Smad4促进成骨分化的机制研究

目的 探讨Smad4基因促进成骨分化的作用机制。方法 采用条件性基因敲除技术Cre/loxp，制备骨细胞特异性敲除Smad4小鼠(Smad4otcko)，小鼠胚胎骨骼透明染色分析胚胎期小鼠长骨生长状况；待小鼠成长至1月龄，X-ray检测突变小鼠与对照组小鼠的骨密度差异；静态骨组织形态学分析检测突变鼠及对照鼠的骨量变化、成骨细胞数量变化等差异；实时荧光定量PCR检测Smad4突变鼠股骨成骨细胞相关因子Runx2、ALP、OSX及OCN；破骨细胞TRAP染色分析Smad4突变鼠破骨细胞形态及数量变化；qPCR检测突变鼠股骨破骨吸收标志基因RANKL、OPG，并计算RANKL/OPG比率。结果 Smad4基因敲除小鼠在胚胎期未出现长骨生长异常。X线结果显示，1月龄时，与对照组小鼠相比，Smad4突变鼠的骨密度降低（P＜0.05），静态骨组织形态学分析表明突变鼠松质骨减少，皮质骨变薄，骨小梁数量减少（P＜0.05）；Smad4突变鼠成骨细胞标志基因表达量显著降低，成骨细胞的数量明显减少（P＜0.05）；RANKL作为破骨吸收标志物表达上调、作为其拮抗剂的OPG表达量下调，RANKL/OPG比率增高（P＜0.05）。结论 Smad4基因通过促进成骨分化，降低破骨吸收从而来维持骨稳态。

Mechanism of the stimulation of osteoblast differentiation by Smad4

Objective To determine the mechanism of promoting effect of osteogenic differentiation by osteocyte-specific gene Smad4. Methods Conditional gene knockout technique Cre/loxp was used to produce osteocytic Smad4 specific knockout mice (Smad4otcko). Embryonic mouse skeletal growth was shown by whole mount skeletal staining of new pups. The difference of bone mineral density was detected in one-month-old mice with X-ray. Changes in bone mass, trabecular number, and osteoblast number were analyzed by static bone histomorphology. The osteoblastic makers ALP, Runx2, OSX, OCN, and osteoclastic markers RANKL, OPG were detected by qPCR. TRAP staining was performed to show osteoclastic absorption. The RANKL/OPG ratio was calculated. Results Smad4otcko mice displayed no gross skeletal abnormalities at birth. X-ray radiography showed reduced bone mineral density, bone mass, and osteoblast number compared with controls (P<0.05). Similarly, the specific markers of osteoblastic differentiation were decreased. Additionally, osteoclast number increased. The expression of RANKL increased while OPG decreased. The radio of RANKL/OPG increased significantly (P<0.05). Conclusion The Smad4 gene regulates bone homeostasis through promoting osteoblastic differentiation and down-regulation of osteoclastic resorption.