联合应用血管内皮生长因子及血小板衍生生长因子BB干预骨髓间充质干细胞的血管化及增殖能力

Effects of vascular endothelial growth factor combined with platelet-derived growth factor-BB on the angiogenic and proliferative abilities of bone marrow mesenchymal stem cells

背景:组织工程骨为修复极限骨缺损提供了新的选择,但只有先形成完善的功能性血管网,保证稳定的成骨和骨整合,才能取得良好的治疗效果。因此,血管化可以说是组织工程骨面临的最大挑战与难题。目的:探讨体外联合应用血管内皮生长因子(vascular endothelial growth factor,VEGF)和血小板衍生生长因子BB(platelet-derived growth factor-BB,PDGF-BB)对骨髓间充质干细胞增殖和成血管能力的影响。方法:体外分离培养SD大鼠骨髓间充质干细胞,分别用不同质量浓度VEGF(20,40,60,80,100,120μg/L)、PDGF-BB(20,40,60,80,100,120μg/L)联合干预以及100μg/L VEGF单独干预、100μg/L PDGF-BB单独干预,CCK-8实验检测2种细胞因子促进细胞增殖的最佳质量浓度,然后在第7天和第14天通过RT-PCR方法检测血管生成素1、缺氧诱导因子1α、肝细胞生长因子、胰岛素样生长因子等相关成血管基因的表达量。结果与结论:①加入生长因子后,细胞增殖能力明显提高,联合作用效果更优,最佳组合为80μg/L VEGF+80μg/L PDGF-BB;②VEGF、PDGF-BB都可以促进血管生成素1、缺氧诱导因子1α、肝细胞生长因子和胰岛素样生长因子的mRNA表达,联合应用时效果最佳;③缺氧诱导因子1α、肝细胞生长因子mRNA表达随时间延长有所升高,差异有显著性意义(P<0.05);而血管生成素1、胰岛素样生长因子mRNA表达量随时间延长有所降低,差异有显著性意义(P<0.05);④体外实验结果证明,当VEGF和PDGF-BB质量浓度均为80μg/L时,能够持续稳定促进整个血管形成过程,且促进作用优于单独一种生长因子。

BACKGROUND:Bone tissue engineering provides a new path to treat critical-sized bone defects.However,a stable osteogenesis and osseointegration can only be insured via formation of an intact vascular network,which can achieve satisfactory therapeutic effects.Thereafter,angiogenesis is a challenge and difficulty in bone tissue engineering.OBJECTIVE:To investigate the effect of combined application of vascular endothelial growth factor(VEGF)and platelet-derived growth factor-BB(PDGF-BB)on proliferation and angiogenesis of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured and isolated in vitro,and interfered with different concentrations of VEGF(20,40,60,80,100,120μg/L)and PDGF-BB(20,40,60,80,100,120μg/L)in combination or 100μg/L VEGF,and 100μg/L PDGF-BB alone.The optimum concentration of promoting cell proliferation was detected by cell counting kit-8 assay.The expression levels of angiopoietin-1,hypoxia-inducible factor-1α,hepatocyte growth factor and insulin-like growth factor were detected by RT-PCR at 7 and 14 days after intervention.RESULTS AND CONCLUSION:(1)After growth factors were added,the cell proliferative ability was significantly improved,and combined application revealed better effect.The optimal concentration grou was 80μg/L VEGF+80μg/L PDGF-BB.(2)Both VEGF and PDGF-BB could promote the mRNA expression levels of angiopoietin-1,hypoxia-inducible factor-1α,hepatocyte growth factor and insulin-like growth factor,and the effect was more obvious in combined application.(3)The mRNA expression levels of hypoxia-inducible factor-1αand hepatocyte growth factor were significantly increased with time(P<0.05).The mRNA expression levels of angiopoietin-1 and insulin-like growth factor were significantly decreased with time(P<0.05).(4)In vitro experimental results suggest that VEGF and PDGF-BB at the concentration of 80μg/L can consistently promote angiogenesis,and the effect of combined application is better than that of single application.