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内源性和外源性甲状旁腺素对小鼠骨折愈合影响的实验研究

Effect of Both Endogenous and Exogenous PTH on Mice Bone Fracture Healing

作者: 专业:骨科学 导师:曹晓建 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

bone fracture healing, parathyroid hormone (PTH), gene knockout, mice

        【目的】:探讨内源性和外源性甲状旁腺素(parathyroid hormone, PTH)是否具有促进成年小鼠骨折愈合的作用。【方法】:选用8周龄PTH野生型和PTH基因敲除(PTH-/-)小鼠作为实验对象,采用髓内针固定的标准股骨干骨折模型。小鼠骨折后1~4周,每天皮下注射PTH(1-34) 80μg/kg作为实验组,或者相应剂量的生理盐水作为对照组。分别于骨折后1周、2周和4周,通过X线摄影、micro-CT、组织学、组织化学和免疫组织化学等方法分析骨痂特征,并且将骨痂组织提取的RNA和蛋白通过RT-PCR和Western blot的方法分析骨形成相关基因和蛋白表达水平。【结果】:骨折后1周,无论给予生理盐水还是PTH处理,相同处理组小鼠之间相比,PTH-/-小鼠软骨性骨痂面积都较野生型小鼠为少;无论是野生型还是PTH-/-小鼠,相同基因型小鼠之间相比,PTH给药组小鼠软骨性骨痂面积较相应生理盐水对照组小鼠为多。骨折后2周,相同处理组小鼠之间相比,PTH-/-小鼠残余的软骨性骨痂比野生型小鼠要多;而同种基因型小鼠之间相比,PTH给药组小鼠软骨性骨痂面积较相应对照组小鼠为少。同时,骨折后2周,与野生型小鼠相比,PTH-/-小鼠骨痂的骨矿密度,骨性骨痂面积,碱性磷酸酶(ALP)、I型胶原的mRNA水平,以及核心结合因子1(Cbfa1)、胰岛素样生长因子I(IGF-I)的蛋白表达水平都是降低的;而相同基因型小鼠之间相比,PTH给药小鼠在这些指标方面都比相应对照组小鼠要高。骨折后4周,相同处理组小鼠之间相比,PTH-/-小鼠在总胶原阳性骨性骨痂面积、成骨细胞数量、ALP阳性面积和I型胶原阳性面积等指标方面,都较野生型小鼠为少;相同基因型小鼠之间相比,PTH给药小鼠在这些指标方面都比相应对照组小鼠要高。骨折后2周和4周,相同处理组小鼠之间相比,PTH-/-小鼠的TRAP阳性破骨细胞数量和破骨细胞表面面积也较野生型小鼠为少;相同基因型小鼠之间相比,PTH给药组小鼠在这些指标方面都比相应对照组小鼠要高。【结论】:内源性和外源性PTH都能通过增加骨痂面积、促进软骨内成骨和成骨细胞骨形成而加速骨折的愈合。
    【Objective】:To demonstrate whether both endogenous and exogenous PTH stimulate bone fracture healing.【Mathods】:Closed mid-diaphyseal femur fractures were created and stabilized with an intramedullary pin in 8-week-old wild-type (WT) and PTH null (PTH-/-) mice. Mice received daily injections of vehicle (V) or of PTH1-34 (80μg/kg) for 1-4 weeks post-fracture, and callus tissue properties was analyzed at 1, 2 and 4 weeks post-fracture by radiography, micro-CT, histology, histochemistry and immunohistochemistry. RNA and proteins were isolated from callus tissues and bone formation related gene and protein expression levels were evaluated by RT-PCR and Western blots.【Results】:At 1 week post-fracture, cartilaginous callus areas were reduced in V-treated and PTH-treated PTH-/- mice compared to V-treated and PTH-treated WT mice respectively, but were increased in both PTH-treated WT and PTH-/- mice compared to V-treated WT and PTH-/- mice respectively. In contrast, at 2 weeks post-fracture, remnant cartilaginous callus areas were increased in V-treated and PTH-treated PTH-/- mice compared to V-treated and PTH-treated WT mice respectively and were reduced in PTH-treated WT and PTH-/- mice compared to V-treated WT and PTH-/- respectively. As well, at 2 weeks post-fracture, the mineral density in callus and bony callus areas, the mRNA levels of ALP, type I collagen, and the protein levels of Cbfa1 and IGF-I were reduced in V-treated and PTH-treated PTH-/- mice compared to V-treated and PTH-treated WT mice respectively and were increased in PTH-treated WT and PTH-/- mice compared to V-treated WT and PTH-treated PTH-/- mice respectively. At 4 weeks post-fracture, total collagen positive bony callus areas, osteoblast number, ALP positive areas and type I collagen positive areas were all reduced in V-treated and PTH-treated PTH-/- mice compared to V-treated and PTH-treated WT mice respectively and were increased in PTH-treated WT and PTH-/- mice compared to V-treated WT and PTH-/- mice respectively. At 2 and 4 weeks post-fracture, TRAP positive osteoclast number and surface were also reduced in V-treated and PTH-treated PTH-/- mice compared to V-treated and PTH-treated WT mice respectively,but were increased in PTH-treated WT and PTH-/- mice compared to V-treated WT and PTH-/- mice respectively.【Conclusion】:These results indicate that both endogenous and exogenous PTH enhance bone fracture healing by increasing callus areas, endochondral bone formation and osteoblastic bone formation.
        

内源性和外源性甲状旁腺素对小鼠骨折愈合影响的实验研究

缩略语4-6
中文摘要6-8
Abstract8-9
前言10-13
材料与方法13-35
结果35-47
分析和讨论47-50
小结50-51
参考文献51-55
综述55-65
    参考文献59-65
攻读学位期间发表文章情况65-66
致谢66
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