长期失神经支配人环杓后肌肌纤维化形态观察及TGF-β_1表达研究

The morphological alteration in myofiber fibrosis and expression in TGF-β_1 in long-term denervated posterior cricoarytenoid muscles

目的：探讨长期失神经环杓后肌肌肉形态学变化及纤维化相关因子TGF-β1蛋白水平表达的变化，为临床开展晚期喉神经修复及时限的选择提供理论依据。方法：按神经损伤时限将喉返神经损伤患者的环杓后肌标本分为4组：6～12个月组（12例）、〉1～2年组（10例）、〉2～3年组（8例）及〉3年组（8例）；正常对照组为正常肌肉（12例）。通过Masson三色染色观察肌肉形态学变化，通过免疫组织化学荧光染色及图像分析、Western blot观察长期失神经后环杓后肌肌纤维化相关因子TGF—β1表达的时间、空间及强度的变化趋势。结果：Masson三色染色显示：随着失神经时限延长，肌纤维细胞逐渐萎缩，截面积减小，胶原纤维细胞逐渐增加，胶原纤维截面积增加。肌肉截面积／胶原截面积比率逐渐下降。失神经0．5～2年肌肉截面积／胶原截面积比率下降最为明显，但失神经〉3年组肌肉相对截面积仍残留36％，为正常对照组的48％。免疫荧光染色显示：TGF—β1在失神经6～12个月组及〉2～3年组肌细胞质有较高表达，〉1～2年组表达最高，后逐渐下降，3年后基本无表达。失神经各组比较均差异有统计学意义（均P〈0．01），失神经〉3年组与正常对照组相比差异无统计学意义（P〉0．05）。TGF—β1的Western blot结果表明：6～12个月组为正常对照组的4．5倍，〉1～2年组达到高峰，为正常对照组的11．4倍，随后缓慢下降；〉2～3年组降为正常对照组的6倍；〉3年组为正常对照组的1．2倍。结论：失神经2年内进行神经修复可能是阻止肌肉纤维化的最佳时期．但失神经3年后作神经修复仍有其肌肉形态学的基础。

Objective：To investigate the morphological alteration and the protein expression of TGF-β1 in long-term denervated posterior cricoarytenoid muscles（PCAMs）and to find the appropriate time point of reinneration in long-term denervated PCAMs. Method：Thirty-eight patients with vocal paralysis were recruited into four groups, the 6-12 months denervation group , the 1- 2 years denervation group, the 2-3 years denervation group and the over 3 years denervation group. Twelve adults sereved as a control group. The morphological alteration was evaluated using Trichrome Masson staining and the change in expression of TGF-β1, a fibrosis related factor, were observed using immunofluorescence stain and Western blot. Result： The secars of myofibers were decreased, but the secars of collagen fibers were increased with the denervated time course extension. The ratio of seears of myofibers to secars of collagen fibers were progressively decrease with increased time course of denervation and more decrease within 2 years. There was still 48 % secar of myofibers remained after 3 years of denervation. TGF-β1 showed no staining in innervated, however, by 6-12 months and 1- 2 years of muscle denervation there was a significant accumulation of TGF-β1 protein in endochylema of myofiber. After 2 year of denervation, expression of TGF-β1 protein in endochylema of myofiber, was decreased significantly, but after 3 year of denervation, there were no expression of TGF-β1 protein in endochylema. In Western blot, the change in protein expression of TGF-β1 was observed an 4.5-fold （P〈0.01） increase from 6-12 months denervated muscles to innervated muscles, 11.4-fold （P〈0.01） increase from 1-2 years denervated muscles to innervated muscles, 11.4-fold （P〈0.01） increase from 2-3 years denervated muscles to innervated muscles （P〈0.01）. After 3 years , the protein expression of TGF-β1 decreased ,0.5-fold to innervated muscles. Conclusion：The morphological alteration and change in expression of TGF - β1 , a important factor of myofibrosis , indicated there was a high amplitude of myofibrosis within 2 years,but there is a morphological basis of muscles to regain their function within 3 years denervated muscles.