DMD/BMD肌细胞抗肌营养不良蛋白免疫荧光组化研究

目的:研究Duchenne/Becker型肌营养不良症(DMD/BMD)患者肌细胞中抗肌营养不良蛋白(dystrophin)的表达及其诊断意义。方法:采用免疫荧光抗体染色技术对5例DMD,2例BMD肌细胞中抗肌营养不良蛋白进行检测,以2例正常人的肌细胞作为以照。结果:对照组肌细胞膜上染色阳性,胞核及胞浆呈阴性;DMD患者肌膜完全无显色;BMD患者染色弱阳性,可见沿肌细胞膜分布的间断斑片状荧光带。结论:抗肌营养不良蛋白缺乏或表达异常是DMD/BMD基本病理基础。应用免疫荧光抗体染色法检测抗肌营养不良蛋白,有助于DMD和BMD的确诊及鉴别诊断     

Dystrophin在不同类型肌营养不良症中的变化及诊断价值

目的研究dystrophin在不同类型肌营养不良症中的变化及分型诊断价值.方法用抗dystrophin抗体对107例肌营养不良症患者肌组织标本行免疫组织化学分析.结果Duchenne型肌营养不良(DMD)患者肌细胞膜上无显色,Becker型肌营养不良(BMD)患者肌细胞膜上显色浅淡、不连续或呈斑片状.肢带型肌营养不良(LGMD)患者肌细胞膜上染色正常.结论dystrophin免疫组化染色对于年龄较小临床不易区分的DMD/BMD患者,可区分开来,以早期预测功能影响程度.该方法也有助于区分临床表现相似的成年散发BMD和LGMD患者,对于正确地进行遗传咨询具有重要意义.     

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     

针吸型肌肉活检联合免疫荧光在假肥大型肌营养不良诊断中的应用

目的 探讨应用针吸型肌肉活检结合免疫荧光染色诊断假肥大型肌营养不良症的应用价值及意义。方法 应用针吸型活检术取533例假肥大型肌营养不良症患者(415例DMD, 118例BMD)的肌组织,采用HE染色观察肌细胞形态,免疫荧光染色技术检测抗肌营养不良蛋白, 以2 例正常人的肌细胞作为对照。结果 正常人肌细胞膜上抗肌萎缩蛋白染色阳性,可见沿肌细胞膜分布完整的荧光条带; DMD 患者肌膜染色阴性,肌细胞膜完全不显色; BM D患者染色弱阳性, 可见沿肌细胞膜分布的间断斑片状荧光带。结论 应用针吸型活检术联合免疫荧光染色可以有效的检测抗肌营养不良蛋白的表达, 有助于DMD 和BMD 的确诊及鉴别诊断。     

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。     

肌营养不良蛋白表达对肌营养不良症的诊断意义

目的：探讨肌营养不良蛋白在肌营养不良症患者肌组织中表达的意义。方法：运用免疫组化法对12例Duchenne型肌营养不良症（DMD）患者及5例Becker型肌营养不良症（BMD）患者的肌组织中肌营养不良蛋白的表达进行分析。并用6例非神经肌肉疾病患者的肌组织作为对照。结果：对照组6例肌组织标本中均可见肌营养不良蛋白表达,其阳性染色勾画出肌细胞的边界,胸及胞浆呈阴性。在DMD中有10例（83．33％）肌细胞膜肌营养不良蛋白不表达。BMD中3例（60）可见沿肌细胞膜分的不连续斑片状弱阳性染色。结论：肌营养不良蛋白的缺失或异常表达,是DMD／BMD型较为特异的改变。运用免疫组化法检测患者肌组织中肌营养不良蛋白的表达,可为DMD／BMD型的病理诊断提供特异指标。     

神经肌肉病患者肌肉中utrophin和dystrophin蛋白表达的相关性

目的探讨utrophin和dystrophin蛋白在神经肌肉病患者肌肉中的表达及其相关性。方法采用免疫荧光方法观察26例共8种神经肌肉疾病患者及2名无神经肌肉疾病者的正常肌肉活检标本冰冻切片utrophin和dystrophin蛋白的表达。结果dystrophin蛋白在Duehenne型肌营养不良（DMD）患者显示为大部分肌纤维荧光圈带缺失、荧光圈带亮度减弱或荧光圈带呈不连续状；在非DMD的肌营养不良、脂肪累积性肌病、强直性肌营养不良、神经源性肌萎缩、多发性肌炎、线粒体脑肌病、肌源性肌萎缩患者肌肉膜上呈现一圈完整的强荧光圈带；在对照组肌肉膜上呈现一圈完整的强荧光圈带。utrophin蛋白在dystrophin蛋白重度减少的DMD患者少部分肌纤维肌膜上呈现不连续的荧光圈带，但强度较弱；在dystrophin蛋白中度减少的DMD患者、非DMD的肌营养不良及其他6种神经肌肉病患者肌肉膜上不显示荧光；在对照组肌肉膜上不显示荧光。结论DMD患者肌肉的dystrophin蛋白表达重度减少的同时，出现utrophin蛋白的表达；而包括DMD患者dystrophin蛋白中度减少、非DMD的肌营养不良在内的其他神经肌肉病患者肌肉的dystrophin蛋白正常表达时，其utrophin未出现表达。     

基因治疗假肥大型肌营养不良症后机体的免疫反应

假肥大型肌营养不良症(DMD)是抗肌萎缩蛋白(dystrophin)基因突变致肌膜上dystrophin完全或部分缺失引起的。基因或干细胞治疗该病的目的是使其表达有功能的dystrophin,因多数DMD患者治疗前体内没有dystrophin,基因治疗后正常的dystrophin会被患者的免疫系统识别并引发免疫反应,进一步破坏病变的肌肉组织。本文介绍了基因治疗DMD后机体产生抗dystrophin的机制及免疫耐受,并探讨了今后临床治疗DMD的策略。     

抗肌萎缩蛋白免疫组化在青少年肌肉疾病中的诊断价值

目的 探讨抗肌萎缩蛋白(dystrophin)表达对青少年肌肉疾病诊断和鉴别诊断的价值.方法 应用免疫组织化学染色,对84例年龄＜25岁的肌肉疾病和2名正常肌肉活检组织进行dystrophin检测,同时用组织化学三磷酸腺苷酶(ATP酶)、还原性尼克酰胺腺嘌呤二核苷酸(NADH-Tr)、改良Gomori、糖原PAS和脂肪苏丹Ⅲ染色法,同时对这些病例进行检测,以作为肌营养不良与其他肌肉疾病的鉴别诊断.结果 正常肌肉组织dystrophin表达强阳性.Duchenne型肌营养不良(Duchenne muscular dystrophy,DMD)中80%病例的肌纤维dystrophin免疫呈阴性反应,20%病例呈不连续点状或不完整线状弱阳性.在Becker型肌营养不良(Becket musculax dystrophy,BMD),所有的病例肌纤维都有不连续点状或不完整线状弱阳性,而其他类型的肌营养不良包括肢带型肌营养不良和强直型肌营养不良的肌纤维均为100%阳性率.另外,除1例糖原累积性肌病出现dystrophin弱阳性反应外,其他的肌肉疾病,包括炎症性肌病、线粒体肌病、神经源性肌萎缩、脂肪累积性肌病、轴空病和中央核肌病dystrophin阳性率为100%.结论 dystrophin免疫组化检测是诊断DMD/BMD可靠和有效的方法,结合形态学改变和组化染色,可以与其他肌肉疾病进行鉴别诊断.     

DMD、BMD患者骨骼肌、皮肤立毛肌肌营养不良蛋白同时欠损

目的研究、对比肌营养不良蛋白(dystrophin)在杜兴型肌营养不良(Duchenne muscular dystrophy,DMD)和贝克型肌营养不良(Becker muscular dystrophy,BMD)患者活检骨骼肌、皮肤立毛肌中的表达。方法用肌营养不良蛋白三个不同区域的单克隆抗体(Dystrophin-N、-C、-R)对11例DMD患者,5例BMD患者和3例其他神经肌病患者同时行活检骨骼肌、皮肤免疫组织化学染色分析。结果与对照例相比,11例DMD患者抗Dystro-phin-N、-C、-R单克隆抗体免疫组织化学染色显示:骨骼肌肌纤维膜Dystrophin-N、-C、-R呈完全欠损;皮肤立毛肌Dystrophin-N、-R完全欠损,Dystrophin-C轻微表达。5例BMD患者抗Dystrophin-N、-C、-R单克隆抗体免疫组织化学染色显示:肌营养不良蛋白在骨骼肌肌纤维膜和皮肤均呈不完全欠损。结论DMD和BMD患者肌营养不良蛋白在骨骼肌肌纤维膜、皮肤立毛肌呈完全/不完全欠损,与骨骼肌活检相同,皮肤活检也是分子病理学诊断DMD、BMD简便、易行、可靠的方法。     

Dystrophin as a diagnostic marker in Duchenne and Becker muscular dystrophy. Correlation of immunofluorescence and western blot

Dystrophin is the gene product of the Duchenne (DMD) and Becker (BMD) muscular dystrophy gene locus on the short arm of the X chromosome. Complete lack of dystrophin is pathognomonic for DMD and variable changes of the molecule may be observed in the milder allelic form of BMD. In the present study the two methods available for dystrophin assessment, immunofluorescence detections on cryosections (IF) and Western blotting (WB) were systematically compared using polyclonal and monoclonal antibodies to various regions along the dystrophin molecule. A total of 95 patients with DMD or BMD were investigated including two female patients. Dystrophin assessment revealed abnormal abundance and/or distribution in all 95 patients with DMD or BMD. Only trace amounts of dystrophin were detected in 29% of the DMD patients and complete lack of dystrophin was found in 71%. In two females with DMD but with normal karyotype single dystrophin-positive fibres were found among more than 90% negative fibres. Out of 26 patients with BMD 19 (73%) had a dystrophin molecule of abnormal molecular weight. The results of IF were largely compatible with those from WB but differences were also observed, e.g. one barely symptomatic BMD patient with dystrophin of increased molecular weight showed normal IF. Out of four carriers of BMD three showed evidence of reduced dystrophin immunostaining in some muscle fibres. In 20 other patients limb girdle muscualar dystrophy with "Duchenne-like" or "Becker-like" phenotype was suspected because dystrophin showed normal abundance and distribution. Focal discontinuity of muscle cell-surface dystrophin staining was observed in one patient with a congenital, autosomal recessive muscular dystrophy and in one out of five patients with polymyositis/dermatomyositis. The study emphasizes the need for, and value of, dystrophin assessment in every case of suspected BMD or DMD.     

A novel approach to identify Duchenne muscular dystrophy patients for aminoglycoside antibiotics therapy

Aminoglycoside antibiotics have been found to suppress nonsense mutations located in the defective dystophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD). However, it is very difficult to find patients that are applicable for this therapy, because: (1) only 5-13% of DMD patients have nonsense mutations in the dystrophin gene, (2) it is challenging to find nonsense mutations in the gene because dystrophin cDNA is very long (14 kb), and (3) the efficiency of aminoglycoside-induced read-through is dependent on the kind of nonsense mutation. In order to develop a system for identifying candidates that qualify for aminoglycoside therapy, fibroblasts from nine DMD patients with nonsense mutation of dystrophin gene were isolated, induced to differentiate to myogenic lineage by AdMyoD, and exposed with gentamicin. The dystrophin expression in gentamicin-exposed myotubes was monitored by in vitro dystrophin staining and western blotting analysis. The results showed that gentamicin was able to induce dystrophin expression in the differentiated myotubes by the read-through of the nonsense mutation TGA in the gene; a read-through of the nonsense mutations TAA and TAG did not occur and consequently did not lead to dystrophin expression. Therefore, it is speculated that the aminoglycoside treatment is far more effective for DMD patients that have nonsense mutation TGA than for patients that have nonsense mutation TAA and TAG. In this study, we introduce an easy system to identify patients for this therapy and report for the first time, that dystrophin expression was detected in myotubes of DMD patients using gentamicin.     

Molecular pathology of Duchenne and Becker muscular dystrophy

The gene for Duchenne (DMD) and Becker (BMD) types of muscular dystrophy has been isolated by Kunkel's and Worton's groups and shown to be the largest one over known in human, spanning more than 65 exons distributed over 2,500 kb in P21 region of X-chromosome. Fourteen kb cDNA encodes 427 kD cytoskeletal protein "dystrophin", supposed to form an anti-parallel homodimer like alpha-actinin and spectrin. The polyclonal antibodies against the synthetic peptides or fusion proteins predicted from dystrophin cDNA disclosed the complete absence of dystrophin at the surface membrane of both skeletal and cardiac muscles of DMD in marked contrast with the continuous and uniform staining in normal muscles. In manifested carriers, the mosaic expression of dystrophin was observed at the surface membrane of the skeletal muscle. BMD, which is thought to be allelic to DMD, revealed a faint or patchy immunostaining along with the abnormal and/or lower amount of dystrophin. In BMD, there is an intimate connection between the amount of dystrophin and the severity of the clinical course. It should be noted that 5 out of 39 patients with clinical diagnosis of limb-girdle (L-G) muscular dystrophy showed a patchy staining pattern, suggesting BMD not L-G. On the basis of dystrophin discovery, a possible therapeutic trial of DMD is discussed.     

Symptomatic nephrolithiasis in prolonged survivors of Duchenne muscular dystrophy

In this study, we describe the association between Duchenne muscular dystrophy (DMD) and symptomatic nephrolithiasis. The DMD patients were matched to non-ambulatory control patients with non-DMD neurological diagnoses via retrospective chart review. All patients with DMD and symptomatic nephrolithiasis were over 20 years old. We found that six of the 29 at-risk DMD patients had nephrolithiasis (20.7%) while only one of the 68 control patients had nephrolithiasis (1.5%) (p<0.0001). Controlling for duration of immobilization with stratified analysis, the risk ratio for nephrolithiasis among DMD patients compared with controls was 9.94. Using rate-based estimates of renal stone development per 10,000 patient-years, the ratio of stone development among DMD patients compared with controls was 18.5. On logistic regression analysis, the corrected odds ratio for nephrolithiasis comparing DMD patients to controls was 14.26. We conclude that, in our study group, DMD was an independent risk factor for symptomatic nephrolithiasis.     

Dystrophin or a “related protein” in Duchenne muscular dystrophy?

Previously we have shown low levels of dystrophin immunoreactivity in muscle from patients with DMD. According to the "frame-shift hypothesis" DMD muscle should not synthesize any dystrophin through to the C-terminus and it has been suggested that the protein detected is not dystrophin, but a related autosomal homologue. We have labelled serial sections of DMD muscle with specific monoclonal antibodies to the amino, rod and C-terminal domains of dystrophin and find labelling on the same individual fibres, allowing us to conclude that the protein detected is Xp21-encoded dystrophin. This has an impact on the interpretation of myoblast transfer experiments. The abundance (on blots) of "C-terminal dystrophin" appears lower than "rod dystrophin" in both BMD and DMD.     

Deletion analysis of the dystrophin gene in Duchenne and Becker muscular dystrophy patients: use in carrier diagnosis

The dystrophin gene was analyzed in 8 Duchenne muscular dystrophy (DMD) and 10 Becker muscular dystrophy (BMD) unrelated families (22 subjects: 18 index cases and 4 sibs) for the presence of deletions by multiplex polymerase chain reaction (mPCR; 27 exons) and Southern hybridization using 8 cDMD probes. Deletions were identified in 5 DMD and 7 BMD patients (6 index cases and 1 sib). The concordance between the clinical phenotype and "reading frame hypothesis" was observed in 11/12 patients (92%). The female relatives of DMD/BMD patients with identifiable deletions were examined by quantitative mPCR. Carriers were identified in 7 families. We also describe a variation in the HindIII pattern with cDNA probe 8 and 11-14. Molecular characterization of the dystrophin gene in this study has been helpful in advising the patients concerning the inheritance of the condition, and carrier diagnosis of female relatives, and should also prove useful for prenatal diagnosis.     

Expression and localization of protein inhibitor of neuronal nitric oxide synthase in Duchenne muscular dystrophy.

In skeletal muscle fibers, nitric oxide is synthesized by neuronal nitric oxide synthase (nNOS), which normally associates with the dystrophin complex in close proximity to the sarcolemma. Many reports have documented that very low levels of nNOS protein exist in muscle fibers of Duchenne muscular dystrophy (DMD) patients. In this study we investigated the functional significance of PIN (protein inhibitor of nNOS) in targeting of nNOS to the sarcolemma and the association between nNOS and the dystrophin complex in normal and dystrophic muscle fibers. Northern blotting for PIN mRNA in normal mouse muscles and muscles of mdx mice (an animal model of DMD) revealed a significant rise in PIN mRNA in dystrophic muscles compared with normal muscles. Immunohistochemical analysis showed that, in normal mouse muscle fibers, PIN expression was localized at the sarcolemma, peripheral nuclei, and the sarcoplasm. By comparison, PIN protein in muscles from mdx mice was more concentrated around the sarcolemma and central nuclei. The presence of PIN protein expression in muscles from mdx mice was evident despite the significant reduction in nNOS and dystrophin protein expressions in these fibers. In muscle sections of DMD patients, the absence of nNOS protein expression was accompanied by maintained PIN expression. Prominent PIN expression was also detectable in macrophages infiltrating dystrophic muscle fibers both in mdx mice and DMD patients. These results suggest that PIN expression in muscles from mdx mice and DMD patients is controlled by factors different from those involved in the regulation of nNOS and dystrophin. Moreover, our results indicate that PIN is not an integral component of the dystrophin complex inside skeletal muscle fibers.     

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。     

Altered regional brain glucose metabolism in Duchenne muscular dystrophy: a pet study

The basis for cognitive impairment in Duchenne muscular dystrophy (DMD) is not well understood but may be related to abnormal expression of dystrophin in brain. The aim of this study was to determine whether regional brain glucose metabolism is altered in children with DMD and whether such metabolic disturbances are localized to regions shown to be normally rich in dystrophin expression. Ten boys (mean age, 11.8 years) with DMD and 17 normal adults as a control group (mean age, 27.6 years) underwent 2-deoxy-2[(18)F]fluoro-D-glucose positron emission tomography (PET) and neuropsychological evaluation. The PET data were analyzed by statistical parametric mapping (SPM). The SPM analysis showed five clusters of decreased glucose metabolism in children with DMD, including the medial temporal structures and cerebellum bilaterally and the sensorimotor and lateral temporal cortex on the right side. At the voxel level, significant glucose hypometabolism was found in the right postcentral and middle temporal gyri, uncus, and VIIIB cerebellar lobule, as well as in the left hippocampal gyrus and cerebellar lobule. The neuropsychological profile of the DMD group revealed borderline nonverbal intellectual functioning, impaired manual dexterity bilaterally, borderline cognitive functioning, and internalizing behavioral difficulties. Our findings demonstrate region-specific hypometabolism, as well as cognitive and behavioral deficits in DMD children. As the regions showing hypometabolism on PET include those normally rich in dystrophin expression, it will be important to determine whether the hypometabolic regions also show cytoarchitectural abnormalities related to the lack of dystrophin.