【作者】 王起；

【导师】 官大威；

【作者基本信息】 中国医科大学 ， 法医学， 2007， 硕士

【摘要】 Caspase-3在大鼠骨骼肌挫伤愈合过程中的表达及其时间规律性研究前言细胞凋亡（apoptosis）是程序化细胞死亡（programmed cell death）的过程，受一系列基因调控的生理性细胞死亡方式。目前，已经研究了多种组织、细胞的凋亡现象，骨骼肌细胞凋亡的研究也取得了一定的进展。一方面，骨骼肌细胞凋亡在维持骨骼肌正常生长发育成熟过程中必不可少；另一方面，细胞凋亡异常与多种骨骼肌损伤和疾病的发生、发展机制有关。研究证明，骨骼肌挫伤后，会发生肌纤维损伤造成的肌源性去神经支配和神经轴突损伤导致的神经源性去神经支配。大量研究已经证实，凋亡在骨骼肌去神经性萎缩中起重要作用。Caspase家族是诱导细胞凋亡的重要的蛋白酶。近年来随着对凋亡信号转导通路研究的进一步深入，caspase-3的作用受到重视。研究发现，caspase-3是导致细胞凋亡的最强大的效应因子。但骨骼肌挫伤愈合过程中是否表达caspase-3，目前国内外尚未见研究报道。本实验在建立大鼠骨骼肌挫伤模型的基础上，应用免疫组织化学染色技术、Western blot技术以及caspase-3活性测定技术对大鼠骨骼肌挫伤愈合过程中caspase-3的表达进行研究，用以推测caspase-3在骨骼肌挫伤愈合过程中的生物学作用，并对其表达时间变化规律在推断损伤时间中的可应用性进行了探讨。材料和方法一、动物模型的建立和分组健康成年的雄性Sprague-Dawley（SD）大鼠30只，体重220-250g，由中国医科大学实验动物中心提供。随机分为10组，每组3只，其中9组为实验组，1组为对照组。实验组大鼠用2％戊巴比妥钠（0.45mg／kg体重）腹腔注射麻醉后，参照Kami等的大鼠骨骼肌急性钝挫伤模型制作方法，将大鼠后肢置于伸膝、踝背屈90°位置，用自制打击器，以重500g金属柱体，从54cm高处自由垂直落下打击动物右小腿骨骼肌（距跟骨1.5cm处），解剖证实挫伤率100％。每只鼠分笼饲养并给予专用饲料及蒸馏水，保持垫料清洁及空气通畅。于损伤后3h、6h、12h、1d、3d、5d、7d、10d和14d将大鼠脱颈椎处死，取右下肢挫伤区及周边区骨骼肌，对照组取相同部位骨骼肌。每组1／2迅速放入4％多聚甲醛／PBS中固定48h后石蜡包埋，制作5μm厚度石蜡切片；1／2冰冻，用于caspase-3的Western blot和活性测定。二、免疫组织化学染色（一）实验步骤切片脱蜡至水；3％H₂O₂，微波法修复抗原；正常羊非免疫血清封闭；兔抗鼠caspase-3多克隆IgG抗体1：600倍稀释，4℃过夜；生物素化山羊抗兔IgG抗体；SP试剂；DAB显色3min，苏木素进行核复染、脱水、透明、中性树胶封片。同时进行常规HE染色。caspase-3抗体购自美国NeoMarkers公司，SP免疫组织化学试剂盒购自福州迈新生物技术有限公司。（二）结果判断caspase-3阳性表达于细胞浆内，呈棕黄色。（三）阳性结果分析免疫组化结果用Motic Images Advanced 3.1图像分析系统采集照片。挫伤周边区：显微镜×400倍下，在每张切片中随机选择10个视野，以骨骼肌细胞胞浆中出现棕色颗粒为阳性信号，测定其阳性面积及积分光密度值。挫伤区及挫伤交界区：显微镜×400倍下，在每张切片中随机选择10个视野，以细胞胞浆中出现棕色颗粒为阳性信号，进行定性分析。定性分析标准如下：-级（阴性）：细胞内无棕黄色颗粒沉着；+级（弱阳性）：阳性面积在0-15％之间；++级（阳性）：阳性面积在15％-25％之间；+++级（中度阳性）：阳性面积在25％-50％之间；++++（强阳性）：阳性面积>50％。（四）统计学分析各损伤时间组数据应用SAS统计软件，采用单因素方差分析方法进行统计学处理，数据以均数±标准差（（?）±s）表示。三、caspase-3的Western blot检测（一）实验步骤Bradford法蛋白质定量；电泳，50μg总蛋白经12％SDS-PAGE分离；转印至PVDF膜上；抗原封闭，采用含0.1％Tween-20的5％脱脂奶粉封闭液；兔抗大鼠caspase-3抗体孵育；山羊抗兔IgG抗体孵育；ECL显色。以GAPDH为内参。（二）结果判断在胶片上，caspase-3的阳性表达为黑色谱带，谱带的浓淡及大小代表表达的强弱，以GAPDH为内参，计算其相对积分光密度值。四、caspase-3活性测定Bradford法进行蛋白定量；将所有样品管用裂解液稀释为统一的蛋白浓度（2.0g／L）；取稀释后液体50μl，各加入50μl反应缓冲液（含有10mmol／L新鲜DTT）；加入5μl caspase-3底物（DEVD-pNA），混匀，37℃孵育1.5h；酶标仪上测定在波长405nm处的OD₄₀₅值；计算各损伤组相对对照组的活性倍增值。实验结果一、挫伤组骨骼肌病理组织学变化伤后3h见挫伤处骨骼肌局部出血、水肿、变性，部分肌纤维断裂，偶见少量多核粒细胞。6h部分骨骼肌胞浆均质化，细胞核消失，骨骼肌间隙内炎细胞增多。12h挫伤的骨骼肌间隙及周边出现较多量的炎细胞。1d挫伤的骨骼肌间隙及周边大量的炎细胞。3d损伤处有大量的细胞核出现。5d天可见肌细胞核增殖，核均位于细胞中央。7d新形成的肌细胞进一步增多，可见胶原形成。10d再生过程更近一步，部分区域可见较多量的胶原形成。14d仍可观察到骨骼肌再生和胶原形成。二、挫伤周边区caspase-3的免疫组织化学染色对照组骨骼肌未见阳性信号；挫伤周边骨骼肌在损伤后3h开始出现弱阳性表达，随时间延长阳性表达逐渐增强，5d达到高峰，随后阳性表达逐渐降低，14d仍然有较强的阳性表达。相邻两组之间caspase-3阳性表达的面积和积分光密度值均有显着差异（P＜0.05）。三、caspase-3在挫伤区的表达损伤后3h可见挫伤区骨骼肌开始出现阳性表达；损伤后6h-1d，可见被炎细胞浸润的骨骼肌表达caspase-3；损伤后3d，可见新形成的骨骼肌核位于细胞中央部位，胞浆内有caspase-3阳性表达；伤后5-7d，可见新形成的肌细胞胞浆内有caspase-3表达；损伤后10-14d，融合的肌细胞内仍然有caspase-3的表达。四、挫伤周边区caspase-3的Western blot结果分析对照组骨骼肌未见表达；随后表达量逐渐上升，5d达到高峰，以后逐渐下降，14d时仍有表达。五、挫伤周边区caspase-3活性表达的倍增值：与对照组相比，caspase-3活性倍增值随损伤时间延伸而增高，损伤后12hcaspase-3的活性明显增高达8.6倍，5d时活性增高值达到最大为17.1倍，以后随时间延伸，caspase-3的活性倍增值逐渐下降。相邻两组之间caspase-3活性表达的倍增值均有显着差异（P＜0.05）。讨论近年来，骨骼肌细胞凋亡的研究取得了一定的进展，在骨骼肌正常生长发育过程中，细胞凋亡对肌细胞的数量起着重要的调节作用。Fidzianska研究表明，包括人类在内的哺乳动物，在胎儿肌纤维发育早期，有超过40％的肌细胞是通过凋亡被清除的。此外，凋亡与多种骨骼肌损伤和疾病的发生机制有关。刘晓蓉等认为，Duchenne型肌营养不良症（Duchenne Muscular Dystrophy，DMD）的疾病进展可能与肌细胞的凋亡的增加有重要关系。晋雯等研究进一步证实在DMD中肌纤维的萎缩、坏死等改变与凋亡有关，并且存在着caspase-3的激活。大强度运动后骨骼肌细胞凋亡已被研究所证实，Arslam等的研究发现大鼠大强度运动后，其骨骼肌细胞凋亡率较非运动组有明显增高。Siu PM等研究表明，大鼠骨骼肌去神经支配和长时间悬挂后肢可造成肌肉萎缩，其肌萎缩的肌肉有凋亡相关基因（Bax、Bcl-2）表达，提示凋亡在骨骼肌去神经和废用性萎缩中起重要作用。有研究表明脊髓损伤后骨骼肌失去神经营养因子及废用而发生萎缩，这种萎缩与骨骼肌的凋亡有关。Hurme等研究证实，骨骼肌挫伤后，会发生肌纤维损伤造成的肌源性去神经支配和神经轴突损伤导致的神经源性去神经支配。本试验观察到在大鼠骨骼肌挫伤愈合过程中，对照组骨骼肌没有caspase-3阳性表达，挫伤周边有caspase-3的表达，caspase-3的活性在挫伤后明显增高。作者认为，在挫伤初期，骨骼肌细胞内维持一定的ATP水平，其水平虽低于正常水平，但仍能保证凋亡过程的能量需要，凋亡细胞及吞噬了凋亡细胞的吞噬细胞释放免疫抑制性细胞因子，产生免疫抑制，阻止坏死的发生，对机体是一种保护作用。但随着时间的延长，挫伤周边骨骼肌去神经支配，导致凋亡的发生。结合上述相关研究报道，提示在骨骼肌挫伤愈合过程中挫伤周边骨骼肌细胞发生凋亡，caspase-3可能是骨骼肌挫伤愈合过程中挫伤周边骨骼肌凋亡的最终执行因子之一。同时在骨骼肌挫伤愈合过程中caspase-3表达与损伤时间有一定的关系，其时序性变化可能作为骨骼肌损伤时间推断的指标之一，服务于法医学实践。有研究提示骨骼肌的分化依赖caspase-3的表达。本实验发现，损伤后5-7d挫伤处骨骼肌被清除干净，新形成的肌细胞大量增生，并表达caspase-3；损伤后10-14d，融合的肌细胞内仍然有caspase-3的阳性表达。而融合的肌细胞是终末分化的肌细胞，正常情况下不会发生凋亡。提示骨骼肌挫伤后，新形成的肌细胞中表达的caspase-3并不是凋亡的效应因子，可能作为骨骼肌分化过程重要的因子。结论1、对照组大鼠下肢骨骼肌中无caspase-3表达；2、大鼠骨骼肌挫伤后，挫伤周边骨骼肌表达caspase-3且caspase-3被激活；提示大鼠骨骼肌挫伤后，挫伤周边骨骼肌发生凋亡；3、大鼠骨骼肌挫伤后，新形成的肌细胞表达caspase-3，提示caspase-3可能在骨骼肌分化过程中起重要作用；4、大鼠骨骼肌挫伤后，周边区caspase-3的表达呈时间规律性变化，提示caspase-3可能作为骨骼肌挫伤损伤时间推断的指标之一。更多还原

【Abstract】 A Study on the Time-Dependent Expression of Caspase-3 During Repair of Contused Skeletal Muscle in RatsIntroductionProgrammed cell death, or apoptosis, is a physiological process of cellular autodestruction, which is governed by series of genes. Lots of researches have been conducted to study the phenomena of apoptosis in different tissues and cells, including skeletal muscle. On one hand, the apoptosis of skeletal muscle is crucial for normal development and tissue homeostasis. On the other hand, untoward apoptosis is associated with some damages and diseases of skeletal muscle. Some studies indicated that apoptosis occurs and plays an important role in denervated skeletal muscle.The members of caspases family are crucial proteases inducing apoptosis. As the most powerful executioner, caspase-3 have been intensively investigated. However little is known about the expression of caspase-3 after skeletal muscle contusion.To investigate the mechanism of caspase-3 after skeletal muscle contusion and explore the applicability of caspase-3 to determination of wound age, the expression of caspase-3 was studied by immunohistochemical, Western blot techniques and colorimetric measurement of proteolytic activity in skeletal muscle contusion at different posttraumatic intervals in rat.Materials and Methods1. Establishment of animal modelA total of 30 male, healthy rats, weighting 220g-250g was anesthesized by intraperitoneal injection of 2% pentobarbital sodium (0.45mg/kg), and received a single impact trauma at the skeletal muscle of the right rear leg. After wounding, each rat was individually housed in a cage and given sterilized chow and redistilled water. Muscles of right lower limb were taken after the animals were anaesthetized and sacrificed by cervical dislocation at 3h, 6h, 12h, ld, 3d, 5d, 7d, 10d, 14d (3 rats in each group) postwounding. The remaining 3 rats were used as control.2. Immunohistochemical detection of caspase-3Immunohistochemistry SP method was applied to investigate the immunoreactivity of caspase-3 in the contused skeletal muscle. After serum incubated, sections were incubated with polyclonal antibody against caspase-3 at 4℃overnight at a dilution 1: 600 followed by incubition with biotinylated goat anti-rabbit IgG antibody at 25℃for 20 minuts. The slides were incubated with SP at 25℃for 20 minutes. DAB was used as chromogen for visualization of the specific reaction. Finally, the sections were counterstained with hematoxylin, dehydrated and mounted. Additionally, H.E staining of the sections was routinely conducted.3. Analysis of caspase-3 by Western blotProteins extracted from whole cells were loaded at 50μg per lane[w/v]on 12% SDS-PAGE. Protein bands were transferred to PVDF membrane, blocked in 5% milk powder and 0.1%Tween-20. Membrane was probed with polyclonal antibody against caspase-3, which was then incubated with secondary antibody. Immunoreactive bands were visualized by ECL for detecting the bands of the protein.4. Determination of caspase-3 activityThe enzymatic reaction for caspase-3 activity is carried out in a 96 well flat bottom microplate that can be read with a microplate reader. Each reaction requires 50μL of cell lysate, 50μL of 2×Reaction Buffer. Prior to using the 2×Reaction Buffer, add 10μL of fresh DTT stock per 1 ml of 2×Reaction Buffer. Then add 5μL of caspase-3 colorimetric substrate (DEVD-pNA). Incubate the plate at 37℃for 1.5 hrs. Read the plate on a microplate reader at 405nm wave length. The results were compared with that of the control group. Fold-increase of caspase-3 activity was calculated and expressed as mean±SD. Results1. Morphological changes of the contused skeletal muscle wound3 houres after wounding, hemorrhage, oedema, degeneration and tom muscle fiber could be detected in the injuried site with a few polymorphonuclear cells were detected. 6 houres after wounding, carcoplasm clarifixation appeared, caryon disappeared. From 12 houres to 1 day after wounding, a large number of inflammatory cells accumulated. 3 days after wounding, lots of cell nucli came out. 5 days after wound, myocyte nucli proliferate, nearly all the nucli located in the middle of the cell. From 7 days to 14 days after wound, skeletal muscle regeneration and collagen emerged.2. Expression of caspase-3 after skeletal muscle contusionIn the control group, caspase-3 could not be detected. In the peripheral zone of the contused skeletal muscle, caspase-3 was expressed at low level in the wound aged 3h-6h, and maximized in the group aged 5d. Thereafter, the expression decreased and minimized in traumatized muscle aged 14d, which was still kept at a certain level as detected by immunostaining and Western blot.Meanwhile, the regenerated skeletal muscle express caspase-3 too.3. Measurement of caspase-3 activityIn comparison with the control, fold-increase of caspase-3 activity was detected in its climax in the contused muscle aged 5d. Thereafter, the enzymatic activity reduced and minimized in the group aged 14d, although it might be detected at a certain level.DiscussionIn recent years, lots of studies have been conducted to investigate the phenomena of apoptosis in skeletal muscle. The apoptosis of skeletal muscle is crucial for maintaining normal development and tissue homeostasis. Fidzianska’s work showed that in mammalian, including human, more than 40% of the myocytes were eliminated by apoptosis in the early period of muscle fiber development. In addition, apoptosis is associated with the mechanism of some injury and pathogenesis of skeletal muscle disorders. Liu’s work showed that the progression of DMD was related to over-apoptosis. Jin’s later work confirmed that the change of atrophy, necrosis in DMD were related to apoptosis. Strenuous exercise would increase the level of apoptosis, Arslam’s work showed that apoptotic index increased after strenuous exercise. Apoptosis in skeletal muscle has also been observed in vivo in suspended hindlimb and after denervation injury. Hurme’s research showed that skeletal muscle contusion would lead to denervation.In the present study, caspase-3 was detected by immunohistochemical, Western blot techniques and caspase-3 activity was determined by colorimetric assay method after skeletal muscle contusion. We found that caspase-3 was detected at the peripheral zone of contused skeletal muscle in rats, which was activated, while caspase-3 can not be detected at non-traumatized zone. We presumed that at the initial stage of contusion, there is certain amount ATP(Adenosine triphosphate) in skeletal muscle, although the level is low, which is enough for apoptotic invents and cells that engulf the apoptotic cells may release immunosuppressive cytokines. Apoptotic cells express specific cell surface molecules on the cell membrane, which are recognized by certain receptors on phagocytes (macrophages or DC) This recognition leads to a rapid and efficient phagocytosis in the early stages of apoptosis. Antigens from phagocytosed cells are processed and presented to the naive T-cells. Since apoptotic cells and phagocytes produce immunosuppressive cytokines, the antigens from apoptotic cells can not elicit an immune response, instead of leading to immunosuppression. Under such a circumstance, the phagocytic antigen-presenting cells do not express co-stimulating molecules. Thus, T-cells become tolerant of antigens from apoptotic cells.Caspase-3 is also expressed in the regenerated skeletal muscle, which may indicate that caspase-3 activity was required for skeletal muscle differentiation.The results suggest that:①The peripheral area of contused rat skeletal muscle express caspase-3, which is activated. It suggests that apoptosis take place in the peripheral area of contused rat skeletal muscle.②Caspase-3 maybe used as a maker for the wound age determination, since caspase-3 was expressed time-dependently after skeletal muscle contusion.③Caspase-3 is required for skeletal muscle differentiation.Conclusions1. Caspase-3 can not be detected at non-contused zone of skeletal muscle.2. The peripheral area of contused rat skeletal muscle expressed caspase-3, which was activated. It is suggested that apoptosis should take place in the peripheral zone of contused skeletal muscle in rats.3. Caspase 3 may be required for skeletal muscle differentiation in rats.4. Caspase-3 may be used as a maker for the skeletal muscle wound age determination.更多还原