本文關(guān)鍵詞：常見(jiàn)進(jìn)行性肌營(yíng)養(yǎng)不良的臨床及病理分析，由筆耕文化傳播整理發(fā)布。

        目的：進(jìn)行性肌營(yíng)養(yǎng)不良癥（progressive muscular dystrophin，PMD）是一組原發(fā)于肌肉組織的遺傳性疾病，其共同表現(xiàn)為緩慢進(jìn)行的肌肉萎縮、肌無(wú)力及不同程度的運(yùn)動(dòng)障礙。常見(jiàn)的類型有杜氏型肌營(yíng)養(yǎng)不良（Duchenne muscular dystrophy，DMD）、貝克型肌營(yíng)養(yǎng)不良（Beckermuscular dystrophy，BMD）、肢帶型肌營(yíng)養(yǎng)不良（Limb girdle musculardystrophy，LGMD）、面肩肱型肌營(yíng)養(yǎng)不良、眼咽型肌營(yíng)養(yǎng)不良、先天型肌營(yíng)養(yǎng)不良、Emery-Dreifuss型肌營(yíng)養(yǎng)不良、遠(yuǎn)端型肌營(yíng)養(yǎng)不良、強(qiáng)直性肌營(yíng)養(yǎng)不良。其中DMD和BMD為最常見(jiàn)的進(jìn)行性肌營(yíng)養(yǎng)不良類型，都是dystrophin基因突變所致的X連鎖隱性遺傳肌病。LGMD是一組具有相似臨床表現(xiàn)但是發(fā)病機(jī)制迥異的疾病。其主要臨床表現(xiàn)為四肢近端肌、腰帶肌肌無(wú)力、肌萎縮進(jìn)行性加重。LGMD可為常染色體顯性（LGMD1型）、隱性（LGMD2型）遺傳。再根據(jù)基因和蛋白缺陷分為不同的亞型。根據(jù)目前明確的受累基因的不同LGMDl分為L(zhǎng)GMDlA～LGMDlH八種類型，LGMD2又分為L(zhǎng)GMD2A～LGMD2Q十七種類型。進(jìn)行性肌營(yíng)養(yǎng)不良的輔助檢查皆示血清CK不同程度增高；肌電圖呈典型肌源性損害特點(diǎn)。肌肉病理表現(xiàn)大致相似：肌纖維大小不同和肌纖維壞死、再生，明顯的結(jié)締組織增生。本研究分別利用肌肉常規(guī)組織染色和免疫組化方法對(duì)擬診PMD的病人肌肉標(biāo)本進(jìn)行檢測(cè)，總結(jié)其臨床和病理特點(diǎn)，對(duì)常見(jiàn)的進(jìn)行性肌營(yíng)養(yǎng)不良類型進(jìn)行鑒別。為日后更準(zhǔn)確分型診斷提供臨床及病理理論依據(jù)。也對(duì)判斷疾病預(yù)后、遺傳咨詢有重要作用。方法：研究分為2組-實(shí)驗(yàn)組和對(duì)照組，實(shí)驗(yàn)組23例，臨床和病理資料保存完整并臨床確診為肌營(yíng)養(yǎng)不良的病人�？偨Y(jié)其發(fā)病年齡、首發(fā)部位、進(jìn)行性肌無(wú)力部位及程度、肌萎縮部位、是否伴有腓腸肌假性肥大、CK值、肌電圖結(jié)果、肌肉常規(guī)染色結(jié)果等臨床病理特點(diǎn)。對(duì)照組2例，臨床和病理資料保存完整，并確診為肌肉病理正常者。經(jīng)液氮預(yù)冷的異戊烷中速凍的冰凍肌肉標(biāo)本制成8um冰凍切片。進(jìn)行H-E染色、改良Gomori三色（MGT）染色、 NADH-TR染色、SDH染色、NSE非特異性酯酶染色、ORO脂肪染色、PAS糖原染色、ATP酶染色光鏡觀察形態(tài)學(xué)變化；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蛋白的八種單克隆抗體做免疫組織化學(xué)染色（immunohistoche-mical staining，IHC）。結(jié)果：1在光鏡下，實(shí)驗(yàn)組23例肌肉常規(guī)病例染色可見(jiàn)肌纖維呈角形或圓形外觀，大小不一，間隙增寬,部分萎縮，明顯核內(nèi)移，結(jié)締組織及脂肪組織不同程度增生，可見(jiàn)肌纖維變性、壞死及部分再生，偶見(jiàn)肌纖維間炎性細(xì)胞浸潤(rùn)，其中15例可見(jiàn)肌裂；NADH染色可見(jiàn)分葉肌纖維、部分肌纖維深染，18例NADH染色可見(jiàn)肌纖維蟲(chóng)噬狀改變；SDH染色示線粒體酶活性出現(xiàn)異常改變；ATP染色示12例兩型肌纖維鑲嵌排列正常分布，I型肌纖維占優(yōu)勢(shì)者5例，II型肌纖維占優(yōu)勢(shì)者6例；16例ORO染色示脂肪成分不同程度增高；16例PAS染色示糖原成分輕度增高。對(duì)照組肌肉病理染色示肌束內(nèi)肌纖維排列緊密，大小相似，呈角形外觀，未見(jiàn)變性、壞死和吞噬現(xiàn)象，無(wú)核內(nèi)移增加，NADH-TR染色、SDH染色、NSE染色、ORO染色和PAS染色未見(jiàn)異常，ATP酶染色兩型肌纖維基本呈鑲嵌排列。2在23例臨床擬診肌營(yíng)養(yǎng)不良病人肌肉中，經(jīng)免疫組織化學(xué)染色共發(fā)現(xiàn)dystrophin染色缺陷10例（dystrophin N/C/R至少一個(gè)有完全缺失（－）的4例，顯色不完整或減弱（±）6例），dysferlin染色缺陷9例（dysferlin完全缺失1例，顯色不完整或減弱8例），α-sarcoglycan染色缺陷11例（α-sarcoglycan完全缺失1例，顯色不完整或減弱10例），β-sarcoglycan染色顯色不完整或減弱10例，γ-sarcoglycan顯色不完整或減弱9例，δ-sarcoglycan顯色不完整或減弱9例。對(duì)照組標(biāo)本肌纖維膜免疫組化染色均無(wú)減弱和缺失。3進(jìn)行性肌營(yíng)養(yǎng)不良患者的臨床特點(diǎn)3.1dystrophinopathyDystrophinopathy為一組疾病，臨床表現(xiàn)多樣。實(shí)驗(yàn)組有10例符合dystrophinopathy診斷，DMD4例，BMD6例。均為男性。其中1例有家族史�；颊叩难錍K水平均增高。DMD患者多為兒童期隱襲起病，多表現(xiàn)為雙下肢近端無(wú)力，鴨步，，易跌倒，也可見(jiàn)單側(cè)肢體無(wú)力。病情進(jìn)展迅速。其中例13青少年起病，臨床癥狀相對(duì)良性，病情進(jìn)展較慢，故臨床診斷為BMD，經(jīng)免疫組化染色示dystrophin完全缺失，sarcoglycan部分減弱，符合DMD診斷標(biāo)準(zhǔn)。BMD患者多見(jiàn)青少年起病，癥狀與DMD相似，肌無(wú)力程度較DMD輕，可伴有擴(kuò)張性心肌病，病情進(jìn)展相對(duì)較緩，肌電圖示肌源性損害。肌肉活檢組織學(xué)檢查示肌源性損害，可見(jiàn)肌纖維萎縮、變性、壞死,肌分裂，肌纖維間質(zhì)增寬，脂肪細(xì)胞侵入，結(jié)締組織增生，核內(nèi)移、增大、深染，部分可見(jiàn)蟲(chóng)蝕現(xiàn)象。3.2sarcoglycanopathy病例6男，22歲，進(jìn)行性加重四肢近端無(wú)力14年，鴨步，無(wú)肌萎縮、腓腸肌肥大，無(wú)感覺(jué)障礙及錐體束征，無(wú)陽(yáng)性家族史，血清CK值4491U/L，心電圖示短PR間期。肌肉病理常規(guī)檢查：肌肉部分區(qū)域脂肪化，肌纖維排列較疏松，大小不一，可見(jiàn)多數(shù)萎縮呈圓形外觀，偶見(jiàn)變性、壞死和吞噬現(xiàn)象，偶見(jiàn)肌分裂、肌再生和核內(nèi)移。免疫組化染色示α-sarcoglycan完全缺失，β/γ/δ-sarcoglycan顯色不完整，dystrophin R/N顯色減弱，dysferlin膜顯色減弱、胞質(zhì)染色加深，考慮為sarcoglycanopathy。結(jié)合其肌肉常規(guī)病例及免疫組化染色，臨床和病理診斷為L(zhǎng)GMD2D。3.3Dysferlinopathy病例21女，38歲，14年前最早出現(xiàn)右下肢遠(yuǎn)端無(wú)力，隨病情進(jìn)展相繼出現(xiàn)雙上肢近端無(wú)力及左下肢遠(yuǎn)端無(wú)力，CK4095.7U/L，肌電圖示肌源性損害，當(dāng)?shù)丶∪饣顧z后診斷多發(fā)性肌炎，于激素治療，病情有加重。肌肉活檢組織學(xué)檢查示結(jié)締組織和脂肪組織明顯增生，部分血管管壁增厚，可見(jiàn)炎性細(xì)胞浸潤(rùn)。肌纖維萎縮、變性、壞死，可見(jiàn)核內(nèi)移和肌分裂現(xiàn)象；可見(jiàn)多個(gè)分葉狀肌纖維，部分表現(xiàn)為蟲(chóng)蝕樣改變。與正常骨骼肌標(biāo)本對(duì)照，免疫組化染色可見(jiàn)dysferlin缺失，dystrophin及sarcoglycan顯色完整。結(jié)合臨床及病例診斷為dysferlinopathy。結(jié)論：1PMD患者中dystrophin蛋白缺失最為常見(jiàn)（臨床擬診PMD的23例中確診dystrophinopathy10例）；2蛋白診斷相對(duì)于臨床診斷更準(zhǔn)確；3BMD臨床上很多地方與LGMD相似，肌肉病理表現(xiàn)無(wú)特異性；BMD散發(fā)病例與LGMD難做出鑒別，需要進(jìn)一步行免疫組化檢測(cè)；4dysferlinopathy容易誤診為多發(fā)性肌炎，應(yīng)用免疫組化鑒別診斷；5肌細(xì)胞膜上dystrophin或sarcoglycan蛋白缺陷，都可能會(huì)造成對(duì)方的繼發(fā)性減少，須同時(shí)染色比較。

    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.

常見(jiàn)進(jìn)行性肌營(yíng)養(yǎng)不良的臨床及病理分析

摘要4-8ABSTRACT8-12前言13材料與方法13-19結(jié)果19-22附圖22-28附表28-31討論31-34結(jié)論34-35參考文獻(xiàn)35-38綜述 dysferlinopathy 的研究概況38-48    參考文獻(xiàn)43-48致謝48-49個(gè)人簡(jiǎn)歷49

  本文關(guān)鍵詞：常見(jiàn)進(jìn)行性肌營(yíng)養(yǎng)不良的臨床及病理分析，由筆耕文化傳播整理發(fā)布。