GDF-5在小鼠肢体骨骼发育中的表达及对肢芽细胞影响的研究

Expression of GDF-5 during the development of limb skeleton system of mice and the effect of GDF-5 on limb bud cell

目的 :初步观察并探讨生长分化因子 5 (GDF 5 )在小鼠肢体发育过程中的表达及对肢芽细胞软骨分化的影响。方法 :RT PCR法检测小鼠肢体发育过程中GDF 5表达的变化情况 ;MTT法测定不同浓度GDF 5对体外微团培养肢芽细胞增殖率的影响 ;Alcian染色在倒置相差显微镜下观察不同浓度GDF 5对肢芽细胞软骨分化的影响 ,探讨GDF 5影响骨骼系统发育的机理。结果 :RT PCR结果提示GDF 5在胚胎发育的第 12、 13d高表达 ,骨骼系统基本形成之后在孕 14、 15d表达渐下降稳定于一较低水平 ;不同浓度GDF 5对肢芽细胞增殖率影响不同 ,5 0ng/ml浓度组促肢芽细胞增殖作用最强 ;Alcian染色结果显示GDF 5促进肢芽细胞软骨分化的作用呈剂量依赖效应 ,以12 0ng/ml组软骨分化最快 ,软骨结节体积最大 ,10 0ng/ml组软骨结节的数量最多。结论 :GDF 5在肢体发育的不同阶段表达水平不同 ,在骨骼系统形成早期及关节系统形成阶段表达最强 ,主要通过影响肢芽细胞增殖及软骨分化而发挥作用。

Objective:To explore the effect of growth differentiation factor-5 (GDF-5) on limb bud cell and mRNA expression during the development of limb skeleton system of mice.Method:The expression of GDF-5 mRNA during the development of limb skeleton system of mice was examined by RT-PCR;The proliferation rate of limb bud cell was investigated by MTT assay;chondrogenesis of limb bud cell was measured at 24,48,72 and 96h after cell cultured and GDF-5 was examined by Alcian stain and phase contrast light microscopy.Result:The expression of GDF-5 mRNA by RT-PCR,was hightest during the early phase of skeletogenesis in E12,E13 and slowly dropped in E14,E15;The MTT assay results showed that GDF-5 could promote limb bud cell proliferation and the effect of 50 ng/ml GDF-5 was most strong;Alcian stain indicated that GDF-5 could increase the size of cartilage condensation and promote chondrogenesis in a dose-dependent manner.Conclusion:The expression level of GDF-5 mRNA is various during different skeletogenesis phase.It is highest during the early phase of skeletogenesis and the formation of joint in E12,13,GDF-5 exerts its action by promote limb bud cell proliferation and chondrogenesis.