肌肉游离移植后MAFbx表达及肌肉功能实验研究

MAFbx EXPRESSION AFTER FREE MUSCLE TRANSPLANTATION AND ITS RELATIONSHIP WITH MUSCLE FUNCTION

目的观察肌肉游离移植后泛素连接酶(MAFbx)mRNA和蛋白表达的变化,以及肌肉萎缩和肌肉功能的动态恢复情况,并探讨三者之间的关系。方法雌性SPF级SD大鼠36只,体重(250±25)g。随机选取一侧行股薄肌原位游离移植手术,作为实验侧;另一侧不行手术作为对照侧。术后观察大鼠一般情况,于术后1、2、4、10、15和30周,分别取6只大鼠测量实验侧和对照侧股薄肌肌肉收缩能力、肌湿重维持率,HE染色观察肌纤维横截面积,实时定量PCR检测MAFbx/Atrogin-1mRNA相对表达量,Western blot检测MAFbx蛋白表达。结果36只大鼠全部成活,切口愈合好,实验侧肢体功能无障碍。实验侧肌纤维横截面积、肌肉湿重维持率、肌肉最大单收缩力维持率和肌肉强直收缩力维持率均呈先降低(术后1～4周)后增加(术后4～30周)的趋势。MAFbxmRNA相对表达量术后2周最高,为对照侧7倍;术后15～30周逐渐下降,为对照侧1.1～1.5倍;各时间点实验侧与对照侧比较,差异均有统计学意义(P<0.05)。Westernblot检测结果显示术后1～15周,实验侧肌肉内MAFbx蛋白相对表达量与对照侧相比,差异均有统计学意义(P<0.05);术后30周差异无统计学意义(P>0.05)。肌湿重维持率、肌纤维横截面积恢复率分别与最大单收缩力维持率和最大强直收缩力维持率的相关系数为0.95、0.75和0.93、0.68(P<0.05)。MAFbxmRNA及蛋白相对表达量分别与肌肉湿重维持率、肌纤维横截面积恢复率、最大单收缩力维持率、最大强直收缩力维持率的相关系数为—0.62(P<0.05)、—0.45(P>0.05)、—0.72(P<0.05)、—0.78(P<0.05)和—0.95(P<0.05)、—0.82(P<0.05)、—0.89(P<0.05)、—0.54(P>0.05)。结论移植肌肉功能降低与肌肉萎缩密切相关。MAFbxmRNA和蛋白表达增高,尤其在获得神经支配后4～15周仍持续升高,可能是肌肉萎缩引起肌肉功能降低的一个联系点。

Objective To study the quantitative changes of ubiquitin ligase MAFbx mRNA and protein expression, muscle atrophy and muscle function following free muscle transplantation and to explore relationships among them. Methods Thirty-six female SD rats, SPF grade, weighing （250 ± 25） g, were used. One hind limb of the rat was randomly selected as experimental side to receive in situ free gracilis muscle transplantation, and the counterlateral hind limb underwent no operation serving as control side. General condition of the rats was observed after operation. Muscle contractive capacity and muscle wet weight maintenance rate of the experimental and the control side were detected 1, 2, 4, 10, 15, and 30 weeks after operation, and 6 rats were killed at each time point. Meanwhile, HE staining was performed to observe muscle fibre cross-sectional area, real-time quantitative PCR was applied to detect relative expression of MAFbx/Atrogin-1 mRNA, and Western blot test was used to observe MAFbx protein expression. Results All rats survived till the end of the experiment, all incisions healed well, and no dysfunction occurred in the experimental sides. The value of muscle contractive capacity, muscle wet weight maintenance rate, muscle＇s maximal force of single contraction, and muscle＇s maximal force of tetanic contraction in the experimental sides dramatically decreased in the first 4 weeks after operation and increased gradually over 4 to 30 weeks. The MAFbx mRNA expression of the experimental sides peaked and was seven times greater than the control sides 2 weeks after operation, then the value gradually decreased over 15 to 30 weeks after operation and was 1.1 to 1.5 times greater than the control sides, and significant difference was evident between the experimental sides and the control sides at each time point （P 〈 0.05）. Significant difference was evident between the experimental sides and the control sides in terms of MAFbx protein expression of the muscle 1 to 15 weeks after operation according to the Western blot result （P 〈 0.05）, and no significant difference was noted at 30 weeks （P 〉 0.05）. The correlation coefficient between muscle wet weight maintenance rate and muscle＇s maximal force of single contraction maintenance rate was 0.95, between muscle wet weight maintenance rate and muscle＇s maximal force of tetanic contraction maintenance rate was 0.75, between muscle fibre cross-sectional area recovery rate and muscle＇s maximal force of single contraction maintenance rate was 0.93, and between muscle fibre cross-sectional area recovery rate and muscle＇s maximal force of tetanic contraction maintenance rate was 0.68 （P 〈 0.05）. The correlation coefficient between MAFbx mRNA expression and the parameter of muscle wet weight maintenance rate, muscle fibre cross-sectional area recovery rate, muscle＇s maximal force of single contraction maintenance rate, and muscle＇s maximal force of tetanic contraction maintenance rate was -0.62 （P 〈 0.05）, -0.45 （P 〉 0.05）, -0.72 （P 〈 0.05） and 0,78 （P 〈 0.05）, respectively; the correlation coefficient between MAFbx protein relative expression and the parameter of muscle wet weight maintenance rate, muscle fibre cross-sectional area recovery rate, muscle＇s maximal force of single contraction maintenance rate, and muscle＇s maximal force of tetanic contraction maintenance rate was -0.95 （P 〈 0.05）, -0.82 （P 〈 0.05）, -0.89 （P 〈 0.05）, and -0.54 （P 〉 0.05）, respectively. Conclusion Decrease of muscle function after transplantation correlates closely with muscle atrophy. The high expression of MAFbx mRNA and protein, especially their persistent increases from 4 to 15 weeks after nerve reinnervation, is a junction between the muscle atrophy and the decrease of muscle function.