重组腺相关病毒载体介导的小基因SMCKA3999治疗对Duchenne肌营养不良肌力的影响

目的研究重组腺相关病毒载体(rAAV)介导的人dystrophin小基因SMCKA3999对DMD病理、肌力改变的治疗作用.方法将dystrophin小基因SMCKA3999克隆至rAAV并包装成rAAVSMCKA3999病毒,以5×109病毒颗粒多点注射于DMD模型鼠mdx腓肠肌,基因治疗4月后免疫荧光法检测肌膜dystrophin基因表达,治疗5月后采用肌肉离体灌注电刺激测定腓肠肌肌力,观察rAAVSMCKA3999对mdx鼠肌力的疗效.结果rAAVSMCKA3999有效稳定表达并使肌膜缺失的dystrophin恢复,明显改善mdx鼠肌力.结论rAAVSMCKA3999对DMD治疗有效,能显著改善mdx鼠肌肉功能,应用重组腺相关病毒载体介导的dystrophin小基因SMCKA3999是治疗DMD有希望的方法.     

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。     

重视Duchenne型肌营养不良症的康复治疗

康复治疗对改善Duchenne型肌营养不良症患者生活质量、延缓病情进展至关重要,一些国家业已制定了康复治疗指南,我国尚缺乏这方面的指导性研究和意见。为了提高临床医师对该病症康复治疗的认识,现对其病情分期、临床特征,以及康复治疗的一般原则、常用方法和注意事项进行概述,为临床康复治疗提供一些参考。     

慢病毒载体介导的截短肌营养不良蛋白cDNA的表达

目的 探讨慢病毒载体介导的截短肌营养不良蛋白cDNA表达的可行性。方法 将PCR克隆所构建的三个截短的肌营养不良蛋白cDNA插入到慢病毒载体，再将此载体转染至293Ad^3＋细胞中包装为重组病毒。用重组病毒感染培养的鼠成肌细胞，通过蛋白免疫印迹检测鼠成肌细胞中截短肌营养不良蛋白cDNA的表达。结果 用PCR克隆所构建的三个截短肌营养不良蛋白cDNA通过慢病毒载体导入鼠成肌细胞后均可表达出特异产物。结论 慢病毒载体可介导截短肌营养不良蛋白cDNA的表达，截短肌营养不良蛋白cDNA和慢病毒载体分别有望作为基因治疗Duchenne肌营养不良的目的基因和载体。     

假肥大型肌营养不良患者dystrophin基因缺失断裂点的分子结构特点

目的分析假肥大型肌营养不良患者dystrophin基因缺失断裂点的分子结构特点，探讨dystrophin基因缺失的发生机制。方法以PCR步移法定位2例DMD患者基因断裂位点，克隆其缺失连接片段并测序，通过Pubmed文献检索获取既往55例缺失连接片段序列资料，对以上57例缺失连接片段5′端和3′端断裂点两侧的序列进行重复序列、基质附着区、TTTAAA序列以及基因缺失后修复方式的分析。结果57例缺失连接片段中40．4％断裂点位于重复序列，其中主要是L1元件和Alu元件；36．3％断裂点在邻近基质附着区5kb之内的区域；15．0％断裂点两侧50bp的范围内发现有，TTTAAA序列；基因缺失后修复的方式仅1例通过Alu元件同源连接，其余56例通过非同源末端连接进行修复，非同源末端连接以形成1～4bp的微小同源序列为主。结论重复序列、基质附着区、TTTAAA序列以及非同源末端连接修复机制均在一定程度上参与dystrophin基因的断裂重组。dystrophin基因缺失可能是由以上多种因素的综合作用所导致，染色体的物理结构可能在基因缺失中起主要作用。     

重组腺相关病毒介导的Dystrophin小基因载体构建及在mdx鼠中表达

目的 :研究重组腺相关病毒载体 (rAAV)介导的人Dystrophin小基因 (SMCKA3 999)载体构建及在DMD模型鼠 (mdx鼠 )的表达。方法 :将SMCKA3 999质粒 ,包装质粒pXX2、腺病毒成分辅助质粒pXX6共转染 2 93细胞 ,包装重组腺相关病毒载体介导的SMCKA3 999基因 (rAAVSMCKA3 999) ,以斑点杂交法测定病毒滴度 ,将rAAVSMCKA3 999单点注射到mdx鼠腓肠肌 ,于注射后 7个月取肌肉提取蛋白质行Westernblot检测。结果 :经三质粒共转染法构建的rAAVSMCKA3 999病毒滴度为 5 0× 10 10 ,在mdx鼠骨骼肌表达持续 7个月以上。结论 :构建的rAAVSMCKA3 999载体为进一步DMD基因治疗研究奠定了基础。     

Duchenne型肌营养不良96例临床及糖皮质激素治疗分析

目的 回顾分析96例Duchenne型肌营养不良(Duchenne muscular dystrophy,DMD)患者的临床、实验室表现,并评判其对糖皮质激素治疗的效果.方法 收集96例DMD患者的临床、实验室及随访资料,按年龄分为≤3岁,4、5、6、7、8、9岁以及≥10岁组(共8组),分析各组激素治疗前后患者血肌酸激酶(CK)和运动功能,采用心肌灌注断层显像评价DMD心肌受累程度,并用韦氏儿童智力量表评价其智能情况.结果(1)血CK(mmol/L)在≤3岁(16547.9±770.9)、5岁(14 371.9±696.7)和8岁组(13 089.8±877.6)分别出现1个高峰;全部患者静脉滴注地塞米松(5～10 mg)10～15 d后血CK显著下降,口服醋酸泼尼松(0.50～0.75 mg· kg-1·d-1)1个月后血CK复升,不同治疗时相之间血CK存在差异(F=6.758,P=0.003).(2)全部患者中有51例长期口服醋酸泼尼松并随访,其中24例反复地塞米松静脉点滴,运动功能较激素治疗前改善.(3)37例DMD行心肌灌注断层显像,显示心室肌放射性核素分布不均匀,呈“花斑样”改变.心肌受损程度与年龄正相关(rs=0.685,P＜0.01).(4)24例DMD行智能评估,全部患者智商均低于健康人群.结论 DMD亚临床阶段存在高CK血症、心肌损害,且心肌损害程度与年龄正相关;糖皮质激素治疗对维持DMD运动功能和心功能有效,建议早期应用糖皮质激素治疗.     

Duchenne型肌营养不良患者肌肉组织myostatin mRNA的表达

目的 探讨肌肉生长抑制素myostatin基因mRNA在Duchenne型肌营养不良(Duchenne muscular dystrophy,DMD)肌肉组织中的表达变化.方法 以甘油醛-3-磷酸脱氧酶基因mRNA表达水平作为内参照,采用逆转录·聚合酶链反应方法扩增7例DMD患者和4例非肌肉病对照者肌肉组织的myostatin mRNA,计算myostatin mRNA表达指数,进行半定量分析.结果 两组肌肉组织均有myostatin mRNA表达;DMD患者肌肉组织中myostatin mRNA的表达指数为0.56±0.16,对照组为0.34±0.15,两组间差异有统计学意义(Z=-2.268,P=0.023).结论 DMD肌肉组织myostatin基因mRNA表达高于对照组水平,myostatin表达水平增高可能与DMD发病机制有关.     

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。     

An octaguanidine-morpholino oligo conjugate improves muscle function of mdx mice

Introduction: Skeletal muscles of mdx mice lack functional levels of dystrophin due to a mutation in Dmd exon 23. Morpholino antisense oligomers can induce expression of a truncated dystrophin by redirecting splicing to skip processing of exon 23. Methods: We tested whether systemic administration of Vivo‐Morpholino, an octaguanidine delivery moiety–Morpholino conjugate that targets exon 23 (VMO23), restored function to muscles of mdx mice. Results: Extensor digitorum longus (EDL) muscles of mdx mice were weaker, less powerful, and showed greater functional deficits after eccentric contractions than normal. VMO23 treatment normalized EDL force and power of mdx mice and eliminated their exaggerated sensitivity to eccentric contractions. Diaphragm muscle strips from mdx mice also produced lower‐than‐normal force and power, and these variables were restored to normal, or near‐normal, levels by VMO23 treatment. Conclusion: These results provide a functional basis for continuing development of VMO23 as a treatment for Duchenne muscular dystrophy. Muscle Nerve, 2011     

Gamma irradiation can reduce muscle damage in mdx dystrophic mice

We report the effects of a single gamma irradiation delivered to the soleus muscle of one limb of normal and mdx mice at the age of 16–20 days. At 45, 75 and 90 days of age transverse cryostat sections from the mid-belly of the muscles were used for microscopic examination. In normal mice the growth of fibres was appreciably reduced by irradiation without fibre loss. In the irradiated soleus of mdx mice the number of the regenerated centrally nucleated fibres was very small and the total number of fibres was remarkably reduced. The number of the peripherally nucleated fibres, presumably surviving since the birth of the animal, was almost consistently larger than in the contralateral non-irradiated limb. The cross-sectional area of the irradiated fibres was smaller. It is well known that proliferation and fusion of satellite cells are required both for regeneration after fibre damage and for the normal postnatal growth of muscle fibres: irradiation appears to reduce regeneration and growth. It is suggested that irradiation reduces damage by reducing fusion associated with growth. Our hypothesis indirectly indicates a significant link between dystrophin deficiency and fibre necrosis and accounts well for many features of mouse dystrophy under natural and experimental conditions. Received: 2 October 1997 / Revised: 26 January 1998, 23 April 1998 / Accepted: 2 June 1998     

Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue

The beta(2)-adrenoceptor agonist (beta(2)-agonist), formoterol, has been shown to cause muscle hypertrophy in rats even when administered at the micromolar dose of 25 micro g/kg/day. We investigated whether a similar low dose of formoterol could improve muscle function in the dystrophic mdx mouse. Ten-week-old male mdx and wild-type (C57BL/10) mice were administered formoterol (25 micro g/kg/day, i.p.) for 4 weeks. Formoterol treatment increased extensor digitorum longus (EDL) and soleus muscle mass, increased median muscle fibre size in diaphragm, EDL, and soleus muscles, and increased maximum force producing capacity in skeletal muscles of both wild-type and mdx mice. In contrast to other studies where beta(2)-agonists have been administered to mice and rats, generally at higher doses, low dose formoterol treatment did not increase the fatiguability of EDL, soleus or diaphragm muscles. Although others have found formoterol can decrease ubiquitin mRNA and proteasome activity when administered to tumour bearing rats at high doses (2mg/kg/day), in the present study low dose formoterol treatment did not alter ubiquitin or the E1 and E3 ubiquitin ligases in diaphragm muscles of wild-type or mdx mice, but it did reduce the level of ubiquitinated proteins in diaphragm of wild-type mice. The findings indicate that formoterol has considerably more powerful anabolic effects on skeletal muscle than older generation beta(2)-agonists (like clenbuterol and albuterol), and has considerable therapeutic potential for muscular dystrophies and other neuromuscular disorders where muscle wasting is indicated.     

Pentoxifylline fails to attenuate fibrosis in dystrophic (mdx) diaphragm muscle

Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy and in mdx diaphragm. The purpose of this study was to determine whether pentoxifylline (PTX) treatment for 4 weeks (16 mg/kg/day) could significantly attenuate the process of fibrosis in diaphragm muscle from mdx mice. PTX treatment had no impact on in vitro diaphragm muscle contractile function. In addition, diaphragm muscle hydroxyproline concentration and the level of type I and III collagen and TGF-beta1 mRNA were unaffected by PTX treatment. These findings do not support the use of PTX as an antifibrotic drug for the treatment of muscular dystrophy.