Mdx小鼠骨骼肌中炎症反应和mpeg1的表达

目的探讨mdx小鼠不同时期骨骼肌的炎性病理改变,观察mpeg1在mdx小鼠及对照鼠中的表达。方法选取雄性C57BL/10ScSn-Dmdmdx/JNju鼠为实验组,对照组为雄性C57BL/6Sc Sn小鼠,根据年龄分为2 w、4 w、8 w、12 w 4个亚组。通过HE染色、MGT染色、ACP染色观察骨骼肌光镜下的形态学改变,总结mdx小鼠骨骼肌炎性病理变化。通过RNA提取,基因芯片的检测及mpegl的qRT-PCR检测,观察mpeg1的表达情况。结果常规组织染色中,2 w的mdx小鼠肌肉偶见高度浓染的肌纤维,未见肌细胞坏死,炎症细胞浸润,4 w可见巨噬细胞吞噬现象散在分布,8 w时炎细胞浸润灶融合成片,12 w时炎性病灶面积减小;利用基因芯片技术筛选出mdx小鼠中有关炎症反应的基因30余个,结果显示与2 w相比,炎症反应相关基因表达量均增加,在8 w时达到峰值,12 w有所下降,但较2 w时仍有升高;qRT-PCR结果显示从4 w开始,mdx小鼠中mpeg1的含量逐渐增加,8 w时含量最高。结论 (1)炎症反应参与mdx小鼠疾病的发生发展:从2 w开始出现,8 w达到高峰,12 w趋于平稳;(2)Mpeg1在mdx小鼠炎症反应中发挥了一定的作用。     

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。     

肌营养不良症模型鼠骨骼肌的组织病理学研究

目的　比较肌营养不良症模型 (mdx)鼠、C57鼠和Duchenne型肌营养不良症 (DMD)患者骨骼肌的组织病理学改变 ,以及dystrophin在肌细胞膜上的分布。方法　取mdx鼠、C57鼠和DMD患者骨骼肌作常规HE染色 ,比较其组织学改变 ;同时对mdx鼠、C57鼠骨骼肌作dystrophin的免疫组化染色 ,比较dystrophin在肌细胞膜上的分布。结果　mdx鼠骨骼肌肌纤维大小不等 ,轮廓变圆 ,肌间隙增宽 ,少量脂肪、结缔组织增生 ,细胞核中心移位增多 ,部分肌纤维变性坏死 ,而DMD患者骨骼肌的改变和mdx鼠基本一致。mdx鼠肌细胞膜缺乏完整环行棕色条带 ,而C57鼠则呈一完整环行棕色条带 ,提示mdx鼠dystrophin蛋白缺乏。结论　mdx鼠有类似于DMD患者的骨骼肌组织病理学改变     

Duchenne肌营养不良模型鼠骨髓移植后dystrophin的表达

目的　观察Duchenne型肌营养不良模型鼠 (mdx鼠 )不同成分骨髓细胞移植后dystrophin的表达。方法　用体外培养的C57BL雄鼠的骨髓细胞、骨髓悬浮细胞、骨髓基质细胞分别经鼠尾静脉注射到放疗后的mdx鼠体内 ,动态观察受体雌性mdx鼠骨骼肌dystrophin的表达 ,并取受体mdx雌鼠血 ,用聚合酶链反应进行Sry基因检测。结果　骨髓细胞、悬浮细胞、基质细胞移植后 1～ 2个月 ,较少肌纤维表达dystrophin(<1 % ) ,3～ 4个月分别约有 7%、6 %、4 %的肌纤维表达dystrophin。Sry基因检测均扩增出受体雌鼠Y染色体上 449bp的DNA片段 ,提示供体细胞在受体内存活。结论　骨髓细胞、悬浮细胞、基质细胞分别移植到mdx鼠体内后 ,mdx鼠骨骼肌均有dystrophin的表达     

mTOR调控自噬在Duchenne肌营养不良症肌纤维损害中的研究

目的 探索哺乳动物雷帕霉素靶点(mTOR)相关自噬调节通路在mdx小鼠骨骼肌纤维中有关线粒体形态学异常以及肌肉萎缩和坏死过程中所处的地位和作用。方法 选取8 w龄雄性mdx小鼠为实验组,同龄C57小鼠为对照组,每组各3只,取股四头肌组织,通过病理染色观察肌肉组织病理学改变,应用电镜技术观察线粒体的形态变化,应用免疫荧光染色技术观察自噬泡标志物微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)和线粒体标志物电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)的共定位情况,利用Western blot技术分析mTOR相关自噬调控通路蛋白的表达,应用Real-Time PCR技术分析自噬相关基因的表达水平。结果 与对照组相比,8w龄mdx小鼠股四头肌组织萎缩,肌纤维内可见核内移、肌再生及炎性细胞浸润;电镜下可见线粒体体积变小,数量增多,并出现线粒体肿胀、嵴消失现象。免疫荧光可见线粒体标记物VDAC和自噬泡标记物LC3的团块状聚集,两者的共定位增加。自噬相关基因蛋白及基因表达上...     

Duchenne肌营养不良模型鼠基因型及肌肉病理的研究

目的研究Duchenne肌营养不良(DMD)模型鼠mdx基因型及肌肉病理改变。方法分别采用光镜、免疫荧光、EvansBlue染料、电镜等方法研究mdx小鼠与正常对照组C57/BL6小鼠腓肠肌病理改变,并检测mdx小鼠的基因型。结果经Dys-3、δ-sarcoglican抗体染色后mdx小鼠肌膜基本未见绿色荧光,正常对照组C57/BL6小鼠肌膜呈明显网状绿色荧光;荧光显微镜观察EvansBlue红色荧光染料,mdx小鼠肌纤维呈明显红色荧光,而肌膜完整的正常对照组C57/BL6小鼠肌纤维不摄取红色荧光染料。mdx模型鼠肌丝排列紊乱,方向不一,肌细胞核位于肌纤维中央,Z盘模糊,肌膜局部不连续,C57/BL6小鼠肌丝排列整齐,Z盘清晰可见。结论mdx小鼠以肌纤维变性、坏死为特征,肌细胞膜缺损是mdx小鼠主要病理改变之一。mdx小鼠dystrophin基因缺陷同时伴有dystrophin相关蛋白缺失,mdx小鼠肌肉病理为DMD进一步治疗研究奠定了基础。     

不同放疗剂量对肌营养不良鼠骨髓干细胞移植的影响

目的　使用不同剂量γ射线对Duchenne型肌营养不良鼠 (mdx鼠 )照射 ,研究骨髓干细胞移植后mdx鼠缺失蛋白表达的情况。方法　将 18只 7～ 8周mdx鼠随机平均分为A、B、C 3组 ,依次给予 7Gy、7.5Gy、8Gyγ射线预处理 ;3天后行骨髓干细胞移植。移植骨髓干细胞取自 4～ 5周的C5 7BL/6鼠 ,体外培养 3d ,按 1 2× 10 7个细胞 /每只剂量移植给 3组预处理后的mdx鼠。连续观察受体鼠的移植物抗宿主病 (GVHD)程度 ;移植 12周后 ,检测受体鼠体内抗肌萎缩蛋白表达情况。结果　C组GVHD反应较明显 ;A、B、C 3组的股骨肌肌纤维dystrophin蛋白表达率分别为 8%、9%和 13 %。各组间缺失蛋白表达率有显著差异。结论　对 7～ 8周mdx鼠 ,给予不同剂量的γ射线预处理 ,骨髓干细胞移植后 ,8Gyγ射线表达最多 ,7Gy射线照射表达量最少 ;但 8Gyγ射线照射GVHD反应较明显。     

骨髓移植治疗Duchenne型肌营养不良症模型鼠后dystrophin相关蛋白表达

目的观察骨髓移植治疗Duchenne型肌营养不良症(DMD)模型鼠—mdx鼠后，β-dystroglycan和α-sarcoglycan在肌膜上的表达情况。方法以正常C57鼠作为供体，以mdx鼠作为受体进行骨髓移植，在移植后2、4、6个月分别进行mdx鼠骨骼肌的β-dystroglycan和α-sarcoglycan免疫荧光染色，计算阳性细胞的荧光积分光密度，与C57鼠及未移植的mdx鼠进行比较。结果在骨髓移植后2、4、6个月mdx鼠骨骼肌肌膜上β-dystroglycan和α-sarcoglycan的表达量均有随时间的延长逐渐增加的趋势．至移植后6个月时，二者的表达量明显高于未治疗的mdx鼠。结论骨髓移植可使mdx鼠的dystrophin相关蛋白在病损骨骼肌细胞膜上表达增加．其对维持肌膜的稳定性、促进肌肉的恢复有重要意义。     

Duchenne型肌营养不良症模型小鼠神经肌肉接头特征研究

目的观察Duchenne型肌营养不良症模型小鼠骨骼肌肌膜抗肌萎缩蛋白(dystrophin)表达变化和神经肌肉接头形态,分析其可能机制。方法 C57BL/6和mdx小鼠各8只,HE染色观察肌细胞组织学形态,免疫荧光染色检测腓肠肌肌膜dystrophin蛋白表达变化和神经肌肉接头形态。结果C57BL/6小鼠腓肠肌肌细胞大小基本一致,呈多角形,胞核位于细胞周边、极少数位于肌纤维中心;肌膜均匀表达dystrophin蛋白;神经肌肉接头形态完好。Mdx小鼠腓肠肌肌细胞大小不一致,呈圆形,部分胞核趋中心化;仅少量或个别肌细胞表达dystrophin蛋白;mdx种鼠突触后膜乙酰胆碱受体断裂成小片段,突触前膜神经末梢突起增多、变细,而mdx幼鼠神经肌肉接头形态与C57BL/6小鼠基本一致;mdx小鼠神经肌肉接头数目明显减少,突触前膜和突触后膜横截面积明显减小,肌细胞间神经轴突明显变细。结论Mdx小鼠骨骼肌肌膜dystrophin蛋白缺失并非导致神经肌肉接头改变的直接因素,可能与病情进展有关。     

重组腺相关病毒（rAAV）介导基因SMCKA3999对Duchenne肌营养不良病理和功能的影响

目的　 研究重组腺相关病毒 (rAAV)载体介导的dystrophin小基因SMCKA3999治疗DMD模型鼠mdx ,从病理和功能观察rAAVSMCKA3999治疗对DMD模型小鼠mdx的疗效。方法　以dystrophin小基因SMCK A3999为目的基因 ,将SMCKA3999克隆至rAAV并包装成rAAVSMCKA3999,以 5× 10 10 病毒颗粒单点注射于DMD模型鼠mdx腓肠肌 ,基因治疗后 4个月及 7个月 ,采用免疫荧光、光镜组织病理、肌电图等方法 ,从形态和功能观察rAAVSMCKA3999治疗对DMD模型小鼠mdx的疗效。 结果　rAAVSMCKA3999使肌膜缺失的dys trophin恢复并稳定表达持续 7个月以上 ,肌肉组织病理改变好转 ,肌病肌电图改变明显改善 ,疗效持续 4个月以上。 结论　rAAVSMCKA3999能改善mdx小鼠骨骼肌的病理及功能 ,采用rAAV介导的dystrophin小基因SMC KA3999对Duchenne肌营养不良基因治疗是有希望的治疗方法。     

Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice

To address whether mdx mice with haploinsufficiency of utrophin (mdx/utrn+/-) develop more severe skeletal muscle inflammation and fibrosis than mdx mice, to represent a better model for Duchenne muscular dystrophy (DMD), we performed qualitative and quantitative analysis of skeletal muscle inflammation and fibrosis in mdx and mdx/utrn+/- littermates. Inflammation was significantly worse in mdx/utrn+/- quadriceps at age 3 and 6 months and in mdx/utrn+/- diaphragm at age 3 but not 6 months. Fibrosis was more severe in mdx/utrn+/- diaphragm at 6 months, and at this age, mild fibrosis was noted in quadriceps of mdx/utrn+/- but not mdx mice. The findings indicate that utrophin compensates, although insufficiently, for the effects of dystrophin loss with regard to inflammation and fibrosis of both quadriceps and diaphragm muscles in mdx mice. With more severe muscle dystrophy than mdx mice and a longer life span than utrophin-dystrophin-deficient (dko) mice, mdx/utrn+/- mice provide a better mouse model for testing potential therapies for muscle inflammation and fibrosis associated with DMD.     

Tenascin-C expression in dystrophin-related muscular dystrophy

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration. © 1996 John Wiley & Sons, Inc.     

Expression profiling in stably regenerating skeletal muscle of dystrophin-deficient mdx mice

The mdx mouse is comparable to Duchenne muscular dystrophy in having an absence of dystrophin. While dystrophic human skeletal muscle undergoes progressive degeneration, in the mdx mouse regeneration and tissue remodeling substantially compensate for the lack of dystrophin. To better understand the molecular events leading to active muscle regeneration in mdx muscles, we have determined the gene expression profiles of wild-type and mdx hind limb muscles using oligonucleotide arrays. Compared to wild-type, 58 genes were found to be differentially expressed in mdx. The molecular signature of actively regenerating skeletal muscle in young adult mdx mice showed upregulation of muscle development genes and genes involved in immune response, proteolysis and extracellular matrix remodeling. Moreover, energy metabolism and mitochondrial function were not compromised. Insights into the processes activated in the mdx muscle to compensate for chronic degeneration may have important implications for therapy in patients with muscular dystrophy.     

Current status and perspective of gene therapy on dystrophic animal model]

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene. An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, though it has a limitation in insertion size up to 4.9 kb. Therefore, a full-length dystrophin cDNA (14 kb) cannot be incorporated into an AAV vector. We previously generated micro-dystrophin transgenic dystrophin-deficient mdx mice. Micro-dystrophin CS1 transgenic mdx mice showed almost complete amelioration of dystrophic phenotypes. We, therefore, constructed an AAV vector expressing micro-dystrophin deltaCS1, a modified version of CS1, driven by skeletal muscle-specific MCK promoter, since the MCK promoter in AAV vector drives longer expression of the LacZ gene than the CMV promoter in skeletal muscle. We injected the AAV-MCK deltaCS1 into anterior tibial (TA) muscles of 5-week-old or 10-day-old mdx mice. Dystrophic phenotypes were largely improved in both injections. Especially in the latter occasion, less than 20% of muscle fibers were microdystrophin positive at 24 weeks after the injection, but specific tetanic force of the injected muscle was not statistically different from that of control normal muscle. In conclusion, deltaCS1 micro-dystrophin introduced by an AAV vector could be a powerful tool for the gene therapy of DMD. A bigger animal model, canine X-linked muscular dystrophy will contribute to pre-clinical study of gene therapy.     

Steroids in Duchenne muscular dystrophy: from clinical trials to genomic research

Steroids represent the only pharmacological palliative treatment for Duchenne muscular dystrophy. However, they do have side effects and despite a large number of published studies showing their efficacy, they are still not universally used. This is largely due to the lack of functional outcome and quality of life measures in most of the published studies and suggests that further trials might be required to answer some of the still unclear aspects of their role. Another important aspect of steroid therapy in Duchenne dystrophy is that we do not know how they work in dystrophic muscle. We have initiated a collaborative study on gene profiling using microarray in steroid-treated mdx mice. cDNA microarray studies were performed to examine the levels of skeletal muscle gene expression in a pool of mdx mice treated with prednisolone for 1 and 6 weeks. Interesting preliminary data on untreated mdx mice suggest that the gene profiling of young (7 weeks) versus older (12 weeks) mice is very significantly different. Furthermore, a large number of genes showed significant changes in expression at the mRNA level on treatment with prednisolone. These included structural protein genes; signalling genes and genes involved in immune response. Hopefully, analysis of this pattern of steroid-induced gene expression will provide some insight into understanding how glucocorticoids improve strength in Duchenne dystrophy, and may help in developing more effective and less toxic therapeutic approaches.     

Gold-labelled dystrophin molecule in muscle plasmalemma of mdx control mice as seen by electron microscopy of deep etching replica

Summary The Duchenne muscular dystrophy product dystrophin has been shown to be located at the inner surface of normal muscle plasma membrane. This study was undertaken to visualize the shape of dystrophin molecules and their topographical distribution at the inner surface of murine skeletal muscle plasma membrane. The immunogold electron microscopy of plastic-embedded quadriceps femoris muscles of six mdx mice and six control mice showed the presence of gold particles along the muscle plasma membrane undercoat of all muscle samples from the control mice without any antibody reaction in the mdx mice muscles. The gold-labelled muscles of six mdx and six control mice were quickly frozen by liquid helium in a rapid-freeze apparatus. High magnification electron microscopy of the quick-freeze, deep-etch, rotary-shadow replicas of the gold-labelled muscles demonstrated the presence of dystrophin molecules associated with gold particles at the cytoplasmic surface of mdx control mice.The dystrophin molecules displayed a variety of shapes, such as rods with a reduction in diameter from one end to the other end and/or with the enlargement of their end(s). These dystrophin molecules were incorporated in the meshowork of muscle plasma membrane-associated cytoskeletons.Supported by Grant (2-A) from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Altered pathological progression of diaphragm and quadriceps muscle in TNF-deficient, dystrophin-deficient mice

We have previously demonstrated a role for T cells in Duchenne muscular dystrophy (DMD) using the mdx mouse and have shown that T cell killing of dystrophic muscle can occur through perforin-dependent and perforin-independent mechanisms. In this investigation, we explore the possibility that one perforin-independent mechanism utilized by the T cells is cytokine-based killing, specifically by tumor necrosis factor (TNF). We tested this hypothesis by generating mice that are TNF-deficient and dystrophin-deficient (TNF−/mdx). Body mass and muscle mass of the TNF−/mdx mice were significantly less than TNF+/mdx mice at 8 weeks of age. Creatine kinase levels and overall muscle strength were unchanged. Histopathology measurements showed different results in the diaphragm and quadriceps muscles. These data suggest that removal of TNF in vivo in dystrophic mice has differential effects on diaphragm and quadriceps suggesting that TNF is an unfavorable target for immunotherapy for DMD.     

Gene therapy for muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene. Although an adeno-associated virus (AAV) vector-mediated gene transfer provides an attractive approach to the treatment of DMD, limitation in insertion size up to 4.9 kb excludes incorporation of a full-length dystrophin cDNA (14 kb) into an AAV vector. We previously generated micro-dystrophin transgenic dystrophin-deficient mdx mice. In 4.9 kb rod-truncated micro-dystrophin CS1 transgenic mdx mice, dystrophic phenotypes were ameliorated almost completely (Biochem Biophys Res Commun 2002; 293:1265-72). We therefore constructed an AAV vector expressing micro-dystrophin CS1 driven by a skeletal muscle-specific MCK promoter, as the expression of the LacZ gene driven by the MCK promoter is longer in an AAV vector than in the CMV promoter in the skeletal muscle (Gene Ther 2002; 9:1576-88). We injected the AAV-MCK delta CS1 into the anterior tibial (TA) muscles of 5-week-old mdx mice, which exhibit active cycles of muscle degeneration/regeneration. At 8 weeks after the AAV vector injection, a large percentage of fibers were dystrophic-positive (10 to 50%). Even 24 weeks after injection, 15 to 75% of myofibers expressed micro-dystrophin. Dystrophin-positive fibers often had centrally located nuclei in the mice, however, the ratio was significantly reduced compared with that of dystrophin-negative fibers. We also measured tetanic force of AAV-MCK delta CS1-treated and non-treated mdx TA to evaluate functional amelioration. Non-treated mdx TA muscles showed marked reduction of specific tetanic force, while AAV-injected muscles showed moderate improvement. In conclusion, our study demonstrated that introduction of delta CS1 micro-dystrophin with an AAV vector successfully protected mdx muscles from progressive dystrophic degeneration.