牵张力促进BMSCs与VECs共培养体系成骨分化的体外研究

Cyclic mechanical strain induces osteogenic differentiation in the coculture system of BMSCs and VECs

目的:研究牵张力对骨髓基质细胞(BMSCs)与血管内皮细胞(VECs)共培养体系成骨分化的作用及相关机制。方法:分离培养大鼠原代BMSCs与VECs。应用Flexcell 5000加力系统,分别对BMSCs与VECs共培养组、BMSCs单独培养组和VECs单独培养组施加6%等轴循环牵张力。加力6、12、24和48 h后,利用实时定量PCR检测Runx2和血管内皮细胞生长因子(VEGF)m RNA的表达量,ELISA法检测细胞培养上清液中VEGF的含量,碱性磷酸酶(ALP)半定量检测ALP活性。通过加入VEGF受体抑制剂Tivozanib,观察VEGF的旁分泌作用。采用SAS 8.0软件包对数据进行统计学分析。结果:1加力6 h时,共培养体系Runx2 m RNA的表达量上调4.3倍(P<0.05);加力48 h时,ALP活性升高1.5倍(P<0.05)。2加力12 h时,共培养体系VEGF m RNA的表达量上调2倍(P<0.05),上清液中VEGF的含量增加10倍(P<0.05),BMSCs分泌大量VEGF,而VECs分泌极少量VEGF。3加入Tivozanib后,共培养组Runx2的表达量下调90%(P<0.05),ALP活性下调48%(P<0.05);而BMSCs单独培养组Runx2的表达量和ALP活性分别下降30%和18%。结论:牵张力促进BMSCs与VECs共培养体系中BMSCs的成骨分化,这种作用可能通过牵张应力诱导BMSCs分泌的VEGF以旁分泌方式由VECs作用于BMSCs来实现。

PURPOSE： To detect the effects of cyclic mechanical strain on the coculture system of bone marrow stromal cells（BMSCs）and vascular endothelial cells（VECs）and to clarify the related mechanism. METHODS： Primary BMSCs and VECs were isolated from Sprague-Dawley rats. BMSCs and VECs coculture group, BMSCs monoculture group and VECs monoculture group were exposed to cyclic mechanical strain（6%） by Flexcell 5000 mechanicl loading system. After mechanical loading for 6, 12, 24 and 48 h, the m RNA expression of Runx2 and vascular endothelial growth factor（VEGF）were tested by real-time quantitative PCR. VEGF protein in cell culture supernate was quantified using Elisa Kit and alkaline phosphatase（ALP）activity was examined. The VEGFR inhibitor Tivozanib was used to analyze the paracrine role of VEGF. Statistical analysis was performed using SAS 8.0 software package. RESULTS： 1The m RNA expression of Runx2 of coculture group subjected to 6% strain was increased significantly at 6h with 4.3 folds（P0.05） and ALP activity at 48 h with 1.5 folds（P 0.05）. 2 The m RNA expression of VEGF of coculture group subjected to 6% stain was increased significantly at 12 h with 2 folds（P0.05） and the content of VEGF in cell culture supernate at 12 h with 10 folds（P0.05）.Plenty of VEGF was secreted by BMSCs but little by VECs. 3 After being subjected to Tivozanib, the expression of Runx2 VECs. 3 After being subjected to Tivozanib, the expression of Runx2 of coculture group was decreased by 90%（P0.05） and ALP activity was decreased by 48%（P0.05）, while those of BMSCs monoculture group were decreased by30% and 18%, respectively. CONCLUSIONS： Cyclic mechanical strain promotes osteogenic differentiation of BMSCs in the coculture system of BMSCs and VECs, possibly by a paracrine VECs-mediated effect of VEGF on BMSCs.