【作者】 张斌；

【导师】 焉传祝；

【作者基本信息】 山东大学 ， 临床医学（神经病学）（专业学位）， 2017， 博士

【摘要】 研究背景Pompe病又称作糖原贮积病Ⅱ型,为常染色体隐性遗传,是由于溶酶体内酸性α-糖苷酶(acid alpha-glucosidase,GAA)缺陷引起,可发生于任何年龄。酸性α-糖苷酶(GAA)主要负责在溶酶体内降解糖原为葡萄糖,其缺陷可导致多种组织内溶酶体糖原不能被降解而蓄积。Pompe病是世界上第一种被确定的溶酶体贮积病,同时也是唯一的既是糖原贮积病又是溶酶体贮积病的致死性疾病。Pompe病临床异质性很强,根据发病年龄、疾病进展速度以及受累器官严重程度将其主要分为两大类:婴儿型和晚发型。婴儿型主要表现为心肺功能障碍,而晚发型临床表现复杂多样,主要表现为进行性加重的肢带肌无力,此外还包括一些特殊的临床表现形式如急性呼吸功能不全和表现为椎基底动脉或颈内动脉系统血管迂曲扩张、多发狭窄、动脉瘤的脑血管病变。一直以来糖原贮积病Ⅱ型的发病机制被认为与肌细胞溶酶体内过多的糖原累积而溶酶体没有足够空间进行扩展,以致溶酶体最终破裂有关即经典的溶酶体肿胀破裂理论。但是随着近年来Pompe病动物模型的建立和酶替代治疗的应用以及电镜技术的发展,自噬在Pompe病发病机制中的作用逐渐受到重视。大量的实验证明,自噬聚集和溶酶体肿胀破裂理论在Pompe病的发病机理中均占有一席之地,但是二者好像并不能同时为主。在婴儿型患者其骨骼肌病理主要是以溶酶体肿胀破裂为主,经过酶替代治疗后自噬才变得更为明显,而在晚发型患者其骨骼肌的主要病理特征则是自噬聚集。Pompe病不再单纯的被认为是一种糖原贮积病,溶酶体不能降解糖原以及肌纤维各种自噬空泡的堆积共同导致肌细胞转运功能障碍、持续的自噬聚集以及明显的细胞骨架结构的异常,所述这些变化都会增强自噬活性以及影响肌纤维收缩性。上调或下调自噬活性都是在寻求一种平衡。所以调控自噬要找到平衡点,从而为寻求新的治疗方法提供依据。自噬最早于1962年由Ashford和Porten提出,是一种进化上高度保守的溶酶体介导的降解过程,又称为溶酶体自噬系统,是一种细胞自我保护机制,主要分为三类:大自噬、微自噬和分子伴侣介导的自噬。在生理状态下自噬于大多数组织内主要提供胞浆内组分的常规循环,清除受损分子和异常聚集的蛋白质,经过代谢后的大分子被再利用,从而维持细胞内组分生成与降解的平衡,而同时自噬更是细胞应对饥饿和其它应激状态的一种生存机制。凋亡即Ⅰ型程序性死亡,定义为自杀式细胞死亡,以核染色质浓缩为主要形态学特征,而自噬的特征为空泡内胞浆内容物被包裹后由溶酶体酶进行降解,又称为Ⅱ型程序性死亡,自噬和凋亡之间关系错综复杂。应激状态下自噬能移除损伤的细胞器利于修复或者通过代谢细胞大分子提供额外的ATP有助于继续生存,而随着应激状态的变化自噬的作用也随之转换。凋亡在衰老、肿瘤、恶病质、溶血、肥胖及烧伤引起的肌萎缩中的作用越来越受到重视。迄今为止有关凋亡在Pompe病发病机理中的作用机制尚不明确。凋亡和自噬的分子机制虽截然不同,但目前已有研究证实在自噬和凋亡的通路中存在一些共同调节分子,在某些条件下自噬能作为凋亡的上游或者是和凋亡一起触发细胞死亡,提示凋亡和自噬在某种通路上存在交叉点,可以相互影响,而且在特定的环境下自噬从保护细胞转化成诱导或直接参与细胞死亡,也可以说是在诱导细胞死亡的过程中细胞凋亡与自噬保持着动态平衡。已有实验证实自噬和凋亡在衰老过程肌肉萎缩的病理机制中发挥着重要作用,而有关二者在Pompe病发病机理的作用机制在国内外尚无相关报道。本课题通过免疫组化、双重免疫荧光等实验方法,首次检测了 caspase-8在晚发型Pompe病患者活检肌组织内有无表达,首次检测了 VMP1和p62在晚发型Pompe病患者活检肌组织内有无共表达,首次证实LAMP-2和catheps in B可在晚发型Pompe病疾病发展的某一阶段共表达于活检肌组织,初步探讨了自噬和凋亡在晚发型Pompe病发病机理中的作用机制,向明确晚发型Pompe病的发病机制又迈进了一步,并为寻找Pompe病的新的治疗靶点提供了实验依据。第一部分观察自噬和凋亡在晚发型Pompe病患者骨骼肌中的发生及分布特点目的观察 LC3B、LAMP-2、VMP1、p62 和 Bcl-2、cathepsinB 在晚发型 Pompe 病患者骨骼肌活检肌肉中的表达及特点。方法总结分析进行肌肉活检的Pompe病患者10例,10例患者均为晚发型。总结Pompe病患者的临床资料和病理学特点,同时另选3例正常对照患者进行肌活检。所有活检肌肉组织均分别进行苏木素-伊红(hematoxylin-eosin,HE)染色、酸性磷酸酶(acid phosphatase,ACP)染色以及溶酶体相关膜蛋白-2(lysosome associated membrane protein-2,LAMP-2)、微管相关蛋白 1 轻链 3B(microtubule-associated protein 1 light chain 3B,LC3B)、空泡膜蛋白1(vacuole membrane protein 1,VMP1)、p62、Bcl-2 和 cathepsin B 的免疫组织化学染色。结果1.10例Pompe病患者骨骼肌病理改变主要表现为肌纤维大小不等,其内可见大量空泡,空泡形态不规则,部分空泡内含嗜碱性颗粒样物质。偶见坏死及再生纤维,肌内膜不同程度增生,肌原纤维间网格状结构紊乱,肌纤维内糖原含量增多。病理表现个体差异较大,即使在同一患者骨骼肌标本可出现部分区域肌纤维轮廓清晰,纤维内也没有空泡形成,仅呈裂隙状,而另一区域却可见大片结构紊乱的肌纤维。2.酸性磷酸酶染色结果在HE染色明显空泡化的区域,酸性磷酸酶染色呈弱阳性;在HE染色呈紫红色颗粒区域,酸性磷酸酶染色呈强阳性;在空泡化不十分严重或者仅仅点状分布区域,酸性磷酸酶染色亦呈弱阳性,且在肌纤维核心区域阳性表达。3.免疫组化染色结果LAMP-2、p62在10例患者肌组织内均呈强阳性表达。而VMP1、LC3B、Bcl-2和cathepsinB在晚发型Pompe病患者活检肌组织内的表达程度变异较大。LC3B在多数肌纤维破坏较重的区域几乎不表达或部分点状弱表达,部分空泡化严重的肌纤维内阳性表达,尚未空泡化肌纤维核心区域呈点状或团块样阳性表达,不完全空泡化肌纤维呈弱表达。VMP1在空泡化较重的肌纤维内不表达,在尚未完全空泡化的肌纤维内可有阳性表达,Bcl-2在空泡化较重的肌纤维肌膜表达阳性,不完全空泡化的肌纤维内弱阳性表达,而尚未空泡化的肌纤维部分弱表达。cathepsin B在破坏较重的肌纤维内不表达或者弱表达,在不完全空泡化或者尚未空泡化的肌纤维内可呈阳性表达;正常对照组各种染色均未见阳性表达。结论在晚发型Pompe病患者受累的骨骼肌组织中,LAMP-2、LC3B、VMP1、p62、Bcl-2、cathepsinB有着不同程度的表达,且分布区域不同,其与骨骼肌肌纤维破坏的程度相关,提示自噬和凋亡可能共同参与了晚发型Pompe病肌纤维结构的破坏,在其不同的发展阶段发挥着不同的作用。第二部分初步探讨自噬和凋亡在晚发型Pompe病发病机理中的作用机制目的检测caspase-8(p18)的表达以及分别通过检测LAMP-2和cathepsinB、LC3B 的共表达,以及空泡膜蛋白 1(vacuole membrane protein 1,VMP1)和 p62的共表达,以探讨自噬和凋亡在晚发型Pompe病发病机理中的作用机制。方法选取行肌肉活检的患者16例,其中晚发型Pompe病患者10例,3例脂质沉积性肌病(lipid storage myopathy,LSM)患者、正常对照组3例。全部活检标本均行 LAMP-2、LC3B、caspase-8(p18)、cathepsinB、VMP1、p62 免疫荧光染色,并用双重免疫荧光方法分别检测LAMP-2和LC3B、cathepsinB,V VMP1和p62的共表达。结果免疫荧光染色显示在空泡化明显的晚发型Pompe病患者肌纤维内主要可见LAMP-2和p62的强阳性表达,在LSM患者肌肉组织内亦有不同程度的表达。LC3B、cathepsin B、VMP1、caspase-8(p18)染色结果接近,表现为在空泡化明显的晚发型Pompe病患者肌纤维内呈弱表达甚至不表达,而在受累肌纤维尚未空泡化或仅仅点状改变时阳性表达。在尚未空泡化或者轻度空泡化的晚发型Pompe病患者肌纤维内可以检测到VMP1和p62的共表达以及LAMP-2和cathepsin B的共表达,共表达主要集中在肌纤维的核心区域。而在明显空泡化的肌纤维内,均未发现共表达。正常对照组各染色均无阳性表达。结论caspase-8介导的细胞凋亡途径在晚发型Pompe病患者肌肉组织病理生理机制中可能发挥着重要作用。VMP1介导的选择性自噬途径在晚发型Pompe病肌肉组织中可能有保护作用,可能成为晚发型Pompe病患者新的治疗靶点。自噬和凋亡相关蛋白在晚发型Pommpe病受累骨骼肌不同的发展阶段可能发挥着不同的作用。更多还原

【Abstract】 BackgroundPompe disease(Glycogen storage disease typeⅡ,GSDII)is a rare hereditary disease caused by genetic defects of GAA(acid alpha-glucosidase)which is a lysosomal enzyme responsible for the degradation of glycogen to glucose.Pompe disease can occur at any age,and is classified into infantile and late-onset forms based on onset age,organ involvement,and disease progression.For a long time the pathogenesis of GSDII is thought to be associated with the accumulation of glycogen in lysosomes leading to lysosome rupture eventually.But in recent years,with the development of the application of animal experiments and enzyme replacement therapy and the development of electron microscopy,the role of autophagy in the pathogenesis has been paid more and more attention.A large number of experiments have proved that the theory of autophagic aggregation and lysosomal swelling and rupture has a place in the pathogenesis of Pompe disease,but the two do not seem to happen simultaneously.Pompe disease is no longer simply considered a lysosomal glycogen storage disease.The accumulation of glycogen and autophagic vacuoles leads to muscle cell transport dysfunction,sustained autophagy aggregation and cytoskeletal structure abnormal,all of changes will enhance autophagy activity and affect the contraction of muscle fiber.Autophagy is proposed by Ashford and Porten as early as 1962,which is a highly conserved evolutionarily degradation process by lysosomes.Autophagy is a self protective mechanism of cells.But it has a perplexing relationship with apoptosis,necrosis and cell death.It is unclear how autophagy induced apoptosis.The main morphological characteristics of apoptosis which is also called type I programmed cell death is nuclear chromatin condensation.The relationship between autophagy and apoptosis is unclear.A large number of experiments confirmed that some common regulatory molecules exist in two pathways.Under certain conditions,autophagy can act as the upstream of apoptosis or trigger cell death.It has been proved that autophagy and apoptosis play an important role in aging muscle atrophy,but there is no report about the mechanism of in the pathogenesis of Pompe disease.In our study,we investigated the mechanism of autophagy and apoptosis in the pathogenesis of Pompe by immunohistochemistry and double immunofluorescence for the first time.Part Ⅰ:The pathological characteristics of autophagy and apoptosis in muscle tissue of late-onset Pompe patientsObjectiveTo study the pathological characteristics of LC3B,LAMP-2,VMP1,p62,Bcl-2,cathepsin B in muscle tissue of late-onset Pompe patients.MethodMuscle biopsies were taken from 10 late-onset Pompe patients and 3 controls.We summarized the clinical data and pathological characteristics in 10 Pompe patients.All the biopsy specimens were processed with hematoxylin-eosin,acid phosphatase staining and immunohistochemical staining for anti-LAMP-2,anti-p62,anti-LC3B,anti-VMP1,anti-Bcl-2 and anti-cathepsin B.Result1.Pathological changes of skeletal muscle in 10 patients with Pompe diseaseThe results showed that the size of muscle fiber was different,and there were a large number of vacuoles in it.The shape of vacuoles was irregular.The necrosis and regeneration fibers were seen occasionally,the proliferation of the endomysium was different,the disordered structure of the myofibrils and the glycogen content increased.Pathological manifestations of individual differences,even in the same skeletal muscle of one patient.2.Acid phosphatase stainingACP staining showed weak positive staining in obvious vacuolar region in HE.ACP staining showed strong positive in purple grain regions.ACP staining was weak in the area of incomplete vacuolization,this positive expression lied in the core area of muscle fibers.3.Immunohistochemical stainingThe expression of LAMP-2,VMP1 in 10 cases was strongly positive,but there was significant variability in the expression of p62,LC3B,Bcl-2 and cathepsin B.Generally the expression of p62,LC3B,Bcl-2 and cathepsin B were positive in non-vacuolated or incomplete vacuolated fibers and negative in vacuolated fibers.ConclusionLAMP-2,LC3B,p62,VMP1,Bcl-2,cathepsin B have different levels of expression and distribution in different course of Pompe,which is related to the degree of destruction of skeletal muscle fibers.It is suggested that autophagy and apoptosis may be involved in the destruction of Pompe muscle fiber structure and play different roles in different stages of development.Part Ⅱ:The role of autophagy and apoptosis in the pathogenesis of 1 ate-onset Pompe patientsObjectiveTo demonstrate the mechanism of autophagy and apoptosis in the pathogenesis of late-onset Pompe by detecting the expression of caspase-8 and the co-expression of LAMP-2 and cathepsin B,LC3B and VMPland p62,respectively.MethodMuscle specimens were obtained retrospectively from 10 Pompe patients and 6 controls,of whom,3 had LSM,and 3 non-myopathic patients.All the samples were stained with anti-LAMP-2,anti-LC3B,anti-caspase-8(p 18),anti-cathepsin B,anti-VMP1,anti-p62,and double immunofluorescence staining was used to detect the co-expression of them.ResultImmunofluorescence staining showed that LAMP-2 and p62 were strong expressed in vacuolated muscle fibers of Pompe and also had different degrees of expression in muscle of patients with lipid storage disease.The expression of LC3B,cathepsinB,VMP1 and caspase-8(p18)staining was weak or not expressed in the muscle fibers with obvious vacuolization.The non-vacuolated or only dot-like changed fibers showed obvious positive expression of these staining.Co-expression of VMP1 and p62,LAMP-2 and cathepsin B were found in the muscle fibers which had not yet been vacuolated completely,and the expression was mainly in the core region of muscle fibers.However,no obvious co-expression was found in the muscle fibers with obvious vacuolization.In contrast,muscle biopsies from 3 non-disease controls showed negative caspase-8(pl8),LAMP-2,cathepsin B,LC3B,VMP1 and p62 staining.Conclusion The apoptosis pathway mediated by caspase-8 may plays an important role in the pathophysiology of late-onset Pompe muscle tissue.VMP1 mediated selective autophagy pathway may play a protective role in late-onset Pompe muscle tissue.Autophagy and apoptosis related proteins may play different roles in different developmental stages.of late-onset Pompe disease.更多还原