不同正畸力值作用下大鼠脑前扣带回皮质层蛋白激酶Mξ的表达

Changes of protein kinases Mζ expression in the anterior cingulate cortex after applying three different magnitude of orthodontic force

目的 探讨不同正畸力值作用下大鼠大脑前扣带回皮质层蛋白激酶Mζ （protein kinasesMζ,PKMζ）表达水平的变化,进一步探讨中枢神经系统的突触可塑性在牙移动疼痛过程中的调控作用.方法 选用200 ～ 250 g雄性SD大鼠136只,使用随机数字表法随机分为加力组（96只）、阴性对照组（20只）和空白对照组（20只）.加力组大鼠安装实验性牙移动装置并分别施加0.39、0.78、1.17 N正畸力（每个加力亚组各32只）,阴性对照组安装相同装置但不加力,空白对照组不予任何处理.观察、记录各组大鼠挠嘴时间,并于术后3d取大脑前扣带回皮质组织（3个加力亚组及两个对照组各20只）,免疫组织化学法和酶联免疫吸附测定法检测PKMζ的蛋白表达水平.术后3d向各加力亚组大鼠前扣带回皮质区域分别注射生理盐水和PKMζ抑制剂（ζ-pseudosubstrate inhibitory peptide,ZIP）（每加力亚组12只）,观察大鼠挠嘴时间的变化.结果 两个对照组挠嘴时间、免疫组化吸光度值和PKMζ蛋白表达差异均无统计学意义（P＞0.05）.随正畸力值的增大,0.39、0.78、1.17 N加力亚组大鼠的挠嘴时间[（58.6±6.9）、（66.3±7.8）、（78.9±8.7）s]、免疫组化吸光度值（4 569±454、6 850±365、8 294±558）和PKMζ蛋白表达水平[（0.32±0.02）、（0.34±0.02）、（0.36±0.02） mg/L]均逐渐升高,3个加力亚组间、各加力亚组与空白对照组、阴性对照组间的差异均有统计学意义（P＜0.05）.注射生理盐水前后各加力亚组大鼠挠嘴时间差异均无统计学意义（P＞0.05）;注射ZIP后各加力亚组大鼠挠嘴时间与注射前相比均显著减少（P＜0.01）.结论 正畸力值增大引起大鼠疼痛程度的增加,可能与大脑前扣带回皮质层PKMζ表达水平升高相关.

Objective To investigate the regulatory effect of central synaptic plasticity on pain induced by experimental tooth movement and to analyzethe expression of protein kinases Mζ（PKMζ） in the anterior cingulate cortex（ACC） after applying different magnitude of orthodontic force.Methods One hundred and thirty-six male Sprague-Dawley（SD） rats（200-250 g） were used in this study.Orthodontic tooth movement devices were placed on the teeth in the experimental group,and different orthodontic forces（0.39,0.78,1.17 N） were applied to move the maxillary first molars,respectively.The same mechanical devices were placed on the teeth in sham-treated group and no orthodontic force was applied.No orthodontic procedure was applied in blank control group.The average time spent on mouth-wiping behavior in each group was recorded after experimental tooth movement.Brain tissue of the anterior cingulate cortex was isolated on day 3 after experiment,and the expression level of PKMζ was analyzed with the method of immunohistochemistry and enzyme-linked immune sorbent assay.ζ-pseudosubstrate inhibitory peptide（ZIP）,a selective inhibitor for PKMζ,was injected into ACC on day 3 after experimental tooth movement,and the effects of ZIP on mouth-wiping behavior were evaluated.Results No statistical difference was found between the blank control group and the sham-treated group in the average time spent on mouth-wiping,value of A and expression level of PKMζ（P〉0.05）.Compared with the sham-treated group and blank control group,the average time of mouth-wiping behavior[（58.6±6.9）,（66.3±7.8）,（78.9±8.7） s],value of A（4 569±454,6 850±365,8 294±558） and expression level of PKMζ[（0.32±0.02）,（0.34±0.02）,（0.36±0.02） mg/L] in 0.39,0.78,1.17 N force group were found to be up-regulated with the increase of orthodontic force（P〈0.05）.LSD test in three experimental sub-group showed statistical difference（P〈0.05）.After microinjection of ZIP,the average time spent on mouth-wiping behavior significantly decreased（P〈0.01）,while microinjecting saline did not change rats＇ mouth-wiping behavior（P〉0.05）.Conclusions More pain caused by increased orthodontic force maybe due to the up-regulation of PKMζ in the anterior cingulate cortex.