去神经对大鼠骨骼肌碳酸酐酶Ⅲ表达和磷酸酶活性的影响

Effects of denervation on expression of carbonic anhydrase Ⅲ and its phosphatase activity in skeletal muscle of rats

目的 观察去神经对骨骼肌碳酸酐酶Ⅲ（carbonic anhydrase Ⅲ,CAⅢ）表达及其磷酸酶（phosphatase）活性的影响,探讨神经冲动受阻是否为重症肌无力（myasthenia gravis,MG）骨骼肌CAⅢ减少的原因.方法 定向切断支配大鼠趾长伸肌（extensor digitorum longus,EDL）和比目鱼肌（soleus,Sol）的神经纤维,术后第7、14、28和56天用Western blot分析EDL和Sol的CAⅢ水平,用固相膜上原位酶活性染色方法评估CAⅢ的磷酸酶活性.结果 （1）正常侧（即去神经对侧）Sol的CAⅢ水平远高于EDL,并且两者都表现出随时间增加（动物年龄增长）而增加的趋势.去神经后,EDL的CAⅢ水平随时间的延长而逐渐增加;Sol的CAⅢ水平则以14 d为分界先增加后降低.（2）正常侧Sol的CAⅢ的磷酸酶活性[随时间增加（动物年龄增长）呈逐渐增加的趋势]均高于EDL（变化不明显）.去神经后,Sol的CAⅢ磷酸酶活性（第14、28、56天分别为14.39±1.93、11.48±1.46、9.04±1.46）明显低于正常侧（22.75±1.80、25.26±3.15、25.82±2.97,t=0.002、0.005、0.002,均P＜0.05）,EDL的CAⅢ磷酸酶活性与正常侧相比亦是降低,但差异无统计学意义.（3）正常侧EDL和Sol的CAⅢ蛋白表达水平和CAⅢ的磷酸酶活性相一致;去神经后CAⅢ蛋白表达水平和CAⅢ的磷酸酶活性发生了背离,即CAⅢ蛋白表达水平增加,但其磷酸酶活性却降低.结论 去神经所致的神经冲动传递障碍与MG自身抗体所致的神经冲动传递障碍对骨骼肌CAⅢ表达水平的影响不同,MG骨骼肌CAⅢ表达水平减少并非是其自身抗体所致的神经冲动传递障碍造成.

Objective To observe the effects of nerve impulses on the expression of carbonic anhydrase Ⅲ （ CAⅢ ） and its phosphatase activity, and to explore whether or not the cause of CAⅢ expressive decreased in skeletal muscles of myasthenia gravis（ MG） is resulted from the obstruction of nerve impulse.Methods The motor nerves of extensor digitorum longus （EDL, mainly composed by fast fibers） and soleus （Sol, mainly composed by slow fibers） were cut off by operation of denervation.Levels and phosphatase activities of CAⅢ were analyzed at 7, 14, 28, and 56 d after denervation by Western blot and specific enzyme staining on the membrane following SDS-polyacrylamide gel electrophoresis, respectively.Results （1） Levels of CAⅢ in Sol of normal side （eg denervated contralateral） were much higher than that in EDL of normal side, and the levels in both Sol and EDL had an enhanced tendency with time （age） increase, especially for Sol.After denervation, the levels of CAⅢ in EDL were gradual increased, however, the level in Sol was 14 d after denervation as the boundary of ascension and then decline.（ 2） The phosphatase activities of CAⅢ in Sol of normal sides were much higher than that in EDL of normal sides, and there were an enhanced tendency with time （age） increase in Sol, but no significant changes were found in EDL The enzyme activities in denervated Sol were lower（in the 14, 28, and 56 days after denervation： 14.39 ±1.93, 11.48 ±1.46, 9.04 ±1.46） much than their contralaterals（22.75 ± 1.80, 25.26 ±3.15, 25.82 ± 2.97; t = 0.002, 0.005, 0.002, all P ＜ 0.05）, the enzyme activities in denervated EDL were also lower than their contralaterals, however, no significant differences were found.（3）It was consistent for CAⅢ levels and phosphatase activities in both Sol and EDL of normal sides.After denervation, however, the deviation of the CAⅢ levels and phosphatase activities happened, the levels of CAⅢ were increased, but the phosphatase activities were decreased.Conclusions The effect of nerve impulse transferring obstructed by denervation on CAⅢ expression of skeletal muscles is different from that by MG auto-antibody.The decrease of CAⅢ protein in the MG muscles may be not resulted from the nerve impulse transferring obstructed by MG auto-antibody.