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目的体外模拟成骨细胞在体内的生存环境,考察活性维生素D3(VD3)、力学拉伸以及两者联合对成骨细胞MC3T3-E1增殖、分化及破骨细胞抑制因子(OPG)和破骨细胞分化因子(RANKL)表达的影响。方法将10 nmol/L VD3、间断性力学拉伸以及两者联合作用于成骨细胞。流式细胞术检测细胞增殖。荧光探针试剂盒检测碱性磷酸酶(ALP)活性。实时定量PCR检测核心转录因子Runx2、OPG、RANKL mRNA水平,Western blotting检测其蛋白表达。结果 VD3抑制成骨细胞增殖,力学拉伸不能改变这种抑制效应。力学拉伸和VD3单独作用成骨细胞均能增加ALP活性及提高ALP、Runx2 mRNA水平,但当联合刺激后这些指标均降低,且成强度依赖性。力学拉伸增加OPG/RANKL比值,增强成骨作用,联合VD3后,OPG/RANKL比值下降。结论力学拉伸能有效诱导成骨分化,增加骨形成。VD3与力学拉伸联合抑制成骨细胞增殖和分化,并通过增加RANKL表达而影响骨重建。研究结果为骨质疏松及相关骨疾病的理论和治疗提供有意义的探索。
OBJECTIVE: To investigate the in vivo survival conditions of osteoblasts in vitro, and to investigate the effects of active vitamin D3 (VD3), mechanical stretching and their combination on the proliferation and differentiation of osteoblasts MC3T3-E1, osteoclast (OPG) and osteoclasts Effect of differentiation factor (RANKL) expression. Methods 10 nmol / L VD3, intermittent mechanical stretching and their combined action on osteoblasts. Flow cytometry was used to detect cell proliferation. Fluorescence probe kit was used to detect alkaline phosphatase (ALP) activity. The mRNA expressions of Runx2, OPG and RANKL, the core transcription factors, were detected by real-time quantitative PCR. The protein expression was detected by Western blotting. Results VD3 inhibited the proliferation of osteoblasts. Mechanical stretching did not change the inhibitory effect. Tensile mechanical stretch and VD3 alone could increase ALP activity and ALP, Runx2 mRNA levels, but these indicators were decreased and intensity-dependent when combined with VD3. Mechanical stretch increased OPG / RANKL ratio, enhanced osteogenesis, combined with VD3, OPG / RANKL ratio decreased. Conclusion Mechanical stretch can effectively induce osteogenic differentiation and increase bone formation. VD3 combined with mechanical stretching inhibits osteoblast proliferation and differentiation and affects bone remodeling by increasing RANKL expression. The results provide a meaningful exploration of the theory and treatment of osteoporosis and related bone diseases.