骨髓间充质干细胞归巢促小鼠骨折愈合的实验研究

Fracture healing improved by homing of BMSCs in mouse model

目的探讨骨髓间充质干细胞（BM SC ）迁移归巢在骨折愈合中的作用。方法6周龄健康成年雄性清洁级C57／BL小鼠65只，体重17～25 g ，采用外力冲击方法制备闭合性股骨骨折内固定模型，7 d后将小鼠分为3组：间充质干细胞（MSC）组（n＝35）尾静脉注射生理盐水0．5 mL ，0．5 h后注射内含1＆#215;106个红色荧光蛋白标记的骨髓间充质干细胞（RFP‐BMSC）溶液0．5 mL ；1，4，8，11‐四氮杂环十四烷（AMD3100）组（n＝15）尾静脉注射1 mmol／L AMD3100（CXCR4受体的特异性阻断剂）0．5 mL ，0．5 h后注射内含1＆#215;106个RFP‐BMSC的溶液0．5 mL ；对照（CON）组（n＝15）尾静脉注射生理盐水0．5mL，0．5h后再次注射生理盐水0．5mL。造模成功后14、28、42d采用冰冻切片荧光示踪法观察M SC组小鼠RFP‐BM SC归巢情况，并在28 d后采用免疫荧光法观察BM SC位置和骨桥蛋白（OPN）及骨钙素（OCN）的表达；14、42 d后分别采用HE染色和Masson三色染色观察3组组织切片骨痂形成情况，并进行组织病理学检查及生物力学检查，评价3组骨折愈合情况。结果骨折后28 d MSC组有大量RFP‐BMSC迁移至骨折处聚集，且高度表达OPN与OCN。14 d后病理组织切片染色结果显示，CON组与AMD3100组以软骨成分为主，而 MSC组多已开始形成骨性骨痂；42 d后CON组与AMD3100组处于骨痂塑形期，皮质骨质量差，MSC组骨痂已基本塑形完成。生物力学检测结果显示， MSC组最大负荷、最大桡度、弹性桡度及刚度显著高于另两组（P＜0．05），接近正常骨组织（P＞0．05）。结论 BMSC归巢能促进骨折愈合，基质细胞衍生因子（SDF）‐1／CXCR4通路在其中发挥重要作用。

Objective To investigate the role of bone mesenchymal stem cells （BMSCs） homing in the fracture healing process of mice .Methods Sixty‐five C57/BL6 male mice with femur fracture were divided into three groups ：saline （0 .5 mL） and red fluorescent protein‐labeled stem cells （RFP‐BMSCs ,1 ＆#215; 106 cells/0 .5 mL） were injected from tail vein 7 days after fracture in the mesenchymal stem cell （MSC） group （n= 35） ,CXCR4 antagonist 1 ,4 ,8 , 11‐tetraazacyclotetradecane （AMD3100 ,1 mmol/L ,0 .5 mL） and RFP‐BMSCs （1 ＆#215; 106 cells/0 .5 mL） were injected in the AMD3100 group（n= 15） ,and saline （1 mL in all） was injected in the control （CON） group（n= 15） .RFP‐BMSCs homing was observed in MSC group by frozen section examination 14 , 28 , 42 days after fracture .Osteopontin （OPN） and osteocalcin （OCN） expressions in each group were observed by immunofluorescence 28 days after fracture .HE staining and Masson trichrome staining were taken in each group 14 and 42 days after fracture , and callus formation was also observed .Histopathological examination and biomechanical examination were executed to evaluate fracture healing 42 days after fracture .Results A large number of RFP‐BMSCs were migrated to fracture site ,and OPN and OCN were highly expressed 28 days after fracture .Pathological staining showed that the fracture sites in CON group and AMD3100 group were mainly composed of cartilage ,and bone callus formed in MSC group . CON group and AMD3100 group were still in callus remodeling period and cortical bone were of poor quality 42 days after fracture ,while callus in MSC group had been almost completed shaping .Biomechanical results showed the maximum load ,maximum radial degree ,elastic radial degree and rigidity of MSC group were significantly higher than that of the other groups .Conclusion MSC homing could promote fracture healing ,and SDF‐1/CXCR4 pathway plays an important role in the process .