大鼠失神经支配骨骼肌萎缩后肌细胞凋亡及Bcl-2、Bax表达的变化

Myocyte apoptosis and Bcl-2 and Bax expression changes in rat atrophic gastrocnemius muscle after denervation

目的 研究失神经支配骨骼肌萎缩后肌细胞凋亡及Bcl-2、Bax表达的变化规律.方法 实验以失神经支配腓肠肌为动物模型.选用体质量为200～250 g的雌性SD大鼠42只,随机分成2组,实验组36只,健康对照组6只.按术后取材时间的不同再将实验组分为6组,每组6只大鼠.分别运用Tunel法及免疫组织化学方法检测失神经后不同时间萎缩腓肠肌细胞凋亡及Bcl-2、Bax的表达变化,同时测量肌湿重比和肌纤维横截面积,透射电镜观察肌细胞的超微结构变化.结果 萎缩的腓肠肌湿重比在失神经4周内和第10周到第12周下降最快,而在第4周到10周和第12周到16周下降较慢,且变化差异无统计学意义（P＞0.05）.Bcl-2仅在失神经早期表达增高,于第4周达高峰;而Bax分别于失神经第2周与第12周达高峰,增高幅度大于Bcl-2.Bcl-2/Bax的比值除对照组（1.522±0.215）和失神经第8周时（1.065±0.165）大于1外,其余各时间点均小于1.肌细胞凋亡率分别于失神经第4周和第12周达到高峰,其变化趋势与Bax相仿.结论 失神经腓肠肌萎缩过程呈现4个阶段;凋亡和凋亡相关基因Bax过量表达在失神经不可逆肌萎缩过程中发挥着重要的作用;靶向作用Bax基因,抑制Bax表达可能延缓失神经骨骼肌萎缩.

Objective To study and analysis changes of apoptosis and expression pattern of Bcl-2 and Bax proteins and their potential role in skeletal muscle denervation atrophy. Methods Gastrocnemius muscle denervation was created in rats. Forty-two female SD rats weighed 200 to 250 g were randomly divided into two groups, 6 for normal control group and 36 for experimental group which were further randomly divided into 6 groups based on different intervals for tissue harvesting. The changes of apoptosis and expression of Bcl-2 and Bax proteins in atrophic gastrocnemius muscles were detected by using Tunel method and immunohistochemical staining at different intervals after denervation. The muscle wet weight ratio and cross-sectional aera of muscle fibers were measured. The ultrastrictiral changes of muscle fiber were also observed. Results There was a rapid decrease and statistically significant difference in the muscle wet weight ratio during the first 4 weeks as well as the time from the 10th week to the 12th week after denervation, but a slow decrease and no statistically significant difference （ P ＞ 0.05） during the time from the 4th week to the 10th week as well as the time from the 12th to the 16th week. Bcl-2 was modestly increased only in the early period after denervation and peaked at the 4th week while Bax was dramatically increased and peaked at the 2nd and 12th week repectively. The Bcl-2/Bax ratio had significantly decreased after denervation and it was smaller than 1 except for the group of 8 week denervation （ 1.065 ± 0.165） and the normal group （ 1.522 ± 0.215）. The apoptotic ratio peaked at the 4th and 12th week after denervation and the changes was similar to the apoptosis and over expression of Bax had an important role in regulating inreversible Bax. Conclusion The process of denervation gastrocnemius atrophy has shown four stages. Apoptosis and Bax over expression play an important in atrophy of denervated skeletal muscle. These findings suggest that interventions targeting Bax gene to inhibit its expression may delay skeletal muscle denervation atrophy.