全身应用神经生长因子对大鼠胫骨干骨折早期愈合作用及骨形态发生蛋白2和血管内皮生长因子表达的影响

Whole body application of nerve growth factor promotes early healing of tibial shaft fracture and improves expression of bone morphogenetic protein-2 and vascular endothelial growth factor in rats

背景:骨折愈合是一种十分复杂的多阶段生理过程,由多种细胞协调参与。有关神经因素对于骨折愈合过程的影响逐渐被重视和深入研究,神经生长因子是一种能够促进细胞生长的生物活性复合蛋白,其促进骨折愈合过程中所发挥的作用机制尚不明确。目的:观察神经生长因子对大鼠胫骨骨折愈合的促进作用及愈合过程中机制。方法:将72只雄性SD大鼠随机分为模型组和神经生长因子组,构建胫骨干闭合骨折模型并应用髓内固定。神经生长因子组给予肌肉注射,注射用鼠神经生长因子;模型组给予肌肉注射生理盐水。实验于2018-05-30经福建医科大学实验动物伦理委员会批准,批准号:FJYKDX-2018-035。结果与结论:①干预2周和4周时,神经生长因子组骨痂体积显著高于模型组,干预6周时,神经生长因子组骨折线消失,髓腔再通,骨折早于模型组达到完全愈合;②苏木精-伊红染色显示干预2周和4周时,神经生长因子组骨小梁生长致密,可见大量成骨细胞生长;③干预2周和4周时,神经生长因子组血清碱性磷酸酶含量明显高于模型组;④免疫组化结果显示,干预2周和4周时,神经生长因子组骨痂内骨形态发生蛋白2和血管内皮生长因子阳性表达均显著多于模型组;⑤定量PCR结果显示,干预2周和4周时,神经生长因子组骨形态发生蛋白2和血管内皮生长因子mRNA表达水平均高于模型组;⑥提示全身应用神经生长因子可增强大鼠胫骨骨折端成骨能力,促进骨折早期愈合;同时神经生长因子可促进骨形态发生蛋白2和血管内皮生长因子的表达,并在不同时期这种促进作用表现出一定的规律,进而诱导成骨细胞分化增殖及细胞外基质的形成和钙化,促进骨形成及骨愈合。

BACKGROUND:Fracture healing is a very complex,multi-stage physiological process with the coordinated participation of multiple cells.The influence of neurological factors on fracture healing has attracted increasing attention.Nerve growth factor is a bioactive complex protein that promotes cell growth.The mechanism of its role in promoting fracture healing is not clear.OBJECTIVE:To observe the effect of nerve growth factor on the healing of tibial fracture in rats and the mechanism.METHODS:Seventy-two male Sprague-Dawley rats were randomly divided into model group and treatment group.The model of tibial shaft closed fracture was established followed by intramedullary fixation.The treatment group was given intramuscular injection of nerve growth factor;the model group was given intramuscular injection of normal saline.The study was approved by the Experimental Animal Ethics Committee of Fujian Medical University on May 30,2018,with approval No.FJYKDX-2018-035.RESULTS AND CONCLUSION:The callus volume in the treatment group was significantly higher than that in the model group at 2 and 4 weeks of intervention.At 6 weeks of intervention,the fracture line disappeared,the medullary cavity was recanalized,and the fracture in the treatment group healed earlier than that in the control group.Hematoxylin-eosin staining showed that the trabeculae grew densely and a large number of osteoblasts were observed in the treatment group at 2 and 4 weeks after intervention.The serum alkaline phosphatase level in the treatment group was significantly higher than that in the model group at 2 and 4 weeks of intervention.Immunohistochemical results showed that the positive expression of bone morphogenetic protein 2 and vascular endothelial growth factor in the callus of treatment group was significantly higher than that of model group at 2 and 4 weeks after intervention.The results of quantitative PCR showed that the mRNA expression of bone morphogenetic protein 2 and vascular endothelial growth factor in the treatment group was higher than that in the model group at 2 and 4 weeks after intervention.To conclude,the whole body application of nerve growth factor can enhance the osteogenesis of tibial fracture end and promote the early healing of tibial fracture in rats.At the same time,nerve growth factor can promote the expression of bone morphogenetic protein 2 and vascular endothelial growth factor,which shows certain regularity in different periods,and in turn induces the differentiation and proliferation of osteoblasts as well as formation and calcification of extracellular matrix to promote bone formation and bone healing.