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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、West-ern 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 changes of muscle morphology and the expression of fibrosis-related factor TGF-β1 in denervated circumflex ciliary muscles and provide a theoretical basis for the clinical choice of laryngeal nerve repair and time-limit. Methods: The posterior circumflex cochlear muscle samples were divided into 4 groups according to the time of nerve injury: 6 to 12 months (12 cases),> 1 to 2 years (10 cases),> 2 to 3 years (8 cases) and> 3 years (8 cases). The normal control group was normal muscle (12 cases). Masson trichrome staining was used to observe the changes of muscle morphology. The changes of the time, space and intensity of TGF-β1 expression in the cochlear tendon after long-term denervation were observed by immunohistochemical staining and image analysis trend. Results: The results of Masson’s trichrome staining showed that with the denervation time prolonged, myofibroblasts gradually atrophied, the cross-sectional area decreased, the number of collagen fibers increased and the cross-sectional area of collagen fibers increased. Muscle cross-sectional area / collagen cross-sectional area ratio decreased. The decrease of muscle cross-sectional area / collagen cross-sectional area ratio from 0 to 2 years was the most obvious in denervated nerve, but the relative cross-sectional area of muscle in 3-year denervated still remained 36%, which was 48% of the normal control group. Immunofluorescence staining showed that the expression of TGF-β1 in the denervated neurons of 6 to 12 months and> 2 to 3 years group was higher than that of the control group, the expression of TGF-β1 was highest in the group of> 1 to 2 years, then decreased gradually, and almost no expression in 3 years . There was significant difference between the groups of denervation (all P <0.01). There was no significant difference between denervation> 3-year group and normal control group (P> 0.05). The Western blot results of TGF-β1 showed that in 6-12 months group, it was 4.5 times higher than that in normal control group and peaked in> 1-2 years group, which was 11.4 times of normal control group, then decreased slowly;> 2-3 years group Down to 6 times the normal control group;> 3-year group was 1.2 times the normal control group. Conclusion: Nerve repair within 2 years may be the best period to prevent muscle fibrosis. However, after 3 years of denervation, nerve repair still has the basis of muscle morphology.