胰岛素对长骨成骨细胞的影响及受体后机制的研究

目的:研究胰岛素对长骨成骨细胞增殖和功能的影响并探讨其受体后分子机制。方法:培养生长期大鼠股骨和胫骨成骨细胞，MTT法检测不同浓度胰岛素和不同作用时间对成骨细胞增殖的影响，并观察骨钙素、胰岛素样生长因子（IGF-1）mRNA、IGF-1蛋白合成及成骨细胞胞内P44/42MAPK及其磷酸化的变化。结果:胰岛素对成骨细胞的增殖和功能的影响具有浓度和时间依赖性，10-7 mol/L时促增殖作用最强，96 h后细胞增殖进入平台期。促进骨钙素合成的最佳胰岛素浓度亦为10-7 mol/L，之后骨钙素合成减少。培养时间对骨钙素合成无明显影响。胰岛素浓度超过10-7 mol/L后IGF-1 mRNA的表达也明显低于对照组。各胰岛素组的IGF-1蛋白浓度均明显高于对照组（P<0.05），但各浓度组间无明显差别（P>0.05）。胰岛素急性刺激成骨细胞后各组的P44/42MAPK表达量无明显差别（P>0.05），但P44/42MAPK磷酸化程度随加入胰岛素浓度的增高而增强(组间P<0.05)；胰岛素慢性处理后各组的P44/42MAPK表达量仍无显著差别（P>0.05），P44/42MAPK磷酸化程度却随着胰岛素浓度的升高而逐渐下降(组间P<0.05)。结论:胰岛素能以生长因子样的作用呈剂量依赖性的方式刺激成骨细胞生长及功能，但高胰岛素状态以及长时间的胰岛素作用后此种增强效应消失。其受体酪氨酸蛋白激酶介导的MAPK信号途径参与了促成骨细胞的生长增殖的作用。

Effects of insulin on osteoblast and its post-receptor mechanism

AIM: To study the effects of insulin on the proliferation and function of osteoblasts and the relationship between insulin post-receptor change in osteoblasts and osteoblastic cell growth.METHODS: The effects of different levels of insulin on osteoblasts were assessed by MTT colorimetry.Osteocalcin in medium was measured by RIM.IGF-1 mRNA expression levels were determined by RT-PCR.The concentrations of free IGF-1 protein in serum-free medium were measured by ELISA.In addition,the protein level and phosphorylated protein of P~(44/42)MAPK were determined by Western blotting analysis.RESULTS: Insulin enhanced the proliferation of osteoblasts,depending on its dose and exposure time.Insulin at concentration of 10~(-7) mol/L showed the strongest effect,and the action attained the plateau phase beyond 96 h.The best concentration that stimulated synthesis of osteocalcin by insulin was 10~(-7) mol/L.When the insulin concentration beyond 10~(-7) mol/L,the osteocalcin concentration was decreased.Exposure time had no effect on insulin-stimulated synthesis of osteocalcin of osteoblastic cells.When the concentration of insulin reaches 10~(-6) mol/L,the IGF-1 mRNA expression stimulated by insulin was also decreased.The concentrations of free IGF-1 protein in insulin-stimulated groups were all higher than that in control group(P<0.05),but there was no statistically significant difference among insulin-stimulated groups(P>0.05).Insulin acute stimulation rapidly induced the activity of tyrosine phosphorylation of P~(44/42)MAPK.The degree of tyrosine phosphorylation of P~(44/42)MAPK was increased step by step along with the increasing doses of insulin from 0 to 10~(-7) mol/L(P<0.05,between groups).After insulin chronicity treatment,there was a marked reduction in the tyrosine phosphorylation of P~(44/42) MAPK(P<0.05,between groups).There was no significant change in protein level of P~(44/42)MAPK.CONCLUSIONS: Insulin enhances the proliferation of osteoblasts as a growth factor at a suitable concentration,but this effect disappears at chronic high insulin stimulation.The MAPK may be involved in the proliferating effect of insulin on osteoblasts.Transient stimulation of insulin activates the P~(44/42)MAPK,however chronic high insulin stimulation results in down-regulation of P~(44/42)MAPK signal activity.