强直性肌营养不良患者肌肉组织中myostatin基因mRNA表达增加

目的 研究肌肉生长抑制素myostatin基因的mRNA在强直性肌营养不良患者肌肉组织中的表达情况.方法 采用半定量RT-PCR检测4例强直性肌营养不良患者和4名非肌肉病对照者肌肉组织中myostatin mRNA的表达.结果 2组肌肉组织中均有myostatin基因的mRNA表达.强直性肌营养不良患者肌肉组织中myostatin基因的mRNA的表达指数为2.25±1.04,明显高于对照组肌肉组织的0.34±0.15,差异有统计学意义(t=3.707,P=0.01).表达指数与肌肉萎缩之间无明显相关(r=-0.719,t=2.95,P＞0.05).结论 强直性肌营养不良患者肌肉组织中myostatin基因mRNA表达显著上调,可能是强直性肌营养不良患者肌肉萎缩的原因之一.     

多发性肌炎患者肌肉组织Myostatin mRNA的表达

目的 探讨肌肉生长抑制素Myostatin基因mRNA在多发性肌炎患者肌肉组织中的表达。方法 采用半定量RT-PCR方法检测5例多发性肌炎肌肉组织和配对非肌肉病对照者肌肉组织Myostatin mRNA表达及其与临床病理特征的关系。结果 两组肌肉组织均有Myostatin mRNA表达;多发性肌炎肌肉组织Myostatin mRNA的表达指数为0.62±0.393,对照组肌肉组织0.34±0.150,二者差异无统计学意义( P = 0.199)。结论: myostatin基因mRNA表达水平在多发性肌炎肌肉组织中与对照组无明显差异。     

Duchenne型肌营养不良症中神经元型一氧化氮合酶和utrophin的表达水平

目的研究神经元型一氧化氮合酶(n NOS)和抗肌萎缩蛋白相关蛋白(utrophin)在Duchenne型肌营养不良症(DMD)中的表达水平及与病情的关系,并探讨DMD中抗肌萎缩蛋白(dystrophin)、n NOS、utrophin 3者间的相关性。方法收集DMD患者26例,并分为轻症组(19例)和重症组(7例),收集正常对照患者21例,利用免疫荧光方法检测n NOS和utrophin的表达并进行统计分析。结果 DMD患者中23例n NOS呈完全缺失、3例明显减少,22例DMD患者utrophin表达增多,4例患者utrophin阴性表达,正常对照中n NOS均为阳性、utrophin均为阴性,DMD患者n NOS表达减少而utrophin表达增多(Z=-6.557、-5.426,P0.05)。轻症组与重症组比较n NOS、utrophin的表达水平均无统计学意义。相关分析DMD中utrophin与dystrophin的表达水平呈负相关(r=-0.419,P0.05),而n NOS与utrophin、dystrophin均无相关性。结论 DMD患者中n NOS的表达减少、utrophin的表达增多,且两者可能参与DMD的病理过程。     

联合应用多重聚合酶链反应和变性高效液相色谱法筛查缺失型Duchenne型肌营养不良

目的 建立准确、快速的缺失型Duchenne型肌营养不良筛查方法.方法 以35个家系中的35例临床诊断明确的Duchenne型肌营养不良男性患者为研究对象,对dystrophin基因(DMD基因)缺失突变热区的11对引物分2组进行多重聚合酶链反应,扩增产物分别进行2%琼脂糖凝胶电泳和变性高效液相色谱法检测,根据检测结果对患者的基因缺失情况进行判断.结果 2%琼脂糖凝胶电泳检测显示,正常对照组共出现11条电泳条带,分别代表11个外显子;DMD基因缺失型突变患者表现为部分电泳条带缺失.变性高效液相色谱法检测显示,正常对照组共出现11个洗脱峰,分别代表11个外显子;DMD基因缺失型突变患者表现为部分洗脱峰消失.35例Duchenne型肌营养不良患者中19例呈缺失型突变,两种检测方法所得结果一致.结论 联合应用多重聚合酶链反应琼脂糖凝胶电泳和变性高效液相色谱法筛查缺失型Duchenne型肌营养不良患者准确、快速,可应用于缺失型Duchenne型肌营养不良患者的基因诊断.     

免疫组织化学dystrophin染色诊断Duchenne型肌营养不良症的研究

目的 探讨Duchenne型肌营养不良症(DMD)肌萎缩蛋白(dystrophin)表达规律和临床意义.方法 收集我院7例DMD患者作为试验组,7例非DMD患者为对照组.使用抗dystrophin杆状结构域单抗、免疫组织化学染色,观察肌膜dystrophin表达.结果 7例DMD患者肌细胞膜dystrophin阴性,7例非DMD患者dystrophin染色阳性.结论 证实DMD患者肌细胞膜dystrophin表达阴性,揭示dystrophin缺失是其发病机制,可以作为确诊DMD手段,对临床诊断DMD有实际意义.     

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.     

自噬在Duchenne型肌营养不良中的研究

目的检测Duchenne型肌营养不良症(DMD)患者骨骼肌中LC3和p62的表达情况,分析自噬在DMD骨骼肌细胞坏死中的作用。方法收集2008年1月~2015年5月在我院就诊的病理确诊为DMD的患者(DMD组,81例),另以怀疑为肌病,但肌肉病理未见明显病变者为对照组(6例)。所有入选者均行心肌酶学、肌电图、骨骼肌活检常规组织学和酶学染色、抗dystrophin-N,-C,-R和抗dysferlin免疫组织化学染色。检测其中6例DMD患者及对照组骨骼肌中LC3和p62的表达。结果 81例DMD患者均为男性,起病年龄(4.60±2.35)岁,首发症状多以双下肢起病为主。血清肌酸激酶值的高峰出现在患者年龄的6~8岁,随着肌细胞明显坏死,肌酸激酶水平下降,但仍高于正常。在DMD患者骨骼肌中,组织病理均示典型肌营养不良改变。半定量Western blot提示DMD患者骨骼肌中LC3-II的表达降低,而p62表达显著升高。结论自噬功能障碍可能参与了DMD骨骼肌细胞坏死的病理生理过程。     

Duchenne型肌营养不良酶学与肌肉脂肪浸润度的研究

目的 研究Duchenne型肌营养不良(DMD)患者血清酶学的相关性和差异性,进一步分析血清酶学与患者年龄、病程及肌肉脂肪浸润程度的相关程度,为诊断疾病、评估病情及康复治疗提供依据.方法 纳入51例确诊为DMD的患者,年龄3～13(7.53±2.91)岁,病程1～11(4.31±2.64)a,发病年龄1～9(3.20±...     

重症肌无力患者骨骼肌中碳酸酐酶Ⅲ蛋白减少的原因

目的 分析重症肌无力患者(MG)骨骼肌中碳酸酐酶Ⅲ(CAⅢ)蛋白和CAⅢ mRNA的表达,并与健康对照组进行比较,探讨MG骨骼肌CAⅢ蛋白缺乏的原因.方法 用Western blot分析17例MG患者与19名健康人骨骼肌CAⅢ蛋白的水平,逆转录-聚合酶链反应(RT-PCR)技术分析CAⅢ mRNA的表达.结果 Western blot显示,MG患者骨骼肌CAⅢ蛋白谱带密度低于健康人骨骼肌;经半定量分析,健康人骨骼肌CAⅢ蛋白水平的相对值为1.70±0.29,MG骨骼肌为0.76±0.08,两者差异有统计学意义(P=0.006).RT-PCR半定量结果为:健康人骨骼肌CAⅢ mRNA表达的相对值为0.29±0.04,MG骨骼肌为0.15±0.02,两组间差异亦有统计学意义(P=0.005).分析同一标本CAⅢ蛋白与CAⅢ mRNA表达,约77%的MG患者骨骼肌中两者呈一致性减少.结论 MG骨骼肌CAⅢ蛋白降低的主要原因是其本身CAⅢ mRNA的表达降低所致,骨骼肌CAⅢ mRNA的表达降低与MG发病的关系还需进一步阐明.     

Duchenne型肌营养不良的临床和病理及抗肌萎缩蛋白表达

目的：归纳总结Duchenne型肌营养不良（DMD）的临床表现,组织病理特点及抗肌萎缩蛋白表达情况。方法：通过临床、病理及免疫组化染色方法,对16例DMD患者的临床表现,肌肉病理改变和肌肉抗肌萎缩蛋白表达情况进行观察分析。结果：年龄〉4岁的14例患儿均有比较典型的DMD临床表现;而年龄〈4岁的2例患儿症状较轻。肌肉病理显示2例为早期改变、11例为中期改变、3例为晚期改变,病理改变严重程度与年龄相关。免疫组化染色显示16例患者的肌肉标本抗肌萎缩蛋白均完全缺失。结论：DMD患者的临床和病理表现的严重程度与年龄有关,检查抗肌萎缩蛋白在肌纤维膜上表达是诊断DMD的金标准。     

Reduced serum myostatin concentrations associated with genetic muscle disease progression

Myostatin is a highly conserved protein secreted primarily from skeletal muscle that can potently suppress muscle growth. This ability to regulate skeletal muscle mass has sparked intense interest in the development of anti-myostatin therapies for a wide array of muscle disorders including sarcopenia, cachexia and genetic neuromuscular diseases. While a number of studies have examined the circulating myostatin concentrations in healthy and sarcopenic populations, very little data are available from inherited muscle disease patients. Here, we have measured the myostatin concentration in serum from seven genetic neuromuscular disorder patient populations using immunoaffinity LC–MS/MS. Average serum concentrations of myostatin in all seven muscle disease patient groups were significantly less than those measured in healthy controls. Furthermore, circulating myostatin concentrations correlated with clinical measures of disease progression for five of the muscle disease patient populations. These findings greatly expand the understanding of myostatin in neuromuscular disease and suggest its potential utility as a biomarker of disease progression.     

Transcriptional activation of the non-muscle, full-length dystrophin isoforms in Duchenne muscular dystrophy skeletal muscle.

Despite promoter tissue specificity, up-regulation of the brain and Purkinje cell type dystrophin isoforms was described in skeletal muscle of X-linked dilated cardiomyopathy (XLDCM) and BMD affected individuals. An extended population of 11 Duchenne muscular dystrophy (DMD) and 11 Becker muscular dystrophy (BMD) patients was investigated to determine whether ectopic muscle expression of the two full-length non-muscular isoforms is a common event in dystrophinopathies and if it has functional significance. Up-regulation of the two non-muscle-specific isoforms was detected in four DMD patients but in none of the BMD affected individuals or non-dystrophic controls. This is the first report of an expression of these two isoforms in DMD skeletal muscle. Ectopic expression is not confined to regenerating or revertant fibers and does not correlate with age at biopsy, clinical phenotype, cardiac involvement, deletion size or location.We consider that muscle ectopic expression of the brain and Purkinje cell-type isoforms has no favorable prognostic significance in DMD and BMD patients.     

Transforming growth factor-beta1 and fibrosis in congenital muscular dystrophies

We evaluated transforming growth factor-beta1 (TGF-beta1) expression in the muscle of four laminin alpha2-negative, four laminin alpha2-positive and seven partial laminin alpha2-deficient congenital muscular dystrophy (CMD) patients, and compared it to Duchenne muscular dystrophy (DMD) patients and controls. TGF-beta1 mRNA levels in skeletal muscle from laminin alpha2-negative and laminin alpha2-positive CMD patients were significantly greater than in controls (P < 0.05 and P < 0.005, respectively), while in partial laminin alpha2-deficient muscular dystrophy patients the amount was not significantly higher than in controls (P > 0.1). The TGF-beta1 values were lower than those found in DMD, although the extent of fibrosis was greater in CMD than in DMD and controls. Our findings suggest that TGF-beta1 is involved in CMD muscle fibrosis, but differently from what we observed in DMD muscles as it seems not to be the major player in connective tissue proliferation.     

多发性硬化患者外周血单个核细胞转录因子Sp3基因表达缺失及其与免疫的关系

目的 研究多发性硬化(Ms)患者外周血单个核细胞(PBMC)转录因子Sp3基因的表达情况及其与机体免疫功能的关系.方法 贵州地区发病的汉族MS患者31例及健康对照30名,采用逆转录PCR(RT-PCR)检测其PBMC的Sp3基因表达情况;双抗体夹心ELISA法检测血清可溶性白细胞介素-2受体(sIL-2R)水平.结果 MS组与健康对照组PBMC cDNA扩增Sp3基因阴性率差异有统计学意义[分别为41.9%(12/31)和6.7%(2/30),x2=7.133,P=0.008].MS组血清sIL-2R含量较健康对照组明显升高[(2788.5±1079.8)、(1270.6±489.4)μg/L,t=6.170,P=0.001],其中Sp3基因阴性[(3364.0±1252.3)μg/L]、阳性[(2450.0±827.0)μg/L]表达患者血清sIL-2R含量较健康对照组均明显升高(F=32.059,P<0.05),Spa基因阴性表达患者比阳性表达患者sIL-2R含量明显升高(q=4.213,P<0.05).结论 贵州地区发病的汉族MS患者PBMC中存在Sp3基因表达缺失,提示汉族MS患者Sp3基因表达无明显的地域差异.sIL-2R参与了MS的发病过程,Sp3基因表达缺失后机体发生了更严重的免疫功能障碍.     

Ezrin蛋白在肌病患者骨骼肌中的表达及意义

目的 研究Ezrin蛋白在肌病患者骨骼肌中的表达及意义.方法 取肌纤维再生活跃的假肥大型肌营养不良(DMD,9例)和多发性肌炎(PM,5例)患者的骨骼肌标本,冰冻连续切片,进行HE染色及抗-Ezrin、抗-神经细胞黏附分子(NCAM)单克隆抗体免疫组化染色,观察被检肌的病理改变和Ezrin蛋白的表达.结果 DMD、PM患者被检肌HE染色所见再生肌纤维直径较小、核位于中央、胞浆嗜碱性;NCAM染色再生肌纤维深染;再生肌纤维Ezrin呈阳性表达,伴随肌纤维成熟Ezrin表达逐渐减弱,成熟肌纤维无Ezrin表达;成肌细胞Ezrin呈阳性表达.结论 Ezrin蛋白与DMD、PM肌病患者骨骼肌纤维再生可能存在密切关系.     

Immunocytochemical analysis of dystrophin in congenital muscular dystrophy.

Using immunocytochemical methods, we examined the intensity and distribution of dystrophin and spectrin immunostaining of skeletal muscles from 51 congenital muscular dystrophy (CMD) patients including 36 Fukuyama congenital muscular dystrophy (FCMD) and 15 non-FCMD (other CMD). 17 age-matched spinal muscular atrophy (SMA) and 5 Duchenne muscular dystrophy (DMD) patient biopsies were studied as controls. All 15 non-FCMD and SMA patients showed normal localization of dystrophin at the surface membrane of each muscle fiber which was undetectable in DMD. In contrast, 34 of 36 FCMD patients exhibited an unusual immunostaining pattern with occasional (17-43%; mean = 28) negative or abnormally immunoreacted (partially deficient, fluffy or intense) fibers for dystrophin. Dystrophin was absent in 2 of 36 patients having a clinical diagnosis of FCMD, and intragenic deletion of the DMD gene was detected in one. Spectrin, a membrane cytoskeletal protein related to dystrophin, also showed an increased number of abnormally immunostained fibers in FCMD (25%), but not so high in age-matched DMD (9%) or SMA patient muscle (0%). Thus, our results suggested the presence of intrinsic factor(s) that produce abnormality of the plasma membrane of FCMD muscle.     

Gene expression profiling of Duchenne muscular dystrophy skeletal muscle

The primary cause of Duchenne muscular dystrophy (DMD) is a mutation in the dystrophin gene, leading to absence of the corresponding protein, disruption of the dystrophin-associated protein complex, and substantial changes in skeletal muscle pathology. Although the primary defect is known and the histological pathology well documented, the underlying molecular pathways remain in question. To clarify these pathways, we used expression microarrays to compare individual gene expression profiles for skeletal muscle biopsies from DMD patients and unaffected controls. We have previously published expression data for the 12,500 known genes and full-length expressed sequence tags (ESTs) on the Affymetrix HG-U95Av2 chips. Here we present comparative expression analysis of the 50,000 EST clusters represented on the remainder of the Affymetrix HG-U95 set. Individual expression profiles were generated for biopsies from 10 DMD patients and 10 unaffected control patients. Two methods of statistical analysis were used to interpret the resulting data (t-test analysis to determine the statistical significance of differential expression and geometric fold change analysis to determine the extent of differential expression). These analyses identified 183 probe sets (59 of which represent known genes) that differ significantly in expression level between unaffected and disease muscle. This study adds to our knowledge of the molecular pathways that are altered in the dystrophic state. In particular, it suggests that signaling pathways might be substantially involved in the disease process. It also highlights a large number of unknown genes whose expression is altered and whose identity therefore becomes important in understanding the pathogenesis of muscular dystrophy. Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Ultrasound imaging of muscles in Duchenne muscular dystrophy

The ultrasound imaging of quadriceps, gastrocnemius and soleus muscles was performed in 30 patients with Duchenne muscular dystrophy (DMD) and 16 control subjects. In controls, the skeletal muscle itself was scarcely echogenic. However, bone surface and fascia were clearly echogenic. The transverse scan of muscle in all DMD patients showed an increased echogenicity, which made the echo from bone or fascia less intense. The abnormal muscle echo was graded according to Heckmatt et al. From the quantitative aspect, there was a significant correlation between disability scale of DMD patients and abnormal echogenicity of the quadriceps muscle. A similar correlation was also observed between results of manual muscle testing the ultrasound imaging. The soleus muscle was usually less abnormal than the gastrocnemius in the ultrasound imaging. Thus, the ultrasound imaging seemed to provide an important information for the clinical assessment of DMD patients.