心肌营养素-1对人脐静脉内皮细胞增殖、迁移、管腔形成及VEGF表达的影响

Effects of cardiotrophin-1 on proliferation,migration,tube formation and VEGF expression of human umbilical vein cells

目的探讨心肌营养素-1(cardiotrophin-1,CT-1)基因对人脐静脉内皮细胞(human umbilical vein cells,HUVECs)迁移、管腔形成及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法利用LipofectamineTM2000将质粒pEGFP-N1-CTF1-GFP及pEGFP-N1瞬时转染至HUVECs;实验分成3组:①HUVECs组:正常对照组;②GFP组:转染质粒pEGFP-N1;③CT-1组:转染质粒pEGFP-N1-CTF1-GFP;MTT法检测HUVECs增殖能力的变化,Transwell法检测HUVECs迁移的变化,体外血管腔形成实验检测血管腔形成能力;Western blot检测CT-1及VEGF蛋白表达的变化。结果 Western blot结果显示,质粒pEGFP-N1-CTF1-GFP转染48h为CT-1蛋白表达高峰;MTT法、Transwell法和血管腔形成实验分别显示,高表达CT-1的内皮细胞的增殖率、迁移指数和血管腔形成数目明显明显高于正常对照组与GFP组(P<0.05),Western blot结果显示,质粒转染48h,CT-1组VEGF的表达明显高于正常对照组与GFP组(P<0.05)。结论高表达CT-1能明显促进HUVECs增殖、迁移及管腔形成,CT-1可能具有促进血管新生的能力;CT-1促进血管新生的能力可能与其上调VEGF的表达有关。

Objective To investigate the effects of cardiotrophin-1 （CT-1） gene on proliferation, migration, tube formation and vascular endothelial growth factor （VEGF） expression of human umbilical vein cells （HUVECs）. Methods pEGFP-N1-CTF1-GFP and pEGFP-N1 were constructed and transiently transfected into HUVECs using LipofectamineTM 2000. HUVECs were divided into three groups： HUVECs group （normal control）, GFP group （transfected with pEGFP-N1）, and CT-1 group （transfected with pEGFP-N1-CTF1-GFP）. Forty-eight hours after transfection, endothelial cells proliferation was evaluated using MTT method, migration assay was performed using Transwell, and tube formation was tested. The protein levels of CT-1 and VEGF expressions were measured by Western blot analysis. Results Western blot demonstrated that 48 hours after transfection was the peak time of CT-1 expression. The MTT assay, Transwell assay and tube formation study indicated that compared with the control and GFP groups, in the up-regulated CT-1 group, the proliferation, migration, and tube formation number of endothelial cells significantly increased. Western blot showed that 48 hours after transfection, the protein level of VEGF expression in the CT-1 group was significantly increased compared with that in HUVECs group and GFP group （P 〈 0. 05）. Conclusion The overexpression of CT-1 significantly increased the proliferation, migration and tube formation of HUVECs. CT-1 may induce angiogenesis by up-regulating the expression of VEGF.