骨形态发生蛋白-4在创伤性骨化性肌炎大鼠模型的表达及意义

Expression and significance of bone morphogenetic protein-4in rat model of traumatic ossifying myositis

目的探讨骨形态发生蛋白-4 (BMP-4)在大鼠创伤性骨化性肌炎(TMO)模型的表达及意义。方法选取60只4~6周龄SD大鼠,随机分为对照组、模型组和药物组,模型组和药物组均建立大鼠右侧跟腱创伤性异位骨化模型,药物组大鼠予塞来昔布10mg/(kg·d)灌胃,模型组、对照组分别灌胃2mL生理盐水,于第5、10周各麻醉30只实验动物,拍片观察异位骨化形成情况,定量分析BMP-4与IL-2基因表达,以Western blot检测BMP-4蛋白的表达。结果模型组与药物组跟腱均有异位骨的形成,药物组跟腱异位骨化形成速率低于模型组,发生成骨时间较晚,差异有统计学意义(P<0.05)。术后实时定量PCR检测数据表明,模型组大鼠右跟腱组织中BMP-4基因表达明显高于左跟腱组织,药物组大鼠右跟腱组织中BMP-4基因表达显著降低。蛋白质免疫印迹检查组织中BMP-4蛋白的表达发现,药物组大鼠骨化组织部位BMP-4蛋白明显低于模型组。免疫组化结果表明,模型组大鼠组织中BMP-4蛋白表达的阳性率明显高于同只大鼠左腿对照组织;药物组大鼠跟腱组织中BMP-4表达的阳性率较模型组降低,并药物组BMP-4表达阳性率随着用药时间的延长而降低,跟腱组织中BMP-4表达的阳性率与模型组比较,差异有统计学意义(P<0.05)。结论 BMP-4在创伤性骨化性肌炎大鼠模型的表达与骨化程度呈正相关,塞来昔布可有效地减少或减轻大鼠跟腱术后炎症反应,抑制BMP-4表达,从而减缓创伤性骨化性肌炎的发生。

Objective To investigate the expression and significance of bone morphogenetic protein-4 in rat model of traumatic ossifying myositis.Methods Sixty SD rats aged 4-6 weeks were randomly divided into control group,model group and drug feeding group.Both model group and drug feeding group established traumatic heterotopic ossification model in the right Achilles tendon of rats.Rats in drug feeding group were given celecoxib 10 mg/(kg·d)gavage,and rats in model group and control group were given 2 mL normal saline respectively.Experimental animals were killed at 5 and 10 weeks respectively.X-ray film was used to examine the formation of new bone.The expression of bmp-4 and IL-2 genes were quantitatively analyzed by taking samples,and the expression of bmp-4 protein was detected by Western blot.Results The ossification degree of the model group was lighter than that of the model group,and the achilles tendon of the model group and the drug group increased gradually with the experimental cycle,and the formation of heterotopic bone was observed in both groups.However,the overall formation rate of the model group was faster than that of the drug group,and the osteogenesis time was earlier than that of the drug group,with a significant difference(P<0.05).QPCR detection showed that the expression of bmp-4 gene in the right achilles tendon tissue of the model group was significantly higher than that in the left achilles tendon tissue of the model group five weeks after surgery,while the expression of bmp-4 gene in the right achilles tendon tissue of the cecb group was significantly lower than that in the model group.Protein expression of bmp-4 in tissue by western blot showed that bmp-4 protein in ossified tissue of rats in the drug feeding group was significantly lower than that in the model group.Immunohistochemical results showed that the positive rate of bmp-4 protein expression in rat tissues of the model group was significantly higher than that in the left leg of the same rat.The positive rate of bmp-4 expression in the achilles tendon tissue of rats in the feeding group was significantly decreased,and with the extension of administration time,the positive rate of bmp-4 expression in the achilles tendon tissue was more and more significantly different from that in the model group.Conclusion Celecoxib can effectively reduce or alleviate the occurrence of traumatic ossifying myositis after Achilles tendon surgery in rats,which may be related to the inhibition of bmp-4 expression by celecoxib.