骨骼肌急性钝挫伤修复过程中自噬相关因子的表达变化

doi:10.12047/j.cjap.5647.2018.024

摘要/Abstract

摘要： 目的：本研究通过观察SD大鼠骨骼肌急性钝挫伤修复过程中自噬相关因子的表达变化，探讨骨骼肌损伤修复可能的生物学机制。方法：30只SD雄性大鼠，随机选取6只作为对照组，其余24只用打击器打击后建立腓肠肌急性钝挫伤模型，然后随机分为4组（n=6），各组分别在造模前及造模后3 d、5 d、7 d、14 d取材，HE染色观察损伤部位腓肠肌形态学变化，透射电镜观察损伤部位腓肠肌超微结构变化，Western blot检测腓肠肌自噬相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）、泛素结合蛋白P62表达水平，RT-PCR检测腓肠肌自噬相关基因（atg） atg7、atg10、atg12、atg16L1 mRNA表达水平。结果：HE染色显示：与对照组相比，骨骼肌损伤后5 d炎细胞浸润达到高峰，7 d可见明显的新生肌细胞，14 d损伤已初步愈合。电镜观察显示：与对照组相比，损伤后3 d，5 d，7 d线粒体肿胀明显、空泡化增多，Z线从消失到飘移增粗，肌质网扩张程度逐渐好转，14 d接近对照组水平。Western blot显示：骨骼肌损伤在自然恢复3 d、5 d、7 d、14 d过程中，LC3-Ⅱ与P62总体呈现先升高后降低的趋势，其中3 d、5 d、7 d组LC3-Ⅱ表达较对照组与14 d组明显升高（P<0.01），同样损伤后第3天P62表达达到高峰（P<0.01），14 d恢复至正常水平。RTPCR显示：骨骼肌损伤自然恢复3 d、5 d、7 d、14 d过程中，atg10 mRNA表达呈现先降低后升高的趋势，其中3 d、5 d、7 d组atg10 mRNA表达较对照组与14 d组明显降低（P<0.01）；atg7、atg12、atg16L1 mRNA表达总体呈现先升高后降低的趋势，其中3 d、5 d、7 d组表达较对照组与14 d组显著升高（P<0.01，P<0.05，P<0.01）。结论：骨骼肌急性钝挫伤后，自噬相关因子表达随损伤的修复而呈现规律性变化，提示自噬参与骨骼肌损伤的修复，推测骨骼肌急性钝挫伤的修复速度可能与细胞自噬水平有关。

关键词: 骨骼肌, 急性钝挫伤, 损伤修复, 自噬, 大鼠

Abstract: Objective:To explore the possible biological mechanism of skeletal muscle contusion repair through researching the changes in expression of autophagy-related genes and proteins in SD rats with acute skeletal muscle contusion. Methods:Six rats were randomly selected as the control group from 30 male SD rats, acute skeletal muscle contusion model were established in the remaining 24 rats with self-made hitter, then the model rats were randomly divided into 4 groups (3 d, 5 d, 7 d, 14 d groups, n=6). On the 3^rd, 5^th, 7^th and 14^th day after injury, injured gastrocnemius of each group was harvested. The morphological and the ultra-microstructure changes of gastrocnemius after injury were observed by HE staining and transmission electron microscope (TEM) respectively. The relative protein levels of (LC3-Ⅱ) and P62 of each group were observed by Western blot. The relative mRNA levels of atg7, atg10, atg12, atg16L1 of each group were observed by RTPCR. Results:The results of HE staining showed that compared with the control group, the inflammation reached its peak on the 5^th day after injury, new muscle fibers were clearly observed in 7 d group. The results of TEM showed that, compared with the control group, oncotic mitochondria could be clearly seen in the 3 d, 5 d, 7 d groups. Also, the Z line changed from disappearing to drift thickening, sarcoplasmic reticulum dilatation gradually improved, there was no evident difference between the 14 d group and the control group, suggesting that the damage has preliminarily healed. The results of Western blot showed that the expressions of LC3-Ⅱand P62 were increased at first and then decreased. The expression of LC3-Ⅱwas markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (P<0.01). Similarly, compared with the control group, the expression of P62 reached its peak on the 3^rd day after injury (P<0. 01), and returned to normal level on the 14^th day. The results of RT-PCR showed that the expression of atg10 mRNA in the natural recovery group of 3 d, 5 d, 7 d, 14 d was firstly decreased and then increased, the atg10 mRNA was markedly down-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (P<0. 01). The expression of atg7, atg12, atg16L1 mRNA was generally increased at first and then decreased, it was markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (P<0.01, P<0.05, P<0.01). Conclusion:The above results indicate that the autophagy is involved in repair of skeletal muscle injury by its autophagyrelated factors,regularly changes after contusion, and the rate of damage repair may be related to the level of autophagy.

Key words: skeletal muscle, acute blunt trauma, damage repair, autophagy, rat

罗翱, 唐成林, 黄思琴, 赵丹丹, 张安宁, 郭全虎, 高睿琦, 曹净. 骨骼肌急性钝挫伤修复过程中自噬相关因子的表达变化[J]. 中国应用生理学杂志, 2018, 34(2): 97-101.

LUO Ao, TANG Cheng-lin, HUANG Si-qin, ZHAO Dan-dan, ZHANG An-ning, GUO Quan-hu, GAO Rui-qi, CAO Jing. Changes in expression of autophagy-related factors during acute contusion repair of skeletal muscle[J]. CJAP, 2018, 34(2): 97-101.

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