成纤维细胞生长因子受体3在脂多糖抑制成骨细胞分化中作用的初步研究

Effect of fibroblast growth factor receptor 3 on inhibiting differentiation of osteoblasts induced by LPS

目的本实验拟通过研究激活成纤维细胞生长因子受体3(FGFR3)信号通路时骨组织成骨细胞分化程度在内毒素血症中的变化,为进一步阐明骨组织在炎症反应中变化的机制奠定基础。方法8周龄雄性C57小鼠和FGFR3功能持续增强的软骨发育不全(Ach)小鼠各6只,分为脂多糖(LPS)组和生理盐水对照组,每组3只。LPS15mg/kg或等量生理盐水腹腔注射4小时后取右侧胫骨提取RNA,定量聚合酶链式反应(PCR)检测白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、骨钙素(OC)的变化。LPS、碱性成纤维生长因子(bFGF)处理前成骨细胞系MC3T3E14小时后定量PCR检测IL-6、TNF-α、OC水平,成骨诱导7天后检测碱性磷酸酶(ALP)活性。结果LPS刺激后,骨组织和MC3T3E1的炎症递质基因IL-6、TNF-α的RNA水平显著增加(P<0.01),成骨标志性基因OC表达下调(P<0.05)。且Ach小鼠相应基因变化幅度显著高于C57的变化幅度(P<0.05)。MC3T3E1同时用LPS与碱性成纤维细胞生长因子(bFGF)处理组的IL-6、TNF-α、OC水平表达水平位于单独用LPS或bFGF组之间。成骨诱导7天后LPS处理组细胞ALP活性显著低于未处理组(P<0.05),bFGF处理组显著增高(P<0.01)。结论LPS刺激骨组织发生炎症反应、抑制成骨细胞分化。在整体动物水平持续性激活FGFR3信号通路加重上述反应,在细胞水平低剂量bFGF激活FGFR3减轻上述反应。

Objective This experiment focuses on the differentiation of osteoblasts in the endotoximia while the FGFR3 pathway is activating, so as to understand the change of bone tissue in inflammation. Methods Six male C57 mice and 6 male Ach mice （ persistently activating function of FGFR3 ） , 8 weeks old, were divided into normal saline（NS） group and lipopolysaccharide（LPS） group,3 mice per group. RNA was extracted from the right tibias 4 hours after LPS or physiological saline injection, and then the levels of interleukin 6 （ IL-6 ） , tumor necrosis factor （x（TNF-o~）,osteocalcin （OC） in tibia and osteoblast were measured by quantitative real-time PCR. Four hours after the preosteoblast cell line MC3T3E1 was cultured with LPS and bFGF,the levels of IL-6 ,TNF-α, OC in tibia and osteoblast also were measured by quantitative real-time PCR. The MC3T3E1 activities of alkaline phospha- tase（ALP） were detected 7 days after culture. Results Both of the levels of IL-6 and TNF-α in tibias and MC3T3E1 were increased while the levels of OC in tibias and MC3T3EI were decreased 4 hours after LPS administration（P 〈0.01）. The expression change was more huge in Ach mice（P 〈0.05）. The expressions of IL-6, IL-1 and OC in MC3T3E1 cultured with LPS and bFGF were higher than those cultured only with LPS and lower than those only with bFGF. Compared with the untreated group,the activity of ALP in MC3T3E1 was decreased in LPS group （ P 〈 0.05 ） and increased in bFGF group （ P 〈 0.01 ）. Conclusion LPS could involve bone tissue in inflam-mation and inhibit the differentiation of osteoblast. Persistently activating the function of FGFR3 in vivo could aggravate these effects. And low dose of bFGF in vitro which activates FGFR3 could relieve these effects.