ADAM10对小鼠颅颌面膜内成骨的影响

The Effects of ADAM10 in Craniofacial Intramembranous Ossification of Mice

目的探索解聚素样金属蛋白酶10(ADAM10)对小鼠颅颌面骨骼膜内成骨的影响。方法应用组织免疫荧光染色及蛋白免疫印迹研究ADAM10在小鼠颅颌面骨中的不同时间点、不同部位的表达水平。通过cre-loxp技术在胚胎小鼠颅颌面骨骼中特异性敲除ADAM10基因,应用体视显微镜观察基因敲除小鼠的颅颌面畸形,X线检查、von Kossa染色检测基因敲除小鼠骨钙化情况,双标免疫荧光观察基因敲除小鼠骨组织细胞增殖、分化、凋亡能力的改变。结果组织免疫荧光染色显示小鼠上、下颌骨成骨活跃区ADAM10表达明显,同时蛋白免疫印迹结果证实,ADAM10在小鼠出生前后表达高,成年后表达明显降低。大体观察基因敲除小鼠身体体型较小,颅颌面畸形表现为面中部发育不足,上下颌骨发育不足,颅顶塌陷。X线片检测及von Kosaa染色显示基因敲除小鼠全身骨骼密度减低,骨组织形成较弱。免疫荧光检测发现ADAM10基因敲除小鼠下颌骨细胞增殖、成骨分化能力减低、凋亡增加。结论 ADAM10广泛表达于小鼠膜内成骨区域,可能通过影响骨组织细胞增殖、分化及凋亡来调控小鼠颅面部膜内成骨过程。

Objective To explore the effects of ADAM10 in craniofacial intramembranous ossification of mice. Methods The expression pattern of ADAM10 in mice mandibular was detected by immunofluorescence assay and western blot. Creloxp technique was used to detect the function of ADAM10 in intramembranous ossification areas during craniomaxillofacial development. ADAM10 conditional knock-out mice was evaluated by X-ray and von Kossa staining. The cell proliferation, apoptosis and osteoblast differentiation were detected by immunofluorescence assay. Results Immunofluorescence assay revealed that ADAM10 was primarily distributed in the maxillary and mandibular skeletal genesis zones. ADAM10 was abundantly expressed from E14.5 to postnatal day 7 （P7） and was significantly decreased during adulthood. On E15.5, the ADAM10 cKO mice exhibited a shorter snout, flatter posterior skull and smaller maxilla and mandible, compared with the control littermates. The X-ray examination and von Kossa staining shown that the ADAM10 cKO embryos exhibited significantly reduced mineral bone deposition in the maxilla and mandible. Immunofluorescence shown that cell proliferation, apoptosis and osteoblast differentiation were impaired in ADAM10 cKO mice. Conclusion ADAM10, widely expressed in osteogenesis area of mice, can affect the intramembranous bone formation by regulating cell proliferation, differentiation and apoptosis.