血管内皮生长因子165转染脐静脉内皮细胞与骨髓间充质干细胞共培养在血管形成中的作用

Co-transplantation of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells transfected with vascular endothelial growth factor 165 gene promotes vascularization

背景:组织工程骨早期血管化是目前亟待解决的难题,细胞共培养以及添加生物活性因子来促进血管生成均是很好地促进早期血管化的方法。目的:探讨人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)过表达血管内皮生长因子165(vascular endothelial growth factor,VEGF165)后与小鼠骨髓间充质干细胞共培养在血管形成中的作用。方法:腺病毒介导VEGF165转染HUVECs,并分离培养小鼠骨髓间充质干细胞,进行共培养。实验分组如下:(1)转染共培养组:Ad VEGF165-HUVECs+骨髓间充质干细胞;(2)转染非共培养组:Ad VEGF165-HUVECs;(3)空载体共培养组:Ad GFP-HUVECs+骨髓间充质干细胞;(4)空载体组:Ad GFP-HUVECs;(5)未转染共培养组:HUVECs+骨髓间充质干细胞;(6)空白对照组:HUVECs。采用CCK8法检测HUVECs的增殖能力,结晶紫染色检测HUVECs的迁移能力,Matrigel法检测HUVECs的血管形成能力。结果与结论:(1)与空白对照组相比,转染共培养组、转染非共培养组、空载体共培养组、未转染共培养组的细胞增殖能力均明显增加(P<0.05);(2)与空白对照组相比,转染共培养组人脐静脉内皮细胞数量最多(P<0.05);(3)与空白对照组相比,转染共培养组形成的小管数目更多形成时间更早,更稳定(P<0.05);(4)结果表明,VEGF165转染HUVECs和骨髓间充质干细胞共同作为种子细胞能更好地促进早期血管生成。

BACKGROUND:Vascularized bone tissue engineering is a hotspot of current research and early vascularization of tissue-engineered bone also becomes an urgent problem to be solved.Addition of bioactive factors and cell co-culture method both contribute to promoting the early vascularization.OBJECTIVE:To investigate the pro-angiogenic effect of human umbilical vein endothelial cells(HUVECs)transfected with vascular endothelial growth factor165(VEGF165)co-cultured with bone marrow mesenchymal stem cells(BMSCs).METHODS:HUVECs were transfected with VEGF165and then co-cultured with BMSCs which were purified from mouse femoral bone marrow.There were six groups in this experiment:(1)AdVEGF165-HUVECs+BMSCs,(2)AdVEGF165-HUVECs,(3)AdGFP-HUVECs+BMSCs,(4)AdGFP-HUVECs,(5)HUVECs+BMSCs,and(6)HUVECs(blank control).Cell counting kit-8detection was applied to analyze the proliferative ability of HUVECs.The abilities of HUVECs migration and vascularization were then detected by crystal violet staining and matrigel determination,respectively.RESULTS AND CONCLUSION:Compared with the blank control group,the proliferation of HUVECs was significantly increased in the other groups except for AdGFP-HUVECs group(P<0.05).Compared with the blank control group,the migration and vascularization abilities of HUVECs were significantly stronger in the group of AdVEGF-HUVECs+BMSCs(P<0.05).To conclude,the co-culture of HUVECs transfected with VEGF165and BMSCs could promote early vascularization effectively.