电磁场干预对异位骨化大鼠模型的抗异位骨化效应及机制探讨

The mechanism by which sinusoidal electromagnetic irradiation inhibits heterotopic ossification

目的观察电磁场干预对异位骨化(HO)大鼠模型的抗异位骨化效应并探讨相关作用机制。方法采用随机数字表法将72只雄性SD大鼠分为对照组、模型组及观察组。采用钳夹、切断跟腱等方式将模型组、观察组大鼠制成HO动物模型,对照组术中仅暴露跟腱组织,未给予钳夹、切断处理。观察组大鼠于制模后每天给予2次50 Hz、1 mT正弦交变电磁场暴露干预,每次持续2 h。于术后3 d、14 d、1个月及3个月时每组各取6只大鼠,提取跟腱组织进行大体、组织学及免疫组化染色观察,并采用Western blot或RT-PCR方法检测标本中母亲DPP同源物7(Smad7)、母亲DPP同源物3(Smad3)、磷酸化母亲DPP同源物3(p-Smad3)、基质金属蛋白酶9(MMP-9)及组织金属蛋白酶抑制剂1(TIMP-1)等蛋白或基因表达情况;于术后1个月、3个月时对各组大鼠患肢跟腱部位进行X线检查以了解异位骨化形成情况。结果术后各时间点对照组大鼠各项检测指标(包括跟腱大体外观、HE染色、免疫组化染色以及X线检查等)均无明显变化;与模型组比较,观察组异位骨化形成被明显抑制。模型组及观察组MMP-9基因表达量均显著高于对照组(P<0.05),且随时间延长呈上升趋势;TIMP-1基因表达量均显著低于对照组(P<0.05),且随时间延长呈下降趋势。与模型组比较,观察组术后各时间点MMP-9表达量均明显降低(P<0.05),TIMP-1表达量均明显升高(P<0.05)。与模型组比较,观察组术后各时间点Smad7蛋白含量均明显增加,p-Smad3蛋白含量均明显减少。结论50 Hz、1 mT正弦交变电磁场干预对HO大鼠模型具有明确的抗异位骨化效应,其作用机制可能与促进Smad7表达,阻止Smad3磷酸化,从而抑制转化生长因子-β(TGF-β)/Smad信号转导通路,促进MMP-9/TIMP-1重新恢复动态平衡有关。

Objective To observe electromagnetic field irradiation′s ability to inhibit heterotopic ossification(HO)and explore the mechanisms involved.Methods A total of 72 male Sprague-Dawley rats were randomly divided into a control group,a model group and an observation group,each of 24.The rats in the model and observation groups were made to model HO by clamping and cutting the Achilles tendon,while among those in the control group the Achilles tendon was only exposed without clamping and cutting.The rats in the observation group were exposed to a 1mT electromagnetic field alternating sinusoidally at 50Hz(SEMF)twice a day for 2h each time.At 3 days,14 days,1 month and 3 months after the operation,Achilles tendon tissue was resected from 6 rats in each group for gross and histological examination and immunohistochemical staining.The protein content and gene expression were detected using western blotting or reverse transcription polymerase chain reaction.X-ray examination was performed at 1 and 3 months after the operation to observe any HO.Results No significant changes were observed in the control group after the operation.Compared with the model group,HO in the observation group was significantly inhibited.The average expression of the MMP-9 gene in the model and observation groups was significantly higher than in the control group,and it increased with time.The average tissue metalloproteinase-1(TIMP-1)gene expression was significantly lower,and it decreased with time.Compared with the model group,the average expression of MMP-9 in the observation group was significantly less,while that of TIMP-1 was significantly greater.Compared with the model group,there was also a significant increase in the expression of decapentaplegic homolog 7(Smad7)protein and a significant decrease in the expression of phosporylated Smad3 protein at each time point.Conclusion Applying 50Hz,1mT SEMF has a definite effect in inhibiting HO,at least in rats.It may be related to promoting the expression of Smad7 and inhibiting the phosphorylation of Smad3,thus inhibiting the TGF-β/Smad signal transduction pathway and promoting the restoration of a dynamic balance of MMP-9 with TIMP-1.