严重烧伤大鼠骨骼肌细胞凋亡的初步研究

目的 探讨严重烧伤后大鼠骨骼肌细胞凋亡状况,以了解其对骨骼肌萎缩的可能作用机制.方法 100只雄性Wistar大鼠被随机分为假伤组和烫伤组;各组又分为伤后1、4、7、10、14 d 5个时间点,每个时间点10只.制备大鼠40%总体表面积m度烫伤模型,于伤后各时间点称体重和胫骨前肌重量.取后肢胫骨前肌,用透射电镜观察骨骼肌细胞凋亡的超微结构变化,用原位末端缺刻标记法(TUNEL)及免疫组化双标检测骨骼肌组织凋亡细胞核.结果 与假伤组比较,严重烫伤后大鼠体重及胫骨前肌重量从伤后1 d起即开始下降,4 d时达到最低(P<0.05和P<0.01),7 d后体重及胫骨前肌莺量开始缓慢上升,但差异仍有统计学意义(P均<0.01).电镜下观察烫伤组骨骼肌可见少部分细胞核核膜皱缩,染色质边集、断裂,核周区域肌丝降解,部分近核胞膜呈泡状突起,而同一骨骼肌细胞的其他核可显示为正常结构.TUNEL检测也发现烫伤组骨骼肌有散在的阳性细胞核,而假伤组骨骼肌细胞核则未见凋亡改变.结论 严重烧伤大鼠骨骼肌细胞存在凋亡现象,这可能是骨骼肌萎缩的原因之一.     

Burn injury upregulates the activity and gene expression of the 20 S proteasome in rat skeletal muscle.

There is evidence that burn injury stimulates ubiquitin-proteasome-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, protein substrates are conjugated to multiple molecules of ubiquitin, whereafter they are recognized, unfolded and degraded by the multicatalytic 26 S protease complex. The 20 S proteasome is the catalytic core of the 26 S protease complex. The influence of burn injury on the expression and activity of the 20 S proteasome has not been reported. We tested the hypothesis that burn injury increases 20 S proteasome activity and the expression of mRNA for the 20 S proteasome subunits RC3 and RC7. Proteolytic activity of isolated 20 S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from burned rats. Northern-blot analysis revealed that the expression of mRNA for RC3 and RC7 was increased by 100% and 80% respectively following burn injury. Increased activity and expression of the 20 S proteasome in muscles from burned rats support the concept that burn-induced muscle cachexia is at least, in part, regulated by the ubiquitin-proteasome proteolytic pathway.     

The effect of melatonin on plasma oxidant-antioxidant skeletal muscle reperfusion injury in rats

We investigated the effects of melatonin administration on skeletal muscle ischaemia-reperfusion injury (IRI) by assessing plasma malondialdehyde (MDA), superoxide dismutase (SOD), total glutathione (GSSH), glutathione peroxidase (GPX) and myeloperoxidase (MPO) concentrations. Male Sprague-Dawley rats (n = 32) were randomized into four groups: group 1 served as time controls; group 2 were the test animals; group 3 received melatonin (30 mg/kg) intraperitoneally prior to the induction of ischaemia; and group 4 received melatonin (30 mg/kg) intraperitoneally prior to the reperfusion period. Administration of melatonin prior to reperfusion significantly decreased the elevated MDA concentration caused by IRI, and significantly elevated GSSH concentrations, which had been reduced by IRI. Ischaemia-reperfusion injury significantly increased activities of GPX, SOD and MPO, and melatonin administration reversed this effect. In conclusion, a pharmacological dose of melatonin showed significant protective effects against IRI by decreasing lipid peroxidation, MPO, SOD and GPX enzyme activities and regulating glutathione content.     

Reperfusion injury induces apoptosis in rabbit cardiomyocytes.

The most effective way to limit myocardial ischemic necrosis is reperfusion, but reperfusion itself may result in tissue injury, which has been difficult to separate from ischemic injury. This report identifies elements of apoptosis (programmed cell death) in myocytes as a response to reperfusion but not ischemia. The hallmark of apoptosis, nucleosomal ladders of DNA fragments (approximately 200 base pairs), was detected in ischemic/reperfused rabbit myocardial tissue but not in normal or ischemic-only rabbit hearts. Granulocytopenia did not prevent nucleosomal DNA cleavage. In situ nick end labeling demonstrated DNA fragmentation predominantly in myocytes. The pattern of nuclear chromatin condensation was distinctly different in reperfused than in persistently ischemic tissue by transmission electron microscopy. Apoptosis may be a specific feature of reperfusion injury in cardiac myocytes, leading to late cell death.     

肌肉运动与骨骼肌细胞的凋亡

背景:不少医学研究表明,细胞凋亡能导致大量自由基增多、Ca~(2+)浓度升高、线粒体膜电位下降引起运动能力的下降.因此,研究细胞凋亡与运动训练的关系意义重要.目的:总结与探索关于肌肉运动与骨骼肌细胞凋亡的相关问题.方法:计算机检索中国期刊全文数据(网址http://dlib.cnki.net/kns50/index.aspx)及PubMed数据库(网址http://www.ncbi.nlm.nih.gov/pubmed/)1990-01/2009-06期间的相关文章,检索词为"肌肉运动,骨骼肌细胞凋亡,muscle exercise,apoptosis in the skeletal muscle".纳入与肌肉运动与骨骼肌细胞的凋亡研究现状与发展密切相关.①有关骨骼肌细胞凋亡的研究.②运动与骨骼肌细胞凋亡研究.③运动诱发骨骼肌细胞凋亡的基因调控研究.④骨骼肌细胞凋亡的分子机制研究.⑤同一领域选择近期发表或在权威杂志上发表的文章.排除重复性研究.结果与结论:运动后,正常肌肉中或是病理状态下的肌肉中骨骼肌细胞都会出现凋亡,凋亡的形态学表现与普通凋亡细胞相似,即核固缩、质膜发泡、细胞器紧缩,凋亡小体形成,其凋亡过程大致可分为3个阶段,即启始阶段、效应阶段和降解阶段.骨骼肌细胞凋亡的增加是导致运动性疲劳的重要原因.目前国内外对骨骼肌细胞凋亡的基因调控研究主要是从凋亡调控因子Bcl-2蛋白、肿瘤坏死因子α及死亡蛋白酶半胱氨酸天冬氨酸酶着手.bcl-2基因蛋白的抗凋亡作用主要是通过阻止线粒体通透性转换孔的开放,阻止线粒体释放促凋亡蛋白、防止线粒体膜脂质过氧化以及线粒体基质Ca2+释放实现的.肿瘤坏死因子家族在启动死亡因子及其受体途径中起重要作用,此途径的启动依赖于死亡配体与死亡受体相结合,激活半胱氨酸天冬氨酸酶,导致细胞凋亡.通过研究探索运动强度与骨骼肌细胞凋亡及坏死的界限关系,有利于在运动中认识运动性疲劳产生的机制及有效消除疲劳.     

乌司他丁对大鼠骨骼肌缺血-再灌注损伤的保护作用

目的 探讨乌司他丁对大鼠骨骼肌缺血-再灌注损伤的保护作用.方法 24只健康成年雄性SD大鼠随机分为3组(每组8只):对照组(C组)、缺血.再灌注损伤组(I/R组)、乌司他丁处理组(U组).C组仅麻醉肢体没有缺血操作;I/R组于再灌注前颈外静脉注入生理盐水0.5 ml;U组于再灌注前同法给乌司他丁0.5 ml(5×104 U/kg).以橡皮带环绕结扎大鼠左后肢根部至趾掌无血流信号达4 h后放开橡皮带,再灌注4 h建立大鼠骨骼肌缺血.再灌注损伤模型.再灌注4 h各组处死动物采集标本,采用逆转录-聚合酶链反应(RT-PCR)法测定骨骼肌组织TNF-α mRNA的表达;酶联免疫吸附试验法(ELISA)测定血浆TNF-α水平;比色法检测血浆乳酸脱氢酶(LDH)、肌酸激酶(CK)、丙二醛(MDA)和组织过氧化物酶(MPO)含量,测定湿质量/干质量均(W/D)及光镜、电镜观察骨骼肌组织结构的变化.应用SPSS 10.0统计软件,用单因素方差分析.结果 骨骼肌组织TNF-α mRNA的表达在I/R组明显高于C组(P＜0.05),而U组则低于I/R组(P＜0.01);血浆TNF-α浓度I/R组明显高于C组(P＜0.01),而U组则低于I/R组(P＜0.05);血浆LDH、MDA、CK和组织MDA、MPO水平以及W/D比值I/R组高于C组(P＜0.05),U组则低于L/R组(P＜0.05);骨骼肌组织形态学和超微结构的损伤U组也较I/R组减轻.结论 乌司他丁通过抑制细胞因子的生成、抑制氧自由基代谢产物MDA的产生和减少MPO的活性而对骨骼肌缺血-再灌注起保护作用.     

肌电图用于评价缺血预处理减轻大鼠骨骼肌缺血再灌注损伤的研究

摘要 目的：探讨针极肌电图（EMG）对缺血预处理（IPC）减轻大鼠骨骼肌再灌注损伤的应用价值。 方法：将75只健康6周龄SD鼠随机分成Ⅰ组（正常值组）,Ⅱ组（实验组或缺血预处理组）,Ⅲ组（对照组或未经预处理组）,每组25只。Ⅱ组为加压止血带捆扎大鼠后肢缺血再灌注损伤的动物模型,连续观察患侧胫前肌损伤即刻及第3、7、14、28天的针极肌电图改变、组织病理变化、后肢神经功能评分。 结果：Ⅱ组肌电图在损伤后第3天出现单个运动单位电位（MUP）,第14天与Ⅰ组比较MUP时限增宽、波幅升高、多相电位增多（P<0.01）,第28天所有参数与Ⅰ组比较差异无显著性（P>0.05）;而Ⅲ组在第7天出现单个MUP,第28天静息状态仍显示+1处正锐波、纤颤电位。病理变化在损伤后第3、7天最明显。后肢神经功能评分：损伤后第7天Ⅱ组有3只达到4级,损伤后第14天Ⅱ组有3只达到5级,第28天全部达到5级,与Ⅲ组比较有显著性差异（P<0.01）。 结论：针极肌电图的动态观察是评价缺血预处理减轻骨骼肌再灌注损伤的客观指标之一。     

Immediate and delayed transplantation of mesenchymal stem cells improve muscle force after skeletal muscle injury in rats

Mesenchymal stem cell (MSC) therapy is a promising approach for regaining muscle function after trauma. Prior to clinical application, the ideal time of transplantation has to be determined. We investigated the effects of immediate and delayed transplantation. Sprague–Dawley rats received a crush trauma to the left soleus muscle. Treatment groups were transplanted locally with 2 × 10⁶ autologous MSCs, either immediately or 7 days after trauma. Saline was used as sham therapy. Contraction force tests and histological analyses were performed 4 weeks after injury. GFP‐labelled MSCs were followed after transplantation. The traumatized soleus muscles of the sham group displayed a reduction of twitch forces to 36 ± 17% and of tetanic forces to 29 ± 11% of the non‐injured right control side, respectively. Delayed MSC transplantation resulted in a significant improvement of contraction maxima in both stimulation modes (twitch, p = 0.011; tetany, p = 0.014). Immediate transplantation showed a significant increase in twitch forces to 59 ± 17% (p = 0.043). There was no significant difference in contraction forces between muscles treated by immediate and delayed cell transplantation. We were able to identify MSCs in the interstitium of the injured muscles up to 4 weeks after transplantation. Despite the fundamental differences of the local environment, which MSCs encounter after transplantation, similar results could be obtained with respect to functional muscle regeneration. We believe that transplanted MSCs residing in the interstitial compartment evolve their regenerative capabilities through paracrine pathways. Our data suggest a large time window of the therapeutical measures. Copyright © 2012 John Wiley & Sons, Ltd.     

骨骼肌生长与适应的机械信号与途径

背景:骨骼肌作为一种机械组织,具有机械收缩的功能,但外界力学机械信号如何转变成生物体的化学信号,从而影响肌肉的生长和代谢仍不是很明确.目的;了解机械刺激与骨骼肌适应的信号途径,为摸索改善骨骼肌质量与适应提供理论依据及实验方案.方法:应用计算机检索Pubmed数据库和中国期刊网1980-01/2008-12相关文献.英文检索词为"skeletal muscle、mechanical stimulation、signal transduction";中文检索词为"骨骼肌,运动,信号传导".纳入标准:①具有原创性,论点论据可靠的实验性文章.②与骨骼肌肥大相关内容的文章.③观点明确,分析全面的文章.排除标准:与文章目的无关的内容和重复性研究.结果与结论:骨骼肌作为一种机械组织,具有机械收缩的功能,但外界力学机械信号如何转变成生物体的的化学信号,从而影响肌肉的生长和代谢仍不是很明确.研究表明,机械刺激通过诱导Akt/mTOR途径,从而促进骨骼肌生长与适应的机制;骨骼肌纤维在机械运动刺激作用下,神经冲动的传递,随后开放Ca2+及相关离子通道,引起胞浆中的钙离子增加,使钙调神经磷酸酶的活性上升,催化NFAT去磷酸化,激活的NFAT进入肌细胞核,作用于相应的靶基因,最终使骨骼肌产生运动适应性变化.结果表明,肌肉机械信号刺激促进胰岛素样生长因子1的释放,进而通过生物信号传导通路来实现.机械刺激促进离子浓度的改变,从而影响钙调磷酸酶/对核因子活化T细胞途径以增加肌肉质量.对于机械运动的物理信号与生物体的化学信号通路的机制研究还有待继续深入探索.     

氧化乐果长期暴露对大鼠骨骼肌氧化损伤的研究

目的探讨氧化乐果对大鼠骨骼肌组织氧化损伤的影响。方法将40只雄性Wistar大鼠随机分为4组,正常对照组,低剂量组,中剂量组和高剂量组,每组10只。低剂量组,中剂量组和高剂量组每天分别给予1/20、1/10、1/5LD50的氧化乐果,采用灌胃的途径给药,连续给药8周,检测大鼠血糖水平,骨骼肌组织中丙二醛（Malondialde-hyde,MDA）含量、超氧化物歧化酶（Superoxide dismutase,SOD）、过氧化氢酶（Catalase,CAT）和谷胱甘肽过氧化物酶（Glutathione peroxidase,GSH-Px）的活性以及骨骼肌的组织病理学检测。结果与正常对照组相比,低剂量、中剂量和高剂量组大鼠骨骼肌MDA含量显著升高（P〈0.05）,SOD、CAT和GSH-PX活性显著降低（P〈0.05）;大鼠的血糖和组织病理学检查未见异常变化。结论长期接触有机磷农药氧化乐果可导致骨骼肌出现氧化应激。     

Oxygen radicals and scavenger enzymes in ischaemia-reperfusion injury of skeletal muscle.

In this study the protective effects of removing oxygen free radicals during ischaemia and reperfusion of skeletal muscle were investigated. The bilateral gracilis muscle model was used in six dogs. Both muscles were made ischaemic for 4 h, followed by reperfusion for 60 min. To remove oxygen free radicals, superoxide dismutase and catalase were given 10 min before ischaemia and during the first 30 min of reperfusion in one muscle; the other muscle served as a control. Muscle blood flow was recorded during the first 35 min of reperfusion. At the end of reperfusion the contents of high-energy phosphates and glycogen were measured. Furthermore, the metabolic burst of leukocytes (chemiluminescence) was determined. Flow as well as creatine phosphate were always highest in the treated muscles as compared with the control muscles (p less than 0.05). Adenosine triphosphate and glycogen were highest in all but one case. Spontaneous leukocyte chemiluminescence was significantly reduced in venous blood from the control muscle and insignificantly reduced in blood from the treated muscle as compared with blood from the aorta. The results are indirect evidence that reactive oxygen metabolites play a role in the genesis of ischaemia-reperfusion injury of skeletal muscle and that treatment with scavenger enzymes may have protective effects.     

茶多酚对力竭运动大鼠骨骼肌组织氧化损伤的保护作用

背景:骨骼肌在急性运动时产生大量自由基,使体内氧化剂与抗氧化剂之间平衡的失调,引起骨骼肌疲劳、肌肉功能障碍和损伤等,使机体运动能力下降.而茶多酚是从茶叶中提取的多酚类物质,具有很强的抗氧化作用,是一种高效低毒的天然抗氧化剂.目的:通过茶多酚对骨骼肌运动性损伤的干预,观察其对骨骼肌氧化损伤的保护作用.方法:雄性SD健康大鼠30只,随机分为对照组、运动组、茶多酚组.茶多酚组大鼠补充茶多酚1周后,与运动组大鼠一起通过一次性力竭游泳建立力竭运动模型,记录大鼠力竭时间,测定力竭运动即刻时大鼠骨四头肌中丙二醛,超氧化物歧化酶谷胱甘肽过氧化物酶和总抗氧化能力.结果与结论:力竭运动使大鼠骨骼肌丙二醛含量增加,超氧化物歧化酶、谷胱甘肽过氧化酶、血浆总抗氧化能力活力下降.茶多酚可以降低运动后丙二醛水平,提高运动后大鼠骨骼肌超氧化物歧化酶、谷胱甘肽过氧化酶、血浆总抗氧化能力活力,延长大鼠力竭时间,由此得出,大鼠力竭运动后骨骼肌组织会发生氧化损伤,茶多酚干预可能会保护骨骼肌氧化损伤,进而提高大鼠运动能力.     

口服Vitamin E对离心运动后大鼠骨骼肌损伤的影响

目的 从骨骼肌超微结构和肌浆网自由基代谢的角度,探讨维生素E(Vitamin E)对离心运动后大鼠骨骼肌损伤的影响.方法 取健康雄性SD大鼠48只,随机分为对照组、运动组、生理盐水组、Vitamin E组,每组各12只,制作相应的动物模型.动物模型采用一次持续性下坡跑运动训练,运动结束后,取大鼠右侧肱三头肌,部分用于电镜标本制做,余下肌肉采用差速离心方法提取肌浆网,并进行丙二醛(MDA)含量和超氧化物歧化酶(SOD)活力测定.结果 Vitamin E组大鼠骨骼肌在离心运动后24 h的损伤超微结构较生理盐水组改善明显,与运动组和生理盐水组比较,丙二醛显著降低,超氧化物歧化酶显著增高,但仍差于对照组.结论 Vitamin E可以降低骨骼肌肌浆网丙二醛含量、增加肌浆网超氧化物歧化酶活性,并使离心运动后骨骼肌损伤形态学改变得以改善.     

Measles virus induces apoptosis in uninfected bystander T cells and leads to granzyme B and caspase activation in peripheral blood mononuclear cell cultures

Background Measles causes lymphopenia and depresses cell‐mediated immunity, but the mechanisms of immunosuppression and cell loss are poorly known. Methods We have used an in vitro model of measles virus (MV)‐infected peripheral blood mononuclear cells (PBMCs) and phytohaemagglutinin‐stimulated PBMCs in order to assess MV–leucocyte interactions. Cell population undergoing apoptosis was measured by flow cytometry and Annexin‐V‐fluos staining. The expression of Fas, FasL, TNRF1, and Bcl‐2 was analyzed by flow cytometry and Western blotting, and activation of caspase cascade was measured using a colourimetric caspase substrate set. The effects of caspase inhibitors were detected by flow cytometry. Results Measles virus was able to infect monocytes, but interestingly induced apoptosis in uninfected T cells, indicating that induction of apoptosis in T cells is mediated by MV‐infected adherent cells. Only 1% of T cells contained MV antigen day 3 p.i. Interestingly the percentage of early apoptotic T cells at the same time was 35%, showing that apoptosis was not the result of MV infection in T cells. Measles virus‐induced Fas but not FasL or TNFR1 expression on PMBC, as well as activation of granzyme B and caspase cascade. Simultaneously, overexpression of Bcl‐2 protein was detected. Caspase inhibitor decreased the amount of apoptotic T cells. Conclusion Measles virus‐infected monocytes induce apoptosis in uninfected T cells, suggesting that infected monocytes probably interact via cell–surface molecules with uninfected T cells and induce apoptosis by indirect mechanisms. Apoptosis of the lymphocytes may contribute to the pathogenesis of MV‐induced immunosuppression and cell loss.     

超氧化物歧化酶模型配合物对大鼠骨骼肌缺血再灌注损伤的保护作用

背景骨骼肌缺血再灌注损伤的治疗方法一直是骨科医生们研究的重点和难点.目的观察超氧化物歧化酶模型配合物(MSODa)对骨骼肌缺血再灌注(I/R)后的保护作用,并对其保护机制进行探讨,为骨骼肌的损伤修复提供理论依据.设计随机对照实验.地点和材料在哈尔滨医科大学附属第二医院完成.清洁级雄性Wistar大鼠96只,体质量(200±20)g,由哈尔滨医科大学附属第二医院动物实验中心提供.干预建立大鼠后肢缺血再灌注模型,将96只大鼠随机分为正常对照组(n=6)、缺血再灌注组(n=30)、生理盐水组(n=30)、MSODa组(n=30).缺血再灌注组,生理盐水组,MSODa组分别在缺血4 h后,再灌注1,2,4,8,12 h末,测定各项指标.主要观察指标测定血浆中的丙二醛、骨骼肌组织中的髓过氧化物酶(MPO),白细胞上β2整合素(CD11b/CD18)和骨骼肌血管中细胞间黏附分子(ICAM)-1的表达情况及各组的组织学改变.结果Ⅱ组各时相点MDA,MPO,CD11b/CD18,ICAM-1的表达均较Ⅰ组有非常显著升高,骨骼肌组织损伤也随再灌注时间的延长而逐渐加重,Ⅳ组则可以明显抑制这种变化.结论MSODa通过减少自由基的生成,抑制黏附分子的表达而减轻骨骼肌I/R损伤.     

胰岛素样生长因子Ⅱ在大鼠骨骼肌运动性损伤再生中的作用

目的:通过观察大鼠骨骼肌运动损伤后具有修复作用的卫星细胞的增殖变化,探讨施加胰岛素样生长因子Ⅱ对肌肉再生的修复作用。方法:选择2个月龄SD大鼠40只。随机分为两组,实验组20只,对照组20只。将两组大鼠建立7d多次力竭跑台训练致骨骼肌损伤的动物模型,即每天力竭1次,连续7d。首次力竭后第1,3,5天实验组于右侧腓肠肌中部的肌膜下分别注射1mL(300ng)外源性胰岛素样生长因子Ⅱ;对照组注射1mL生理盐水。两组分别在第1次力竭后的第7,9,11,17,24,30天各处死大鼠3只,对两组大鼠骨骼肌行苏木精-伊红染色,以光镜和电镜超微结构观察增殖细胞核抗原表达(增殖细胞核抗原以细胞核出现棕黄色颗粒,增殖细胞核抗原指数以随机记数50个高倍视野下共计500个细胞百分率表示),并进行χ2检验。结果:①两组大鼠腓肠肌光镜观察结果:第1次力竭后的第7,11,17天实验组卫星细胞明显增多;对照组胶原纤维增多;24d实验组肌纤维塑形良好,对照组可见瘢痕愈合。②两组大鼠腓肠肌超微结构电镜观察结果:实验组线粒体形态较正常,糖原颗粒增多,可见新生的肌纤维;对照组可见肌浆网扩张,线粒体肿胀,还有凋亡细胞出现。③两组大鼠腓肠肌免疫组织化学染色观察增殖细胞核抗原的表达:实验组明显高于对照组(χ2=9.023,P<0.05)。结论:胰岛素样生长因子Ⅱ可以促进大鼠运动性骨骼肌损伤后的卫星细胞增殖,并减少瘢痕愈合,表明其对大鼠骨骼肌运动性损伤有加快损伤后修复和促进再生的作用。