健骨颗粒含药血清培养成骨细胞整合素/黏着斑激酶信号通路相关因子的表达

Effects of serum containing Jiangu granules on the expression of correlative factors in osteoblast integrin/focal adhesion kinase signal pathway

背景:目前有关成骨细胞整合素的功能状态及整合素信号转导调控的研究较少。健骨颗粒对成骨细胞整合素表达水平及信号转导通路调控的作用未被阐明。目的:观察健骨颗粒含药血清对成骨细胞黏附的影响,揭示健骨颗粒对成骨细胞整合素表达水平及信号转导通路的调控作用。方法:健康雄性3月龄SD大鼠用于制备健骨颗粒含药血清,选取最佳剂量的含药血清干预第3代大鼠成骨细胞,ELISA法检测培养液中纤连蛋白、Ⅰ型胶原、玻连蛋白的水平,Western blot法检测成骨细胞黏着斑激酶的磷酸化情况,实时荧光定量PCR法检测成骨细胞纤连蛋白、Ⅰ型胶原、玻连蛋白、黏着斑激酶mRNA的表达。结果与结论:浓度为20%的健骨颗粒含药血清能明显增强体外培养成骨细胞的增殖活力。随着含药血清干预时间的延长,成骨细胞表达及分泌的纤连蛋白、Ⅰ型胶原、玻连蛋白、黏着斑激酶mRNA及蛋白水平逐渐上升,黏着斑激酶的磷酸化水平也逐渐增高(P<0.05);均明显高于同期的生理盐水血清干预水平(P<0.05或P<0.01)。说明健骨颗粒能作用于成骨细胞整合素激活的粘着斑激酶转导通路各相关因子,使成骨细胞进入"分泌增加→黏附增加→功能增强、分裂增殖→分泌增加"的良性循环,对成骨细胞的成骨效应起正性调节作用。

BACKGROUND：Few studies addressing functional status of osteoblast integrin and regulation of integrin-signal transduction pathway.The effects of Jiangu granules on the expression of osteoblast integrin/focal adhesion kinase signal pathway remain poorly understood.OBJECTIVE：To observe the effects of serum containing Jiangu granules on osteoblast adhesion,and to explore Jiangu granules on the expression of osteoblast integrin and regulation of signal transduction pathway.METHODS：Male healthy Sprague Dawley rats,aged 3-month,were prepared for serum containing Jiangu granules and intervented on the 3rd passages of rat osteoblasts.The expressions of fibronectin,type Ⅰ collagen and vitronectin were measured by ELISA.Phosphorylation of osteoblast focal adhesion kinase was detected by Western blot.And the mRNA expressions of fibronectin,type Ⅰ collagen,vitronectin and focal adhesion kinase were determined by real-time fluorescence quantitative PCR.RESULTS AND CONCLUSION：The proliferation rate of osteoblasts was the fastest when the concentration of the serum containing Jiangu granules was 20%.The expressions of type Ⅰ collagen,fibronectin,vitronectin,focal adhesion kinase,as well as focal adhesion kinase phosphorylation were gradually increased with time prolonged（P 0.05）,which were obvious greater than those of the normal saline control group（P 0.05 or P 0.01）.Jiangu granules-containing serum has direct or indirect role on osteoblast integrin signal transduction pathway relevant factors,promotes osteoblasts entering a virtuous circle of ＂secretion increasing→adhesion enhancing→activity enhancing,proliferation increased→secretion increasing＂,and has a positive regulatory effect on the osteogenesis of osteoblasts.