成肌细胞移植阻止mdx鼠运动诱导的肌损害

目的:观察过量运动对肌营养不良症模型鼠(mdx鼠)骨骼肌的损害作用,以及成肌细胞移植对运动诱导损害肌纤维的保护作用。方法: 采用分离消化法对C57新生鼠的成肌细胞进行体外培养、纯化鉴定后,肌肉注射到mdx鼠左后肢,右后肢肌注DMEM作对照。成肌细胞移植后1个月,让mdx鼠作运动试验3 d后,静脉注射Evans蓝,次日取骨骼肌作冰冻切片,行dystrophin免疫荧光检测。荧光显微镜下,观察Evans蓝和dystrophin阳性纤维数,图像分析比较。 结果: 未移植肢体有26.82%±14.85%的肌纤维显示Evans蓝染色,而移植侧骨骼肌只有10.37%±2.87%的肌纤维显示Evans蓝染色。两者相比有显著差异(P＜0.05)。移植侧肢体有48.32%±6.54%的肌纤维dystrophin阳性,对照侧几乎没有dystrophin阳性肌纤维。Evans蓝染部分没有dystrophin表达。结论: 成肌细胞移植对运动诱导损害的肌纤维具有防护作用。     

mdx小鼠骨骼肌组织成肌、成脂、成骨基因的特异性表达

背景：干细胞移植是治疗肌营养不良症的有效方法之一,但移植的干细胞在病理骨骼肌中成肌表达较低。 目的：通过比较mdx小鼠和C57小鼠的骨骼肌形态及成肌、成脂、成骨基因表达的差异,探讨mdx小鼠骨骼肌病理改变的可能机制。 方法：取mdx小鼠与C57小鼠的骨骼肌组织行冰冻切片,苏木精-伊红染色和Vonkossa染色观察两种小鼠肌肉组织的形态特征;提取mdx小鼠和C57小鼠骨骼肌组织总RNA,real-time PCR检测成肌、成脂、成骨相关基因的表达。 结果与结论：mdx小鼠骨骼肌有肌纤维坏死和再生,伴有轻度脂肪、纤维结缔组织增生,Vonkossa染色可见钙结节沉积,而C57小鼠的骨骼肌细胞形态清晰,核位于细胞周边。与C57小鼠比较,mdx小鼠肌肉组织成骨、成脂基因表达有不同程度的上调(P < 0.05),而成肌基因表达下调(P< 0.05)。dystrophin基因缺失及成肌基因表达下调、成骨和成脂基因上调是造成mdx小鼠肌肉组织变性坏死的原因。     

骨髓移植治疗Duchenne型肌营养不良模型鼠的实验研究

目的：观察骨髓移植治疗Duchenne型肌营养不良（DMD）模型鼠（mdx鼠）后,骨骼肌中抗肌萎缩蛋白的表达，以及病理、生理和运动功能的变化情况，从而正确评价骨髓移植治疗DMD的疗效。 方法： 以正常C57鼠作为供者，以致死剂量放疗后的mdx鼠作为受者进行骨髓移植，在移植后4个月和6个月分别用荧光免疫组化法观察抗肌萎缩蛋白表达情况，用HE染色观察并计算骨骼肌细胞核中心移位纤维比例（CNF），同时进行mdx鼠腓肠肌电生理检查和运动功能检测。 结果： 骨髓移植后的mdx鼠骨骼肌细胞膜上有部分抗肌萎缩蛋白表达，CNF比例和肌肉湿重下降，mdx鼠的被动运动功能增强。 结论： 骨髓移植后，骨髓干细胞在mdx鼠体内的骨骼肌和骨髓中定居，并分化成可以表达抗肌萎缩蛋白的骨骼肌细胞，使mdx鼠病理、生理得到部分改善，最终导致了肌肉的运动功能的增强。说明骨髓移植治疗DMD是有一定价值的，为临床应用提供实验基础。     

mdx小鼠与C57小鼠骨髓基质细胞体外分化能力的比较

背景:肌营养不良症是一种渐进性致死性X连锁隐性遗传性肌肉疾病,目前无特效治疗。肌营养不良症模型鼠(mdx小鼠)的骨髓基质细胞增殖及定向分化能力是否正常,自身骨髓移植是否合适还有待研究。目的:观察mdx小鼠骨髓基质细胞体外培养时的增殖及多向分化能力。方法:取mdx小鼠与C57小鼠胫股骨骨髓基质细胞体外培养,经吉姆萨染色后观察其形成成纤维细胞集落形成单位的能力;通过不同诱导液使骨髓基质细胞定向分化为成骨、成脂、成肌细胞,观察其形态学特性;并分别用Vonkossa染色、油红O染色、免疫荧光检测desmin阳性细胞对已分化细胞进行鉴定和分化率比较;培养1周时,提取分化细胞总RNA,反转录后,用real-time PCR检测各分化细胞相关基因表达。结果与结论:mdx小鼠骨髓基质细胞形成的成纤维细胞集落形成单位数目和体积均小于C57小鼠。其成骨、成肌分化的效率均明显低于C57小鼠(P0.01),两种小鼠的骨髓基质细胞成脂分化效率差异无显著性(P0.05)。real-time PCR检测结果显示,与C57小鼠相比,mdx小鼠的骨髓基质细胞成骨、成肌基因表达均有不同程度下降,而成脂基因表达无明显差异。结果提示,mdx小鼠的骨髓基质细胞体外培养时的增殖及定向分化能力较C57小鼠下降,与Dystrophin基因缺失有关,mdx小鼠自体骨髓移植将会受限。     

骨髓间充质干细胞移植治疗mdx鼠：3个月疗效

背景：干细胞移植治疗肌营养不良症是目前的研究热点,相对造血干细胞移植,间充质干细胞移植风险较小。 目的：观察骨髓间充质干细胞移植治疗Duchenne型肌营养不良鼠(mdx鼠)的疗效。 方法：4周龄mdx鼠16只,随机分为治疗组与对照组,每组8只,经静脉移植及肌肉局部注射C57BL/6小鼠的骨髓间充质干细胞或等量生理盐水。 结果与结论：移植3个月后,治疗组较对照组血清肌酸激酶水平下降,骨骼肌肌膜部分有dystrophin蛋白表达,而对照组检测不到dystrophin蛋白表达。但是两组的运动功能无明显改善。结果初步表明骨髓间充质干细胞移植对mdx鼠有一定的治疗作用,可能使肌细胞膜破坏减少,延缓病情发展。     

骨髓干细胞移植治疗不同年龄mdx鼠的疗效研究

目的：研究不同年龄的Duchenne型肌营养不良鼠（mdx鼠）与骨髓干细胞移植后缺失蛋白表达的关系。 方法： 获取4-5周C57BL/6小鼠的骨髓干细胞，体外培养3 d，静脉移植到7Gy γ射线预处理的6周龄、8周龄两组各6只mdx鼠。移植12周后，对移植鼠骨骼肌dystrophin蛋白表达情况进行检测。 结果： 6周龄、8周龄两组mdx鼠，静脉移植1.2×107骨髓干细胞，3个月后，分别有16%和7%的骨骼肌纤维表达了dystrophin蛋白。 结论： 静脉移植同种、同系鼠骨髓干细胞的mdx鼠，3个月之后, 不同年龄mdx鼠骨骼肌细胞dystrophin蛋白表达的阳性率不同，幼年鼠骨髓干细胞移植有较高比率的缺失蛋白表达。     

骨髓干细胞移植后mdx鼠腓肠肌病理变化

目的 研究骨髓干细胞移植后mdx鼠腓肠肌组织病理变化. 方法 7～9周龄mdx鼠20只平均分为4组,放射处理后移植1.2×107细胞/只同种异基因全骨髓干细胞,于移植后4、8、12及16周用HE染色观察腓肠肌组织细胞形态及核中心移位纤维(CNF).C57鼠和未治疗mdx鼠各5只作阳性和阴性对照. 结果 CS7鼠腓肠肌横切面可见肌细胞大小形态基本一致,无核中心移位现象.各细胞移植治疗组和阴性对照组mdx鼠均有大量的炎细胞浸润,核中心移位明显.未治疗mdx鼠CNF最高,约达70%;移植后4、12和16周,CNF分别为55%、50%和44%. 结论 骨髓干细胞移植后mdx鼠腓肠肌CNF随移植时间延长逐渐减少,提示骨髓干细胞移植后长久持续参与受损骨骼肌的修复与再生.     

C57BL/6小鼠胫骨前肌和趾长伸肌肌梭解剖学分析

目的 定位C57BL/6小鼠胫骨前肌(TA)和趾长伸肌(EDL)肌梭分布,分析肌梭在骨骼肌中的固定方式,并统计肌梭各区域长度和赤道横截面积(CAS)等参数分析肌梭形态学共性,为肌梭的形态和功能研究提供解剖学基础.方法 5只正常成年C57BL/6小鼠取TA和EDL,利用改良的骨骼肌冷冻技术得到无冰晶样本,样本连续冷冻切片...     

C57胎鼠、乳鼠及成年小鼠心室肌细胞分离、培养及鉴定

背景：培养的心肌细胞被广泛应用于心肌细胞的生理特性、毒性实验、基因工程、疾病模型和药物筛选等方面的研究。获得纯度较高活性良好的品系小鼠心肌细胞是研究的关键前提。 目的：分离和培养C57小鼠胎鼠、乳鼠及成年小鼠心室肌细胞。 方法：应用机械切碎心室肌后,胰蛋白酶消化不同发育阶段的C57小鼠心室肌细胞,差速贴壁1 h纯化心室肌细胞,锥虫蓝染色判定心肌细胞活力,体外分别培养48~72 h后分别行倒置显微镜、扫描及透射电镜观察细胞形态,微电极阵列评价细胞电生理指标,免疫组化鉴定。 结果与结论：经3~6次消化后,心室组织消化完全,即刻细胞存活率大于85%。倒置显微镜下观察,细胞呈梭形、多角形。12 h有少部分细胞搏动,48 h细胞交织成网,搏动呈同步性,搏动频率30~90次/min。说明用胰蛋白酶组织消化法可以成功地分离、培养,并获得形态、活力良好的胎鼠、乳鼠及成年C57小鼠心室肌细胞。     

自体骨骼肌瓣移植于心脏的实验形态学研究

用成年杂种狗带血管神经蒂的背阔肌瓣植入人工缺损的心室壁进行实验观察，结果显示，肌瓣与心肌愈合良好，其间有一明显的疤痕带，有血管通性。近疤痕和缝线部，绝大多数心肌和骨骼肌纤维的形态，结构正常，少数纤维萎缩和纤维化。部分骨骼肌纤维呈心肌样改变，肌原纤维重新排列组合。重组的肌纤维呈心肌样分支，胞核大致位于肌纤维中央。本实验说明自体骨骼心肌成形术有可行性。     

Aquaporin 4 Expression in the mdx Mouse Diaphragm

Expression of aquaporin (AQP) 4 in the surface membranes of skeletal myofibers is well established; however, its functional significance is still unknown. The alterations of AQP4 expressions in dystrophic muscles at RNA and protein levels have been reported in various dystrophic muscles such as dystrophinopathy, dysferlinopathy, and sarcoglycanopathy. We are interested in the relationship between the severity of dystrophic muscle degeneration and the expression of AQP4. Here we compared the AQP4 expression of the limb muscles with that of diaphragms in both mdx and control mice. The dystrophic muscle degeneration, such as rounding profile of cross sectional myofiber shape, dense eosin staining, central nuclei, and endomysial fibrosis in mdx mice, were more marked in diaphragms than in limb muscles. The decrease of AQP4 expression at protein level was more marked in diaphragms than in the limb muscles of mdx mice. However, the expression of AQP4 mRNA in the diaphragms of mdx mice was not reduced in comparison with limb muscles of mdx mice. The present study revealed that AQP4 expression at protein level was correlated with the severity of dystrophic changes in muscle tissues of mdx mice.     

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd ^mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6–30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.     

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.     

Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice

Duchenne muscular dystrophy (DMD) is caused by the absence of a functional dystrophin protein and is modeled by the mdx mouse. The mdx mouse suffers an early necrotic bout in the hind limb muscles lasting from approximately 4 to 7 weeks. The purpose of this investigation was to determine the extent to which dystrophin deficiency changed the proteome very early in the disease process. In order to accomplish this, proteins from gastrocnemius from 6-week-old C57 (n = 6) and mdx (n = 6) mice were labeled with fluorescent dye and subjected to two-dimensional differential in-gel electrophoresis (2D-DIGE). Resulting differentially expressed spots were excised and protein identity determined via MALDI-TOF followed by database searching using MASCOT. Proteins of the immediate energy system and glycolysis were generally down-regulated in mdx mice compared to C57 mice. Conversely, expression of proteins involved in the Kreb’s cycle and electron transport chain were increased in dystrophin-deficient muscle compared to control. Expression of cytoskeletal components, including tubulins, vimentin, and collagen, were increased in mdx mice compared to C57 mice. Importantly, these changes are occurring at only 6 weeks of age and are caused by acute dystrophin deficiency rather than more chronic injury. These data may provide insight regarding early pathologic changes occurring in dystrophin-deficient skeletal muscle.     

Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the mdx mouse

The pattern of postnatal growth and development of skeletal muscle in mdx mice was studied by light and transmission electron microscopy and by autoradiography and was compared with that in their normal age-matched controls at 4 and 32 weeks of age. The muscle weights of both the extensor digitorum longus (EDL) and soleus muscles of mdx mice were significantly greater than those in control mice at both ages. Body weights of male and female mdx mice were also increased over controls up to 12 weeks of age. At 4 weeks, both the EDL and soleus muscles exhibited focal areas of degeneration, necrosis, and regeneration of centrally nucleated extrafusal fibers resulting in a wide range of fiber sizes. By 32 weeks, the majority of fibers in both muscles were centrally nucleated, and focal areas of recent regeneration were observed. By electron microscopy, the course of macrophage infiltration into areas of degenerating fibers and the ongoing regeneration of myofibers within redundant cylinders of external lamina were noted. This pattern was frequent in 4-week-old mdx muscles and was present to a lesser degree at 32 weeks. A notable lack of both adipose tissue infiltration and fibrotic change in the endomysium were observed in muscles at both ages. Autoradiograms of muscles from 4-week-old mdx mice injected with tritiated thymidine showed an increased proportion of labeled sublaminal nuclei at 24 and 48 hours after injection compared to controls. At 32 weeks of age, labeling of nuclei in muscles of mdx mice was also greater than in controls, but was reduced compared to muscle labeling in 4-week-old mdx mice. The observed features of mdx muscle tissue suggest that this animal model is more applicable to the study of regeneration dynamics than to Duchenne-type human muscular dystrophy.