常见进行性肌营养不良的临床及病理分析

  本文关键词：常见进行性肌营养不良的临床及病理分析，由笔耕文化传播整理发布。

        目的：进行性肌营养不良症（progressive muscular dystrophin，PMD）是一组原发于肌肉组织的遗传性疾病，其共同表现为缓慢进行的肌肉萎缩、肌无力及不同程度的运动障碍。常见的类型有杜氏型肌营养不良（Duchenne muscular dystrophy，DMD）、贝克型肌营养不良（Beckermuscular dystrophy，BMD）、肢带型肌营养不良（Limb girdle musculardystrophy，LGMD）、面肩肱型肌营养不良、眼咽型肌营养不良、先天型肌营养不良、Emery-Dreifuss型肌营养不良、远端型肌营养不良、强直性肌营养不良。其中DMD和BMD为最常见的进行性肌营养不良类型，都是dystrophin基因突变所致的X连锁隐性遗传肌病。LGMD是一组具有相似临床表现但是发病机制迥异的疾病。其主要临床表现为四肢近端肌、腰带肌肌无力、肌萎缩进行性加重。LGMD可为常染色体显性（LGMD1型）、隐性（LGMD2型）遗传。再根据基因和蛋白缺陷分为不同的亚型。根据目前明确的受累基因的不同LGMDl分为LGMDlA～LGMDlH八种类型，LGMD2又分为LGMD2A～LGMD2Q十七种类型。进行性肌营养不良的辅助检查皆示血清CK不同程度增高；肌电图呈典型肌源性损害特点。肌肉病理表现大致相似：肌纤维大小不同和肌纤维坏死、再生，明显的结缔组织增生。本研究分别利用肌肉常规组织染色和免疫组化方法对拟诊PMD的病人肌肉标本进行检测，总结其临床和病理特点，对常见的进行性肌营养不良类型进行鉴别。为日后更准确分型诊断提供临床及病理理论依据。也对判断疾病预后、遗传咨询有重要作用。方法：研究分为2组-实验组和对照组，实验组23例，临床和病理资料保存完整并临床确诊为肌营养不良的病人。总结其发病年龄、首发部位、进行性肌无力部位及程度、肌萎缩部位、是否伴有腓肠肌假性肥大、CK值、肌电图结果、肌肉常规染色结果等临床病理特点。对照组2例，临床和病理资料保存完整，并确诊为肌肉病理正常者。经液氮预冷的异戊烷中速冻的冰冻肌肉标本制成8um冰冻切片。进行H-E染色、改良Gomori三色（MGT）染色、 NADH-TR染色、SDH染色、NSE非特异性酯酶染色、ORO脂肪染色、PAS糖原染色、ATP酶染色光镜观察形态学变化；用抗dystrophin Rod、抗dystrophin C-terminal、抗dystrophin N-terminal、抗dysferlin、抗α-sarcoglycan、抗β-sarcoglycan、抗γ-sarcoglycan、抗δ-sarcoglycan蛋白的八种单克隆抗体做免疫组织化学染色（immunohistoche-mical staining，IHC）。结果：1在光镜下，实验组23例肌肉常规病例染色可见肌纤维呈角形或圆形外观，大小不一，间隙增宽,部分萎缩，明显核内移，结缔组织及脂肪组织不同程度增生，可见肌纤维变性、坏死及部分再生，偶见肌纤维间炎性细胞浸润，其中15例可见肌裂；NADH染色可见分叶肌纤维、部分肌纤维深染，18例NADH染色可见肌纤维虫噬状改变；SDH染色示线粒体酶活性出现异常改变；ATP染色示12例两型肌纤维镶嵌排列正常分布，I型肌纤维占优势者5例，II型肌纤维占优势者6例；16例ORO染色示脂肪成分不同程度增高；16例PAS染色示糖原成分轻度增高。对照组肌肉病理染色示肌束内肌纤维排列紧密，大小相似，呈角形外观，未见变性、坏死和吞噬现象，无核内移增加，NADH-TR染色、SDH染色、NSE染色、ORO染色和PAS染色未见异常，ATP酶染色两型肌纤维基本呈镶嵌排列。2在23例临床拟诊肌营养不良病人肌肉中，经免疫组织化学染色共发现dystrophin染色缺陷10例（dystrophin N/C/R至少一个有完全缺失（－）的4例，显色不完整或减弱（±）6例），dysferlin染色缺陷9例（dysferlin完全缺失1例，显色不完整或减弱8例），α-sarcoglycan染色缺陷11例（α-sarcoglycan完全缺失1例，显色不完整或减弱10例），β-sarcoglycan染色显色不完整或减弱10例，γ-sarcoglycan显色不完整或减弱9例，δ-sarcoglycan显色不完整或减弱9例。对照组标本肌纤维膜免疫组化染色均无减弱和缺失。3进行性肌营养不良患者的临床特点3.1dystrophinopathyDystrophinopathy为一组疾病，临床表现多样。实验组有10例符合dystrophinopathy诊断，DMD4例，BMD6例。均为男性。其中1例有家族史。患者的血清CK水平均增高。DMD患者多为儿童期隐袭起病，多表现为双下肢近端无力，鸭步，，易跌倒，也可见单侧肢体无力。病情进展迅速。其中例13青少年起病，临床症状相对良性，病情进展较慢，故临床诊断为BMD，经免疫组化染色示dystrophin完全缺失，sarcoglycan部分减弱，符合DMD诊断标准。BMD患者多见青少年起病，症状与DMD相似，肌无力程度较DMD轻，可伴有扩张性心肌病，病情进展相对较缓，肌电图示肌源性损害。肌肉活检组织学检查示肌源性损害，可见肌纤维萎缩、变性、坏死,肌分裂，肌纤维间质增宽，脂肪细胞侵入，结缔组织增生，核内移、增大、深染，部分可见虫蚀现象。3.2sarcoglycanopathy病例6男，22岁，进行性加重四肢近端无力14年，鸭步，无肌萎缩、腓肠肌肥大，无感觉障碍及锥体束征，无阳性家族史，血清CK值4491U/L，心电图示短PR间期。肌肉病理常规检查：肌肉部分区域脂肪化，肌纤维排列较疏松，大小不一，可见多数萎缩呈圆形外观，偶见变性、坏死和吞噬现象，偶见肌分裂、肌再生和核内移。免疫组化染色示α-sarcoglycan完全缺失，β/γ/δ-sarcoglycan显色不完整，dystrophin R/N显色减弱，dysferlin膜显色减弱、胞质染色加深，考虑为sarcoglycanopathy。结合其肌肉常规病例及免疫组化染色，临床和病理诊断为LGMD2D。3.3Dysferlinopathy病例21女，38岁，14年前最早出现右下肢远端无力，随病情进展相继出现双上肢近端无力及左下肢远端无力，CK4095.7U/L，肌电图示肌源性损害，当地肌肉活检后诊断多发性肌炎，于激素治疗，病情有加重。肌肉活检组织学检查示结缔组织和脂肪组织明显增生，部分血管管壁增厚，可见炎性细胞浸润。肌纤维萎缩、变性、坏死，可见核内移和肌分裂现象；可见多个分叶状肌纤维，部分表现为虫蚀样改变。与正常骨骼肌标本对照，免疫组化染色可见dysferlin缺失，dystrophin及sarcoglycan显色完整。结合临床及病例诊断为dysferlinopathy。结论：1PMD患者中dystrophin蛋白缺失最为常见（临床拟诊PMD的23例中确诊dystrophinopathy10例）；2蛋白诊断相对于临床诊断更准确；3BMD临床上很多地方与LGMD相似，肌肉病理表现无特异性；BMD散发病例与LGMD难做出鉴别，需要进一步行免疫组化检测；4dysferlinopathy容易误诊为多发性肌炎，应用免疫组化鉴别诊断；5肌细胞膜上dystrophin或sarcoglycan蛋白缺陷，都可能会造成对方的继发性减少，须同时染色比较。

    Objective: Progressive muscular dystrophy (PMD) is a group of geneticdiseases which happen at muscle tissue. Their common features are slowmuscle atrophy, the gravis and movement disorders at different degrees. Thecommon types are Duchenne muscular dystrophy (DMD), Becker musculardystrophy (BMD), limb-girdle muscular dystrophy (LGMD), facioscapulohu-meral muscular malnutrition, oculopharyngeal muscular dystrophy, congenitalmuscular dystrophy, Emery-Dreifuss, muscular dystrophy, distal musculardystrophy and myotonic dystrophy.DMD and BMD are the most common types of the progressive musculardystrophy, which are X-linked recessive genetic myopathy caused bydystrophin gene mutations. LGMD is a group of diseases with similar clinicalmanifestations but totally different pathogenesis. The disease is clinicallycharacterized by progressive severe muscles weakness and atrophy ofproximal limb muscles and belt muscle. LGMD can be autosomal dominant(LGMD1type) or recessive (LGMD2type) genetic and can be divided intodifferent subtypes based on gene and protein defects. According to the specificaffected gene, LGMDl can be divided into LGMDlA~LGMDlH, totally8types, and LGMD2can be divided into GMD2A~LGMD2Q, totally17types.The auxiliary examination includes serum CK increasing in variousdegrees, EMG showing typical myogenic damage characteristics and musclepathology. The muscle pathology has broadly similar performance: the size ofthe muscle fibers and muscle fiber necrosis, regeneration, the apparentproliferation of connective tissue.In this study, we use muscle routine histological staining and immun-ohistochemical methods to detect patient muscle dystrophin, dysferlin andsarcoglycan expression, summarize the clinical and pathological features, andidentify the common type of progressive muscular dystrophy. This makes clinical and pathological theoretical basis for future more accurate genotypingdiagnostic and plays an important role in determining the prognosis of thedisease and genetic counseling.Methods: The study is divided into two groups-the experimental groupand the control group. The experimental group has23cases: the clinical andpathological data preserved intact and clinical diagnosis of musculardystrophy patients. I summarized their age of onset, the starting position,location and degree of progressive muscle weakness, muscle atrophy parts,whether associated with clinical and pathological features of thegastrocnemius muscle pseudo-hypertrophy, CK values, the results of the EMG,muscle routine staining results. There are2cases in the control group: theclinical and pathological data preserved intact and clinically diagnosed asmuscle pathology normal.Deep-frozen in liquid nitrogen-precooled isopentanethe frozen muscle the specimens made8um frozen sliced with muscle routinehistological staining,resistant of dystrophin Rod, the anti of dystrophinC-terminal, anti of dystrophin N-terminal anti-dysferlin, anti-α-sarcoglycan,anti-β-sarcoglycan, anti-γ-sarcoglycan, the five kinds of theanti-δ-sarcoglycan protein monoclonal antibodies do immunohistochemicalstaining(IHC).Results:1In the light microscope,23cases of muscle routine case staining musclefibers were angular or round appearance, large and small, partial atrophyobvious nuclear transfer, the gap widened, connective tissue hyperplasia, a lotof degeneration, necrosis and regeneration of muscle fiber degeneration andnecrotic muscle fibers inflammatory cell infiltration,15cases of visiblemuscle fibers split; of NADH, SDH enzyme activity limitations increased orreduce, of which18cases NADH staining visible insect bite-like muscle fibers;12cases of ATP staining two types of muscle fiber distribution normal persons,five cases have the advantage of the type I muscle fibers, six cases of type IImuscle fibers advantage; ORO staining part of the fat composition of musclefibers increased in16cases; PAS stained sections of muscle fiber increased glycogen constituents of the16cases. Muscle pathology staining of thecontrol group, showing the arrangement of muscle fibers within the musclebundle close of similar size, muscle fibers were angular appearance, nodegeneration, necrosis and phagocytosis, increased nuclear-free transfer, noabnormal staining NADH-TR and SDH staining. NSE staining, ORO stainingand PAS staining were normal the basic ATPase staining two types of musclefibers arranged in a mosaic.2Altogether10dystrophin,9dysferlin and11α-sarcoglycan can deficiencywere found in the group by IHC.there were4absence,6defect indystrophinN/C/R staining,1absence and8defect in dysferlin staining,1absence and10defect in α-sarcoglycan.10β-sarcoglycan,9γ-sarcoglycan and9δ-sarcoglycan are defected in the group by IHC. IHC staining of thespecimen muscle fiber membrane had no control group weakened andmissing.3The clinical characteristics of progressive muscular dystrophy sufferers3.1DystrophinopathyDystrophinopathy is a group of diseases with clinical manifestation.Experimental group has10cases which meet dystrophinopathy diagnosis,including4DMD cases and6BMD cases. It contains10male cases and thereis a family patient history. The patients’ creatine kinase levels are increased.DMD patients are insidious onset in childhood, generally with performance ofproximal weakness of the lower limbs, duck step-by-step, easy to fall, andunilateral limb weakness. The disease has a rapid progress. BMD patients areusually onset at adolescent, which has similar symptoms with DMD: muscleweakness lighter than DMD, maybe associated with dilatationcardiomyopathy, the progress of the disease is relatively moderate, theelectromyography icon myogenic.Muscle biopsy histologic examinationshowed myogenic damage, visible to varying degrees of muscle fiber atrophy,degeneration, necrosis, muscle split muscle fibers interstitial widened invasivefat cells, connective tissue proliferation, nuclear transfer increases, deeplystained, some visible worm-eaten phenomenon. 3.2sarcoglycanopathyCase6is a male of22years old. The symptoms are progressive increaseproximal limb weakness14years, duck step, no muscle atrophygastrocnemius muscle hypertrophy, no sensory disturbances and pyramidaltract signs, no positive family history. His serum CK levels4491U/L, andECG showed a short PR interval. Muscle biopsy histological examinationshowed the muscle part of regional fat, muscle fibers arranged looser, largeand small, rounded appearance majority is shrinking, occasionallydegeneration, necrosis and phagocytosis, occasional muscle split muscleregeneration and nuclear transfer. Immunohistochemical staining showed thecomplete absence of α-sarcoglycan, β/γ/δ-sarcoglycan staining is incomplete,dystrophin R/N staining are weakened. The dysferlin staining is weakensd onmembrane, and deepen in the cytoplasmic.According to the IHC staining,case6is sarcoglycanopathy. In combination with muscle routine case andimmunohistochemical staining, clinical and pathological diagnosis areLGMD2D.3.3DysferlinopathyCase21is a female of38years old.14years ago, the first performancewas the weakness of distal right lower limbs. With the progress of thedisease,the weakness have appeared in the proximal of the upper limbs andthe left distal lower extremity.CK is4095.7U/L. Myogenic electromyographyicon showed myogenic muscle fiber atrophy. Muscle biopsy histologicalexamination showed myogenic muscle fiber atrophy, degeneration andnecrosis, visible nuclear shift and muscle splitting, the muscle bundle clothingand muscle underwear connective tissue hyperplasia, muscle fibers visiblesmall focal or scattered distribution of inflammatory cell infiltration.Comparing with normal skeletal muscle specimens, immunohistochemicalstaining visible dysferlin missing, dystrophin and sarcoglycan staining showedcomplete. Combined with clinical and pathological,the diagnosis isdysferlinopathy. Conclusions:1. Dystrophinopathy is the most type in PMD.2. Protein is more accurate diagnosis than the clinical diagnosis.3. BMD patients’condition is relatively light with different clinicalmanifestations and many similar features with LGMD. Muscle pathologymanifested as changes of varying degrees of muscular dystrophy, non-specific.It is difficult to make the identification of sporadic cases with LGMD needfurther line IHC detection.4. Dysferlinopathy is usually misdiagnosed as polymyositis.It should bediagnosised by IHC.5. Muscle cell membrane dystrophin or sarcoglycan protein deficienciesare possible to cause secondary missing each other.

常见进行性肌营养不良的临床及病理分析

摘要4-8ABSTRACT8-12前言13材料与方法13-19结果19-22附图22-28附表28-31讨论31-34结论34-35参考文献35-38综述 dysferlinopathy 的研究概况38-48    参考文献43-48致谢48-49个人简历49

  本文关键词：常见进行性肌营养不良的临床及病理分析，由笔耕文化传播整理发布。