血管内皮前体细胞通过旁分泌功能促进间充质细胞成骨分化  

Endothelial progenitor cells promote osteogenic differentiation of marrow stromal cells in a paracrine manner

作　　者：张猛[1] 张宏伟[2] 冯文磊 王艳杰[1] 印双红[3] 陈雪玲[4] 吴向未[2] 

机构地区：[1]石河子大学医学院,832000 [2]石河子大学医学院第一附属医院肝胆外科 [3]铜仁学院材料与化学工程学院 [4]石河子大学医学院免疫学教研室

出　　处：《中华医学杂志》2015年第16期1253-1257,共5页National Medical Journal of China

基　　金：国家自然科学基金（31271458）;人力资源和社会保障部留学回国人员科技活动项目（RSLX201201）;科技支疆计划项目（2014AB047）

摘　　要：目的 探讨血管内皮前体细胞（EPC）促进间充质细胞（MSC）成骨分化的机制.方法 分离、培养与扩增6-8周龄C57 BL/6小鼠EPC、MSC,提取血管内皮前体细胞的条件培养基（EPC-CM）并检测血管内皮生长因子（VEGF）、转化生长因子β1（TGFβ1）、血小板源性生长因子（PDGF）、胰岛素样生长因子1（IGF-1）、基质细胞衍生因子1（SDF-1）、碱性成纤维细胞生长因子（bFGF）的水平;为探讨EPC和EPC-CM对MSC成骨分化的影响,实验分为MSC/EPC组和MSC组,并根据加入的EPC-CM浓度不同分为0 EPC-CM组、25％ EPC-CM组及50％ EPC-CM组,诱导14d后茜素红染色检测各组钙盐沉积情况;在50％血管内皮前体细胞条件培养基下,分别加入上述6个细胞因子的中和抗体,检测血管内皮前体细胞条件培养基中参与促进间充质细胞成骨分化的细胞因子.结果 血管内皮前体细胞表面抗原VEGFR2、CD34、CD133的阳性率分别为79.3％、79.5％和76.4％,并且能在基质胶上形成血管样结构;血管内皮前体细胞分泌VEGF、TGFβ1、PDGF、IGF-1、SDF-1、bFGF;MSC/EPC组矿化结节形成明显多于MSC组,钙盐半定量检测示MSC/EPC组吸光度（A）值大于MSC组A值（0.733±0.032与0.236±0.020,P＜0.001）;50％ EPC-CM组矿化结节形成多于25％EPC-CM组,25％ EPC-CM组多于0 EPC-CM组;半定量分析显示50％ EPC-CM组A值高于25％EPC-CM组（0.637±0.028与0.336±0.024,P＜0.001）,25％ EPC-CM组A值高于0 EPC-CM组（0.336±0.024与0.239±0.013,P=0.004）;在50％血管内皮前体细胞条件培养基下,加入VEGF、TGFβ1、IGF-1抗体的3组矿化结节相对于PBS组明显减少,差异均有统计学意义（均P＜0.01）.结论 血管内皮前体细胞可能通过分泌VEGF、TGFβ1、IGF-1促进间充质细胞成骨分化.Objective To explore the mechanisms of endothelial progenitor cells （EPCs） on promoting osteogenic differentiation of marrow stromal cells （MSCs）.Methods EPCs and MSCs were isolated and cultured successfully from C57BL/7 murine bone marrow by in vitro amplification.EPC-conditioned medium （CM） was extracted to detect the concentrations of vascular endothelial growth factor （VEGF）,transforming growth factor-beta 1 （TGFβ1）,platelet-derived growth factor （PDGF）,insulin-like growth factor 1 （IGF-1）,stromal cell-derived factor 1 （SDF-1） and basic fibroblast growth factor （bFGF）by enzyme-linked immunosorbent assay （ELISA）.After 14 days of induction,alizarin red staining was used to detect every group＇s calcium salt deposition.And analyses were conducted for the effects of above mentioned antibodies of cytokines on osteogenic differentiation of MSCs.Results Positive rates of EPCs were 79.3%,79.5%,76.4% for VEGFR2,CD34 and CD133 respectively,and EPCs could form tube-like structure on matrigel.EPCs secreted VEGF,TGFβ1,PDGF,IGF-1,SDF-1 and bFGF.MSC/EPC group formed more mineralized nodules than MSC group,and semi-quantitative results showed the optical density of MSC/EPC group was higher than that of MSC group （0.733 ± 0.032 vs 0.236 ± 0.020,P 〈 0.001）.The number of formed mineralized nodules of 50% EPC-CM group were more than those of 25% EPC-CM group,and semi-quantitative results showed that the optical density of 50% EPC-CM group was higher than that of 25% EPC-CM group （0.637 ± 0.028 vs 0.336 ± 0.024,P 〈 0.001）,the number of formed mineralized nodules of 25% EPC-CM group were more than those of 0 EPC-CM group,and semi-quantitative results showed that the optical density of 25% EPC-CM group was higher than that of 0 EPC-CM group （0.336 ±0.024 vs 0.239 ± 0.013,P =0.004）.On the basis of 50% of EPC-CM,the number of formed mineralized nodules significantly declined in the presence of anti-VEGF antibody,anti-TGFβ1 antibody,anti-IGF-1 antibody（P 〈 0.01）.C

关 键 词：血管内皮前体细胞 骨髓间充质细胞 血管内皮生长因子 转化生长因子Β1 胰岛素样生长因子1 

分 类 号：R543[医药卫生—心血管疾病]