周围神经损伤修复后早期骨骼肌细胞凋亡的初步研究
摘要
目的:探讨神经修复后早期大鼠骨骼肌萎缩中骨骼肌细胞凋亡情况。
方法:选择健康雄性SD大鼠54只,体重200±10g,随机分为3组,失神经对照组(A组,18只),神经吻合组(B组,18只),正常对照组(C组,18只)。应用10%水合氯醛(0.3ml/100g)对大鼠行腹腔麻醉,麻妥后,A组,于左侧股二头肌前缘肌间隙进入暴露分离坐骨神经,以坐骨神经分为腓总神经和胫神经处以近1.5cm为中点切除上下各0.5cm之坐骨神经,远近两端自然回缩;B组,于相同位置在未经任何处理情况下应用快刀片将坐骨神经截断,随即在手术显微镜下应用10-0医用尼龙线以外膜血管为参照将两断端外膜吻合,神经处理完毕后,应用生理盐水冲洗手术野,逐层闭合,术后未应用抗生素,未对患肢进行固定;C组不做任何处理。分别在2d,14d,28d在A,B,C三组中随机取出6只大鼠,测量体重,应用颈椎脱臼法将其处死,将大鼠左侧腓肠肌自肌肉起止点完整取出,测定腓肠肌肌湿重(GAS)后将其分成两份,一部分(大于200g)加入冰细胞裂解液100μl,应用超声波匀浆仪低温下(4℃)组织裂解。一部分置于4%中性多聚甲醛溶液中固定12小时,随后在不同浓度蔗糖溶液中上行脱水直至沉底。随后,在-20℃条件下,制作腓肠肌横断面冰冻切片,切片厚度15μm,对切片进行封闭(室温下浸入3%H2O2甲醇溶液,10分钟)与通透(冰上浸0.1%TritonX-100柠檬酸钠溶液,2分钟)。随后将冰冻切片置于荧光素(FITC)标记的脱氧核苷酸末端转移酶(TdT酶)混合液中,在37℃避光湿润条件下行60分钟标记反应。同时制备阳性对照样本——切片在脱氧核糖核酸酶I(DNase I)反应液中37℃处理10～30分钟;阴性对照样本——切片在不含有TdT酶的反应液中进行处理。标记反应完毕后,选择4,6-二氨基-2-苯基吲哚(DAPI)对细胞核进行复染,加入500μL的DAPI工作液(DAPI浓度为1-2μg/mL的甲醛溶液),37℃孵育15分钟,甲醇漂洗后防荧光淬灭封片剂封片。应用激光共聚焦显微镜对切片进行观察,计数凋亡细胞核。将制备好的组织匀浆液转移到1.5mL预冷离心管, 10000转/分,4℃离心5分钟;保留上清液于冰上待用。应用Braford法测定蛋白浓度,当蛋白含量达标后(>2μg/μl),吸取50μL上清液加入50μL反应缓冲液和5μL Caspase-3底物或Caspase-8底物于370C避光孵育4小时,应用荧光分光光度计在波长405 nm处测吸光度值(OD),以不加底物的样本作为阴性对照。通过计算OD反应组/OD阴性对照来确定各组Caspase-3或Caspase-8活化程度。数据以均数±标准差(±s)表示,用SAS统计学软件处理。
结果:在SD大鼠神经损伤(离断伤)修复(神经缝合)后早期骨骼肌相对正常神经支配骨骼肌,骨骼肌细胞凋亡现象有所增加,凋亡相关蛋白Caspase-3和Caspase-8活性较正常神经支配骨骼肌亦有所上升,但凋亡细胞核数量,Caspase-3和Caspase-8活性均弱于完全失神经骨骼肌。
结论:骨骼肌细胞凋亡是SD大鼠周围神经损伤修复后早期骨骼肌萎缩的机制之一,死亡受体信号通路参与到了神经修复后早期骨骼肌凋亡过程中。
Objective: To explore the role of apoptosis in skeletal muscle at the early stage of peripheral never regeneration in rats.
Methods: Atotal of 54 male Sprague-Duwley rats were randomly assigned to 3 groups (n = 18/group): denervated(A) group, neurorrhaphy group(B), the rats were not treated in group C as control.Under sterile conditions,Denervation was performed by surgically exposing the sciatic nerve in the thigh and then removing a 1-cm-long section of the nerve to prevent reinnervation. In the B group, sciatic nerve was transected 15 mm above its bifurcation and immediately sutured two ends’epineurium refering to its blood vessel. All operating procedure had been operated in the operating microscope.Rats were subjected to 2, 14, 28 days of unilateral denervation or neurorrhaphy of the sciatic.The gastrocnemius were harvested ,weighed muscle-wet weight(GAS) and body mass (BM), gastrocnemius mass served as the degree of muscle atrophy.A search for marker of apoptosis, nuclear DNA fragmentation, using terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (the TUNEL method) in situ, then detected in the confocal microscopy. A another portion of gastrocnemius muscle was homogenized and then analysed the activity of caspase-3 and -8 by spectrophotometry.
Results: TUNEL labeling of fragmented DNA on histological sections in neuorrhaphy group revealed higher levels of apoptotic nuclei than control ,lower than denervated group’s at the earl stage(<28d). The activity of caspase-3 and -8 in posted neuorrhaphy group is also higher than control,lower than denervated group.
Conclusion: At the initial stage of peripheral never regeneration, apoptosis may contribute to muscle atrophy and extrinsic apoptotic pathway may takes part in it.
方法:选择健康雄性SD大鼠54只,体重200±10g,随机分为3组,失神经对照组(A组,18只),神经吻合组(B组,18只),正常对照组(C组,18只)。应用10%水合氯醛(0.3ml/100g)对大鼠行腹腔麻醉,麻妥后,A组,于左侧股二头肌前缘肌间隙进入暴露分离坐骨神经,以坐骨神经分为腓总神经和胫神经处以近1.5cm为中点切除上下各0.5cm之坐骨神经,远近两端自然回缩;B组,于相同位置在未经任何处理情况下应用快刀片将坐骨神经截断,随即在手术显微镜下应用10-0医用尼龙线以外膜血管为参照将两断端外膜吻合,神经处理完毕后,应用生理盐水冲洗手术野,逐层闭合,术后未应用抗生素,未对患肢进行固定;C组不做任何处理。分别在2d,14d,28d在A,B,C三组中随机取出6只大鼠,测量体重,应用颈椎脱臼法将其处死,将大鼠左侧腓肠肌自肌肉起止点完整取出,测定腓肠肌肌湿重(GAS)后将其分成两份,一部分(大于200g)加入冰细胞裂解液100μl,应用超声波匀浆仪低温下(4℃)组织裂解。一部分置于4%中性多聚甲醛溶液中固定12小时,随后在不同浓度蔗糖溶液中上行脱水直至沉底。随后,在-20℃条件下,制作腓肠肌横断面冰冻切片,切片厚度15μm,对切片进行封闭(室温下浸入3%H2O2甲醇溶液,10分钟)与通透(冰上浸0.1%TritonX-100柠檬酸钠溶液,2分钟)。随后将冰冻切片置于荧光素(FITC)标记的脱氧核苷酸末端转移酶(TdT酶)混合液中,在37℃避光湿润条件下行60分钟标记反应。同时制备阳性对照样本——切片在脱氧核糖核酸酶I(DNase I)反应液中37℃处理10～30分钟;阴性对照样本——切片在不含有TdT酶的反应液中进行处理。标记反应完毕后,选择4,6-二氨基-2-苯基吲哚(DAPI)对细胞核进行复染,加入500μL的DAPI工作液(DAPI浓度为1-2μg/mL的甲醛溶液),37℃孵育15分钟,甲醇漂洗后防荧光淬灭封片剂封片。应用激光共聚焦显微镜对切片进行观察,计数凋亡细胞核。将制备好的组织匀浆液转移到1.5mL预冷离心管, 10000转/分,4℃离心5分钟;保留上清液于冰上待用。应用Braford法测定蛋白浓度,当蛋白含量达标后(>2μg/μl),吸取50μL上清液加入50μL反应缓冲液和5μL Caspase-3底物或Caspase-8底物于370C避光孵育4小时,应用荧光分光光度计在波长405 nm处测吸光度值(OD),以不加底物的样本作为阴性对照。通过计算OD反应组/OD阴性对照来确定各组Caspase-3或Caspase-8活化程度。数据以均数±标准差(±s)表示,用SAS统计学软件处理。
结果:在SD大鼠神经损伤(离断伤)修复(神经缝合)后早期骨骼肌相对正常神经支配骨骼肌,骨骼肌细胞凋亡现象有所增加,凋亡相关蛋白Caspase-3和Caspase-8活性较正常神经支配骨骼肌亦有所上升,但凋亡细胞核数量,Caspase-3和Caspase-8活性均弱于完全失神经骨骼肌。
结论:骨骼肌细胞凋亡是SD大鼠周围神经损伤修复后早期骨骼肌萎缩的机制之一,死亡受体信号通路参与到了神经修复后早期骨骼肌凋亡过程中。
Objective: To explore the role of apoptosis in skeletal muscle at the early stage of peripheral never regeneration in rats.
Methods: Atotal of 54 male Sprague-Duwley rats were randomly assigned to 3 groups (n = 18/group): denervated(A) group, neurorrhaphy group(B), the rats were not treated in group C as control.Under sterile conditions,Denervation was performed by surgically exposing the sciatic nerve in the thigh and then removing a 1-cm-long section of the nerve to prevent reinnervation. In the B group, sciatic nerve was transected 15 mm above its bifurcation and immediately sutured two ends’epineurium refering to its blood vessel. All operating procedure had been operated in the operating microscope.Rats were subjected to 2, 14, 28 days of unilateral denervation or neurorrhaphy of the sciatic.The gastrocnemius were harvested ,weighed muscle-wet weight(GAS) and body mass (BM), gastrocnemius mass served as the degree of muscle atrophy.A search for marker of apoptosis, nuclear DNA fragmentation, using terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (the TUNEL method) in situ, then detected in the confocal microscopy. A another portion of gastrocnemius muscle was homogenized and then analysed the activity of caspase-3 and -8 by spectrophotometry.
Results: TUNEL labeling of fragmented DNA on histological sections in neuorrhaphy group revealed higher levels of apoptotic nuclei than control ,lower than denervated group’s at the earl stage(<28d). The activity of caspase-3 and -8 in posted neuorrhaphy group is also higher than control,lower than denervated group.
Conclusion: At the initial stage of peripheral never regeneration, apoptosis may contribute to muscle atrophy and extrinsic apoptotic pathway may takes part in it.
引文
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