健骨颗粒促进成骨细胞增殖的分子机制**☆

摘要

BACKGROUND:Extracel ular signal-regulated kinase is a protein kinase dependent on Ras pathway activation, and plays an important role in the proliferation and differentiation of osteoblasts. PD98059 is a specific MEK inhibitor, and inhibits phosphorylation of extracel ular signal-regulated kinase through inhibition of MEK activity, thus blocking extracel ular signal-regulated kinase signaling pathway. At present, the role of extracel ular signal-regulated kinase in the process of proliferation and differentiation of rat osteoblasts is reported less. It is stil unclear about the role of extracel ular signal-regulated kinase in the regulation of osteoblast proliferation and differentiation promoted by Jiangu granules. OBJECTIVE:To observe the role of extracel ular signal-regulated kinase signaling pathway in the promotion of osteoblast proliferation and differentiation after intervention with Jiangu granules. METHODS:Passage 3 osteoblasts from the skul of Sprague-Dawley rats were harvested and divided into four groups. Control group was intervened with saline serum. Drug group was intervened with the best concentration of serum containing Jiangu granules. Inhibitor group was intervened by PD98059. Combination group was intervened with PD98059 plus serum containing Jiangu granules. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine proliferative capacity of osteoblasts. Alkaline phosphatase and hydroxyproline levels were measured by colorimetric method. Expression of core binding factor alpha 1, type Ⅰ col agen, Osterix mRNA was measured by PCR-SYBR GREEN. Expression of extracel ular signal-regulated kinase in osteoblasts was detected using western blot method. RESULTS AND CONCLUSION:After blocking extracel ular signal-regulated kinase signal pathway, the expression of extracel ular signal-regulated kinase in the inhibitor and combination groups was significantly lower than that in the control and drug groups. Levels of alkaline phosphatase and hydroxyproline, as wel as core binding factor alpha 1, type Ⅰ col agen, Osterix mRNA expression in osteoblasts, in the inhibitor group were significantly lower than those in the control and drug groups. It is indicated that extracel ular signal-regulated kinase exerts an important role in promoting osteoblast proliferation and differentiation after intervention with Jiangu granules.%　　背景：ERK是依赖于Ras途径激活的一个蛋白激酶，在成骨细胞增殖和分化过程中发挥重要作用。而PD98059是MEK特异性抑制剂，它可通过抑制MEK的活性来抑制ERK的磷酸化，从而起到阻断ERK信号通路的作用。目前有关ERK在大鼠成骨细胞增殖和分化过程中的作用研究甚少。ERK在健骨颗粒促进大鼠成骨细胞增殖和分化过程中调控的作用更未被阐明。　　目的：观察ERK信号转导通路在健骨颗粒促成骨细胞增殖和分化过程中的作用。　　方法：取第3代SD大鼠头盖骨成骨细胞，空白组加入生理盐水血清；中药组加入最佳浓度的健骨颗粒含药血清；阻滞剂组添加 PD98059阻断剂，中药加阻断剂组添加 PD98059阻断剂与健骨颗粒含药血清。用MTT 法测定细胞的增殖能力，比色法测碱性磷酸酶、羟脯氨酸水平。收集细胞实时荧光定量 PCR-SYBR GREEN法检测Cbfa1、Ⅰ型胶原、OSX mRNA的表达，Westren blot法检测成骨细胞ERK的表达情况。　　结果与结论：添加PD98059阻断剂后，阻滞剂组和中药加阻滞剂组中ERK的表达显著低于空白组和中药组。阻断剂组的成骨细胞内碱性磷酸酶、羟脯氨酸的表达以及Cbfa1、Ⅰ型胶原、OSX mRNA表达显著低于空白组和中药组。ERK 在健骨颗粒促进大鼠成骨细胞增殖和分化过程中发挥重要作用，ERK信号通路可能是健骨颗粒促进成骨细胞增殖和分化的主要途径。