骨抑素对大鼠骨髓间充质干细胞增殖、成骨分化和促进血管生成能力的影响

Effects of ostestatin on proliferation,osteogenic differentiation and angiogenic ability of bone marrow mesenchymal stem cells

目的 探讨骨抑素(osteostatin,OST)对缺氧环境下骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖、成骨分化和促进血管生成能力的影响及其在骨缺损修复中的应用前景。方法 从大鼠骨髓中分离培养BMSCs。BMSCs经OST处理后,在常氧(21%氧气)或缺氧(1%氧气)条件下培养。采用cellcountingkit-8(CCK-8)法检测细胞增殖,台盼蓝染色法检测细胞死亡率。通过碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红S(alizarin red S,ARS)染色评价成骨分化。western blot检测ALP、成骨转录因子(runt-related transcription factor 2,RUNX2)和骨钙素(osteocalcin,OCN)的蛋白水平。ELISA检测培养基中血管生成素1(angiopoietin-1,Ang-1)和血管内皮生长因子(vascular endothelial growth factor,VEGF)浓度。小管形成试验评估促进血管生成能力。结果 OST剂量依赖性增强大鼠BMSCs的增殖(F=4.89,P=0.006)。缺氧诱导下,BMSCs的增殖、小管形成量增加(F=28.98、76.68,P均<0.001),Ang-1和VEGF的浓度提高(F=183.7、585.6,P均<0.001);ALP、RUNX2和OCN的蛋白水平降低(F=646.8、432.7、257.5,P均<0.001)。OST处理后,BMSCs增殖(常氧+OSTvs常氧,P均<0.001;缺氧+OSTvs缺氧,P=0.005)、小管形成量(常氧+OSTvs常氧,P=0.002;缺氧+OSTvs缺氧,P=0.005)进一步增加,Ang-1浓度(常氧+OST vs常氧,P<0.001;缺氧+OST vs缺氧,P<0.001)和VEGF的浓度(常氧+OST vs常氧,P<0.001;缺氧+OSTvs缺氧,P<0.001)进一步提高;ALP、RUNX2和OCN的蛋白水平升高(常氧+OSTvs常氧,P均<0.001;缺氧+OST vs缺氧,P均<0.001)。结论 OST可增强缺氧环境中BMSCs的增殖和成骨分化能力,促进血管生成。

Objective To investigate the effect of osteostatin(OST)on the proliferation,osteogenic differentiation,and angiogenesis of bone marrow mesenchymal stem cells(BMSCs)under hypoxia,and to explore its applicability in bone defect repair.Methods BMSCs were isolated from bone marrow of rats before being treated with OST and cultured under normoxic(21%oxygen)or hypoxic(1%oxygen)conditions.CCK-8 assay was used to evaluate cell proliferation,while trypan blue staining was adopted to detect cell mortality.Osteogenic differentiation was evaluated via alkaline phosphatase(ALP)staining and alizarin red S(ARS)staining.Levels of ALP,RUNX2(runt-related transcription factor 2)and OCN(osteocalcin)were measured by Western blot.Concentrations of Ang-1(angiopoietin-1)and VEGF(vascular endothelial growth factor)in the medium were detected by ELISA.Angiogenesis was evaluated by tube formation assay.Results The proliferation of BMSCs in rats was enhanced in an OST dose-dependent manner(F=4.89,P=0.006).Under hypoxic inductions,the proliferation and tubule formation of BMSCs increased(F=28.98,76.68,both P<0.001),so did the concentrations of Ang-1 and VEGF(F=183.7,585.6,both P<0.001).The protein levels of ALP,RUNX2,and OCN decreased(F=646.8,432.7,257.5,all P<0.001).After OST treatment,the proliferation(normoxia+OST vs normoxia,both P<0.001;hypoxia+OST vs hypoxia,P=0.005)and tubule formation of BMSCs(normoxia+OST vs normoxia,P=0.002;hypoxia+OST vs hypoxia,P=0.005)continued to increase.The concentrations of Ang-1(normoxia+OST vs normoxia,P<0.001;hypoxia+OST vs hypoxia,P<0.001)and VEGF(normoxia+OST vs normoxia,P<0.001;hypoxia+OST vs hypoxia,P<0.001)further increased.The protein levels of ALP,RUNX2,and OCN increased(normoxia+OST vs normoxia,both P<0.001;hypoxia+OST vs hypoxia,both P<0.001).Conclusion OST can enhance proliferation,osteogenic differentiation and angiogenesis of BMSCs under hypoxic conditions.