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目的:探讨硝酸甘油(Nitroglycerin,NTG)对人骨髓间质干细胞(Human Bone Marrow-derived Mesenchymal Stem Cells,HBMSCs)的增殖及向成骨细胞分化的影响及其作用机制。方法:建立HBMSCs体外模型,诱导HBMSCs向成骨细胞分化。给予不同浓度的NTG,测定一氧化氮(NO)含量,考察不同浓度NTG对HBMSCs的NO生成的影响;用BrdU细胞增殖试剂盒测定反映细胞的增殖情况;测定细胞内碱性磷酸酶(ALP)活性与钙沉积量,反映不同浓度NTG对HBMSCs向成骨细胞分化的影响,探讨NO和ALP及钙沉积之间的关系;根据以上实验选出NTG最小有效浓度,再合用内皮型一氧化氮合酶(Endothelial nitricoxide synthase,eNOS)拮抗剂以及诱导型一氧化氮合酶(Induciable nitric oxide synthase,iNOS)拮抗剂探讨NTG作用于HBMSCs的机制是否与NOS有关。结果:NTG呈剂量依赖性的增加原代HBMSCs的增殖、NO含量、ALP活性及钙沉积量。NTG 10μM对HBMSCs增殖及成骨型分化的诱导作用以及对HBMSCs的NO产量和NOS活性的影响不能被eNOS抑制剂LNAME及iNOS拮抗剂1 400 W所阻滞。结论:NTG可通过NO途径促进HBMSCs的增殖及其向成骨细胞的分化,但其释放NO的效应并不依赖NOS。
OBJECTIVE: To investigate the effects of Nitroglycerin (NTG) on proliferation and osteoblast differentiation of human bone marrow mesenchymal stem cells (HBMSCs) and its mechanism. Methods: HBMSCs in vitro models were established to induce HBMSCs to differentiate into osteoblasts. The concentration of NTG was measured to determine the content of nitric oxide (NO). The effects of different concentrations of NTG on the NO production of HBMSCs were observed. The proliferation of HBMSCs was detected by BrdU cell proliferation kit. The levels of intracellular alkaline phosphatase (ALP) Activity and calcium deposition, reflecting the different concentrations of NTG on HBMSCs differentiate into osteoblasts, to explore the relationship between NO and ALP and calcium deposition; NTG selected based on these experiments the minimum effective concentration, combined with endothelial nitric oxide Endothelial nitric oxide synthase (eNOS) antagonist and inducible nitric oxide synthase (iNOS) antagonist were used to investigate whether the mechanism of NTG acting on HBMSCs is related to NOS. Results: NTG increased the proliferation, NO content, ALP activity and calcium deposition of primary HBMSCs in a dose-dependent manner. The effect of NTG 10μM on the proliferation and osteogenic differentiation of HBMSCs and the effect on NO production and NOS activity of HBMSCs can not be blocked by the Lonicellin inhibitor NOS and iNOS antagonist 1400 W. Conclusion: NTG can promote the proliferation of HBMSCs and its differentiation into osteoblasts through the NO pathway, but the effect of releasing NO is not dependent on NOS.