严重烧伤后大鼠骨骼肌超微结构、微区Ca2＋含量及钙蛋白酶变化规律的研究

目的：探讨严重烧伤后大鼠骨骼肌超微结构、骨骼肌细胞微区Ca2＋含量及钙蛋白酶（calpain-1和calpain-2）的变化规律。方法选取48只雄性SD大鼠按照速记数字表法分为对照组（n＝8）和实验组（n＝40，5个时相点，每时相点8只）。实验组大鼠在麻醉后背部浸入94℃热水12 s，造成30％总体表面积（TBSA ）Ш度烫伤；对照组大鼠背部同面积浸入37℃温水行假烫伤，其余操作同实验组。于伤后即刻、24h、3d、7d、14d采集两组大鼠腹主动脉血离心获取血清并收集各组大鼠胫骨前肌组织。透射电镜观察胫骨前肌组织超微结构，电子探针X射线显微分析法（EPMA）检测骨骼肌细胞微区Ca2＋相对含量，酶联免疫吸附测定（ELISA）法测定血清中乳酸脱氢酶（LDH）、肌酸激酶（CK）活性，酶法检测calpain的活性，蛋白印迹方法检测胫骨前肌中calpain-1和calpain-2的含量。对数据进行单因素方差分析和独立样本t检验。结果对照组大鼠胫骨前肌肌原纤维排列整齐；实验组大鼠伤后24 h胫骨前肌肌原纤维排列混杂，伤后3 d肌丝轻度溶解，Z线不规则，伤后7 d局部Z线消失。实验组骨骼肌细胞细胞质、线粒体Ca2＋相对含量伤后即刻开始升高，伤后24 h 达到峰值，其Ca2＋相对含量分别为0．96±0．06、1．08±0．11，与对照组0．27±0．03、0．60±0．07相比差异有统计学意义（P＜0．05），同时伤后24 h肌浆网Ca2＋相对含量迅速降低0．37±0．06，与对照组1．34±0．11相比差异有统计学意义（P＜0．05）；血清LDH和CK活性实验组伤后即刻和伤后24 h均明显升高，伤后24 h实验组血清LDH活性（3067．45±482．55）U／L显著高于对照组（735．00±291．30）U／L，差异有统计学意义（P＜0．05）；伤后24 h～14 d实验组calpain活性显著升高，伤后7 d达到峰值（10．59±0．18）μmol／L与对照组（7．62±0．19）μmol／L相比差异?

Variation in ultrastructure of skeletal muscle,Ca2＋ content of micro area,and calcium-dependent protease activity after severe burn in rats

Objective To study the relationship between ultrastructure of skeletal muscle,Ca2＋content in cell micro area,activity of calpain-1 and calpain-2,and skeletal muscle damage after severe burn in rats.Methods Forty eight male Sprague-Dawley rats were randomized to control and experimental groups （n=8/group at each time point）.Rats in the burn group were anesthetized and subjected to scald injury （30% total body surface area：Ⅲ）.At different time points,tibialis anterior muscle tissue and serum specimens were collected from both the groups. Transmission electron microscopy, electron probe microanalysis,Western blot,enzymatic analysis,and enzyme-linked immunosorbent assay were used to detect study parameters.Results Under electron microscopy,the muscle fiber bundle of skeletal muscle ranked neatly in control group,mixed arrangement and curled filaments appeared 24 h post injury in experimental group,Z-line irregular and mild dissolution appeared 3 d post injury,partial disappearance of Z lines occurred 7 d post injury in experimental group;The activities of serum lactic acid dehydrogenase and creatine kinase were increased significantly;the muscle tissue damage began to appear.During the post injury period,from 24 h to 14 d,the changes in Ca2＋ levels and calpain-1 and calpain-2 content and activity were consistent with both ultrastructural changes in skeletal muscle and the changes in the dynamics of muscle tissue damage.Conclusions After severe burn injury,Ca2＋was redistributed in skeletal muscle cell micro areas.The cytoplasmic region was overloaded with Ca2＋ to promote calpain-1 and calpain-2 protein biosynthesis and activity,which may play an important role in muscle fiber degradation and skeletal muscle injury.