tCNTF grew actively. TAT-tCNTF de- creased HUVEC intracellular free calcium concentration enriched by CD. Distribution of F-actin in HUVECs with CD massively re- duced while crowded together after subsequent treated with TAT-tCNTF. Western blot showed F-actin protein had almost no change. Conclusion TAT-tCNTF can confront and reverse the injury caused by CD, and such mechanism might be associated with the increas- ing of cell viabilitv and maintaining of actin cvtoskeleton.