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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。
Objective: To investigate the effect and mechanism of stretch on osteogenic differentiation of bone marrow stromal cells (BMSCs) and vascular endothelial cells (VECs). Methods: Primary rat BMSCs and VECs were isolated and cultured. Flexcell 5000 force system was used to apply BMSCs and VECs co-culture group, BMSCs alone group and VECs alone culture group to exert 6% isokinetic cyclic strain. The expression of Runx2 and VEGF mRNA was detected by real-time quantitative PCR at 6, 12, 24 and 48 h after loading. The content of VEGF in the cell culture supernatant was detected by ELISA, the activity of alkaline phosphatase (ALP) semi-quantitative detection of ALP activity. The paracrine effect of VEGF was observed by adding the VEGF receptor inhibitor Tivozanib. Data was statistically analyzed using the SAS 8.0 software package. Results: The expression of Runx2 m RNA in co-culture system was up-regulated by 4.3-fold (P <0.05) at 1 h after 6 h, while ALP activity increased by 1.5 times (P <0.05) at 48 h. 2 at 12 h, the expression of VEGF m RNA in co-culture system was up-regulated by 2-fold (P <0.05), the content of VEGF in supernatant increased by 10-fold (P <0.05) A small amount of VEGF. 3 After adding Tivozanib, the expression of Runx2 in co-culture group was reduced by 90% (P <0.05) and ALP activity by 48% (P <0.05), while Runx2 expression and ALP activity in BMSCs alone group decreased by 30% and 18 %. Conclusion: Stretching can promote osteogenic differentiation of BMSCs in BMSCs co-culture system with VECs. This effect may be mediated by distraction stress-induced VEGF secretion from BMSCs in a paracrine manner by VECs on BMSCs.