生长分化因子-5影响肢芽细胞分化的机制

The mechanism of growth differentiation factor-5 on limb bud cell differentiation

目的研究生长分化因子-5(growthdifferentiatifoanctor-,5GDF-5)对肢芽细胞分化的影响机制及在骨形成过程中的作用机制。方法取妊娠第12d胎鼠肢芽,行肢芽细胞微团培养,以不同浓度(0ng/ml、10ng/ml、50ng/ml、100ng/ml、120ng/ml)重组鼠GDF-5,干预培养中的肢芽细胞。于细胞培养第1d、3d,用扫描电镜观察不同浓度GDF-5对肢芽细胞生长与分化的影响。于细胞培养第3d,采用免疫组织化学和实时定量PCR等方法观察GDF-5对肢芽细胞核结合转录因子(Cbfa1)与Ⅹ型胶原蛋白表达的影响。结果在细胞培养第1d,扫描电镜观察结果显示,GDF-5可使肢芽细胞连接更为紧密,高浓度GDF-5组有成熟软骨基质和软骨结节形成,低浓度组也有软骨结节形成,该效应与GDF-5浓度呈正相关。在细胞培养第3d,免疫组织化学检测结果显示,Ⅹ型胶原蛋白表达以120ng/ml浓度组最为广泛和强烈,0ng/ml浓度组表达最弱,其表达趋势接近于Cbfa1的表达,表明GDF-5有促进肢芽细胞向成熟软骨方向分化的作用。实时定量PCR结果显示,GDF-5在mRNA水平同样促进Cbfa1表达,其促进作用呈剂量依赖效应。结论GDF-5通过上调Cbfa1和Ⅹ型胶原蛋白表达的途径促进肢芽细胞向软骨分化和促进骨形成。

Objective To study the mechanism in the effect of growth differentiation factor-5(GDF-5) on bone formation and limb bud cell differentiation. Methods Micromass cultures were carried out using limb buds of day 12 mouse embryos, they were treated with different concentration of recombinant mouse GDF-5 (0 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 120 ng/ml). On day 1 after recombinant mouse GDF-5 treatment, scanning electron microscopy was used to observe the morphology of cartilage nodule. On day 3, expression of Cbfa1 protein in limb buds was examined by immunohistochemistry and mRNA of Cbfa1 and collagen Ⅹ by real time PCR. Results Scanning electron microscopy showed that limb buds cell junction was tighter, there was matured cartilaginous matrix and nodules formation under higher concentration of GDF-5 on day 1, and also cartilaginous nodules under lower concentration. GDF-5 could promote limb buds cell chondro-differentiation and cartilage nodules formation. The effects were positively correlated with concentration of GDF-5. Immunohistochemistry displayed that GDF-5 could promote expression of Cbfa1 and collagen Ⅹ. The expression of collagen Ⅹ was more spreading and powerful with GDF-5 in 120 ng/ml, but weakest with GDF-5 in 0 ng/ml, it tended to express as closely as that of Cbfa1. Real time PCR exhibited that GDF-5 at the level of mRNA could accelerate expression of Cbfa1 mRNA. These effects were in a dose-dependent manner. Conclusion GDF-5 is able to promote limb bud cell differentiation into cartilage and bone formation through up-regulation of the expression of Cbfa1 and collagen Ⅹ.