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-timePCR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。     

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Von kossa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(P<0.01),两种小鼠的骨髓基质细胞成脂分化效率差异无显著性(P>0.05).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。     

骨髓间充质干细胞移植后迪谢内肌营养不良模型鼠的肌肉病理学改变

目的：观察骨髓间充质干细胞移植治疗迪谢内肌营养不良模型鼠后肌肉病理改变，及迪谢内肌营养不良致病基因dystrophin蛋白表达变化。方法：实验于2003-07／2004-07在华中科技大学同济医学院同济医院神经科实验室进行。取4-6周龄C57BL／6雄性小鼠的股骨和胫骨，采用密度梯度分离和贴壁培养相结合的方法分离培养骨髓间充质干细胞。④正常组：2周龄C57BL／6小鼠4只，不干预。②对照组：2周龄mdx鼠4只，左后腿腓肠肌注射50μL磷酸盐缓冲液。③移植组：2周龄mdx鼠4只，左后腿腓肠肌注射50μL骨髓间充质干细胞细胞悬液（约1&;#215;10^6）。16周后3组小鼠麻醉下取左腿腓肠肌中部肌肉，苏木精-伊红染色后观察核内移肌纤维占总肌纤维百分率，及dystrophin免疫组化染色计算阳性纤维与总肌纤维百分率。结果：12只小鼠均进入结果分析。④核内移肌纤维占总肌纤维百分率：正常组肌纤维少核内移（1.2％）；对照组肌纤维大小不等，大量核内移（75％）；移植组肌纤维核内移明显减少（23．3％）。②dystrophin染色阳性纤维与总肌纤维百分率：正常组为100％，移植组显著高于对照组（75％，23.3％）。结论：骨髓间充质干细胞移植治疗迪谢内肌营养不良模型小鼠mdx，可使其骨骼肌细胞膜骨架蛋白dystrophin恢复，肌肉病理改变好转，且疗效持续到治疗后4个月以上，提示骨髓间充质干细胞移植对mdx小鼠治疗有效。     

系统性红斑狼疮小鼠骨髓间充质干细胞的成骨、成脂分化能力下降

背景：系统性红斑狼疮患者的骨髓间充质干细胞与正常人相比是否有不同,相关报道较少。 目的：对比系统性红斑狼疮模型小鼠与正常小鼠的骨髓间充质干细胞多向分化能力的差别。 方法：分离培养系统性红斑狼疮模型小鼠与C57BL小鼠的骨髓间充质干细胞（对照组）,分别进行成骨、成脂诱导分化,观察两种小鼠的骨髓间充质干细胞的分化能力。 结果与结论：C57BL小鼠的骨髓间充质干细胞经传代后,为长梭形,呈均匀分布生长;系统性红斑狼疮模型小鼠的骨髓间充质干细胞表现为生长缓慢,细胞相对C57BL小鼠要少一些。经过成骨诱导显示系统性红斑狼疮模型小鼠的钙结节和钙盐明显少于对照组,PCR检测成骨基因Runx2,碱性磷酸酶,骨钙素表达也明显下降。经过成脂诱导显示系统性红斑狼疮模型小鼠的脂滴明显少于对照组,PCR检测成脂基因PPARγ2,脂蛋白酯酶表达也明显下降。说明系统性红斑狼疮模型小鼠的骨髓间充质干细胞成骨与成脂能力都低于C57BL小鼠,系统性红斑狼疮模型小鼠的骨髓间充质干细胞的多向分化能力受损。     

骨髓间充质干细胞移植后迪谢内肌营养不良模型鼠的肌肉病理学改变

目的:观察骨髓间充质干细胞移植治疗迪谢内肌营养不良模型鼠后肌肉病理改变,及迪谢内肌营养不良致病基因dystrophin蛋白表达变化。方法:实验于2003-07/2004-07在华中科技大学同济医学院同济医院神经科实验室进行。取4～6周龄C57BL/6雄性小鼠的股骨和胫骨,采用密度梯度分离和贴壁培养相结合的方法分离培养骨髓间充质干细胞。①正常组:2周龄C57BL/6小鼠4只,不干预。②对照组:2周龄mdx鼠4只,左后腿腓肠肌注射50μL磷酸盐缓冲液。③移植组:2周龄mdx鼠4只,左后腿腓肠肌注射50μL骨髓间充质干细胞细胞悬液(约1×106)。16周后3组小鼠麻醉下取左腿腓肠肌中部肌肉,苏木精-伊红染色后观察核内移肌纤维占总肌纤维百分率,及dystrophin免疫组化染色计算阳性纤维与总肌纤维百分率。结果:12只小鼠均进入结果分析。①核内移肌纤维占总肌纤维百分率:正常组肌纤维少核内移(1.2%);对照组肌纤维大小不等,大量核内移(75%);移植组肌纤维核内移明显减少(23.3%)。②dystrophin染色阳性纤维与总肌纤维百分率:正常组为100%,移植组显著高于对照组(75%,23.3%)。结论:骨髓间充质干细胞移植治疗迪谢内肌营养不良模型小鼠mdx,可使其骨骼肌细胞膜骨架蛋白dystrophin恢复,肌肉病理改变好转,且疗效持续到治疗后4个月以上,提示骨髓间充质干细胞移植对mdx小鼠治疗有效。     

成体脂肪来源的Flk-1+间充质干细胞用于杜氏肌营养不良症治疗的可行性研究

为了探讨成体脂肪来源的间充质干细胞（adipose derived mesenchymal stemcell,AD-MSC）移植治疗杜氏肌营养不良症（Duchenne muscular dystrophy,DMD）的可行性,从成年GFP小鼠脂肪组织中分离得到间充质干细胞（mesen-chymal stem cell,MSC）,用流式细胞术分析其细胞表型和细胞周期;在体外分别以成肌和成内皮诱导体系诱导AD-MSC的定向分化,通过免疫荧光染色和RT-PCR进行鉴定;经尾静脉移植AD-MSC到CTX肌肉损伤模型小鼠和mdx小鼠（DMD动物模型）体内,通过RT-PCR和免疫荧光染色检测供体细胞的分布和分化情况,并进行统计分析。结果表明：从GFP小鼠脂肪组织中分离得到的Flk-1＋MSC可以在体外分化为肌肉细胞和血管内皮细胞;细胞移植后,可在损伤肌肉部位检测到GFP阳性的肌纤维、血管内皮细胞和肌肉干细胞;mdx小鼠移植后肌膜上抗肌萎缩蛋白（dystro-phin）的表达部分恢复,骨骼肌中心核肌纤维比例也大大降低。结论：AD-MSC是一种较为理想的供移植治疗DMD的干细胞,它不仅可以定向归巢到损伤的肌肉组织中,参与肌纤维重建,而且能改善组织供血,缓解DMD病征,同时部分供体细胞以干细胞形式存在,可以代替原来肌肉干细胞的功能,不断修复损伤的肌肉组织。     

骨髓间充质干细胞移植治疗Duchenne肌营养不良型mdx小鼠后的肌电图和dystrophin蛋白表达改变

目的 研究骨髓间充质干细胞（MSCs）移植治疗Duchenne肌营养不良（DMD）模型鼠mdx鼠后肌电图改变及dystrophin蛋白表达变化。方法 分离培养正常小鼠MSCs局部肌肉注射移植入DMD模型鼠mdx鼠，数周后观察肌电图改变及dystrophin蛋白表达变化。结果 MSCs移植后mdx鼠肌电图明显改善，dystrophin蛋白阳性表达肌纤维增加明显。结论 MSCs局部骨骼肌肌肉内细胞移植治疗mdx鼠有一定效果。     

肌营养不良症模型鼠神经元型一氧化氮合酶抑制蛋白的表达及定位

目的:观察肌营养不良症模型鼠(mdx)肌肉组织神经元型一氧化氮合酶抑制蛋白(proteininhibitorofnNOS,PIN)的表达及定位,以及与dys-trophin复合物及神经元型一氧化氮合酶(nNOS)的关系。方法:取mdx鼠(18只,实验组)和C57BL/10ScSn鼠(15只对照组)骨骼肌作常规染色,比较其组织学改变;同时对mdx鼠,C57BL鼠骨骼肌作Northernblot和固定化蛋白质印迹法(Westernblot)及免疫组化分析。结果:Northernblot分析发现,mdx鼠肌肉组织PINmRNA显著高于C57BL/10ScSn鼠,对照组骨骼肌有少量PINmRNA表达,其杂交斑点A值为1352.4±219.7,mdx组杂交斑点灰度A值为2724.2±394.6,两者差异有非常显著性意义(t=11.985,P<0.05);固定化蛋白质印迹法(Westernblot)显示,PIN蛋白的杂交信号在mdx组与对照组均出现在约8ku处,两者的吸光度平均值(A)几乎相等,mdx组的杂交斑点灰度A值为2378.2±486.7,对照组PIN蛋白的杂交斑点灰度A值为2438.5±494.9,两者差异无显著性意义(t=0.352,P>0.05);免疫组化及激光共聚焦扫描显微镜均揭示,在C57BL/10ScSn鼠肌纤维中,PIN蛋白在肌膜、周边核附近呈强阳性表达,肌质中弱表达,而mdx鼠的PIN蛋白在肌膜及中心核附近呈强阳性表达,细胞质中亦弱表达;PIN于mdx鼠肌纤维表达明显,而nNOS及dystrophin复合物表达明显减少     

Micro-dystrophin基因修饰的MSCs在mdx小鼠体内分化为肌卫星细胞的研究

目的：探讨携带抗肌萎缩蛋白小基因（Micro-dystrophin）的自体骨髓间质干细胞（MSCs）在Duchenne型肌营养不良症（DMD）模型鼠（mdx小鼠）体内分化为肌卫星细胞的潜能。方法：采用逆转录病毒介导micro—dystrophin基因转染mdx小鼠MSCs,通过尾静脉注射移植治疗mdx小鼠,移植后12周免疫荧光法检测受体鼠腓肠肌micro-dystrophin的表达,并分离培养肌卫星细胞进行鉴定。结果：受体鼠腓肠肌成功检测到micro-dystrophin表达;分离培养肌卫星细胞发现部分细胞可同时表达micro—dystropbin和c—met及micro—dystrophin和desmin。结论：自体mdx小鼠MSCs可携带外源性micro—dystrophin基因,并在体内分化为micro—dystrophin阳性肌细胞,少数细胞可作为肌卫星细胞长期存在。     

Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the dystrophin gene. Transplantation of autologous myogenic cells genetically corrected ex vivo is a possible treatment for this disorder. In order to test the regenerative efficiency of freshly isolated satellite cells, we purified quiescent satellite cells from limb muscles of 8-12-week-old green fluorescent protein-transgenic (GFP-Tg) mice using SM/C-2.6 (a recently developed monoclonal antibody) and flow cytometry. Freshly isolated satellite cells were shown to participate in muscle regeneration more efficiently than satellite cell-derived myoblasts passaged in vitro do, when transplanted into tibialis anterior (TA) muscles of 8-12-week-old cardiotoxin-injected C57BL/6 mice and 5-week-old dystrophin-deficient mdx mice, and analyzed at 4 weeks after injection. Importantly, expansion of freshly isolated satellite cells in vitro without passaging had no detrimental effects on their regenerative capacity. Therefore we directly isolated satellite cells from 5-week-old mdx mice using SM/C-2.6 antibody and cultured them with lentiviral vectors expressing micro-dystrophin CS1. The transduced cells were injected into TA muscles of 5-week-old mdx mice. At 4 weeks after transplantation, the grafted cells efficiently contributed to regeneration of mdx dystrophic muscles and expressed micro-dystrophin at the sarcolemma. These results suggest that there is potential for lentiviral vector-mediated ex vivo gene therapy for DMD.     

Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-kappaB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKbeta in mdx mice elucidated that NF-kappaB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-kappaB in the progression of muscular dystrophy and suggest the IKK/NF-kappaB signaling pathway as a potential therapeutic target for DMD.     

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAV-CMVLacZ) was introduced, beta-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product. We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle degeneration. AAV vector administration, however, evoked substantial immune responses in CVBA3' muscle. Importantly, AAV vector using muscle-specific MCK promoter also elicited responses in mdx muscle, but at a considerably later period. These results suggested that neo-antigens introduced by AAV vectors could evoke immune reactions in mdx muscle, since increased permeability allowed a leakage of neo-antigens from the dystrophin-deficient sarcolemma of muscle fibers. However, resident antigen-presenting cells, such as myoblasts, myotubes and regenerating immature myofibers, might also play a role in the immune response.     

Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.     

Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice

Adeno-associated virus (AAV)-mediated microdystrophin gene therapy holds great promise for treating Duchenne muscular dystrophy (DMD). Previous studies have revealed excellent skeletal muscle protection. Cardiac muscle is also compromised in DMD patients. Here we show that a single intravenous injection of AAV serotype-9 (AAV-9) microdystrophin vector efficiently transduced the entire heart in neonatal mdx mice, a dystrophin-deficient mouse DMD model. Furthermore, microdystrophin therapy normalized the heart rate, PR interval, and QT interval. The cardiomyopathy index was also significantly improved in treated mdx mice. Our study demonstrates for the first time that AAV microdystrophin gene therapy can ameliorate the electrocardiographic abnormalities in a mouse model for DMD.     

Aquaporin 4 mRNA levels in neuromuscular tissues of wild-type and dystrophin-deficient mice

Aquaporin (AQP) 4 is a water-specific channel protein and is abundant in central nervous tissues and skeletal muscles. Recently, the AQP4 molecule has been increasingly highlighted in its pathophysiological role of several neurological diseases, such as stroke, muscular dystrophy and neuromyelitis optica. We therefore measured the levels of AQP4 mRNA and glyceraldehyde-3 phosphate dehydrogenase mRNA (an internal control) in muscle and brain tissues of wild-type mice (C57BL10/ScSn) and age-matched dystrophin-deficient mdx mice (C57BL10/ScSn mdx) by real-time quantitative RT-PCR. The relative AQP4 mRNA level was highest in the spinal cord among the neuromuscular tissues examined in wild-type mice. Among the muscle tissues of wild-type mice, the relative AQP4 mRNA level was higher in extensor digitorum longus (EDL) muscles, and its descending order was EDL, quadriceps femoris, soleus and heart muscles. It is noteworthy that there was no difference in the relative AQP4 mRNA levels in the brain tissues between wild-type mice and age-matched mdx mice. In contrast, the AQP4 mRNA level in the quadriceps femoris muscle was significantly lower in mdx mice than in wild-type mice. The fact that the spinal cord contains the highest AQP4 mRNA may be related to the pathogenesis of neuromyelitis optica, in which AQP4 protein is the target antigen. In addition, the low expression level of AQP4 mRNA in the mdx mouse muscle suggests a functional link between AQP4 and dystrophin in the muscle tissue. We suggest that a similar pathomechanism may underlie the phenotypic consequences of the mdx mouse and Duchenne muscular dystrophy.