线粒体病基因型—临床表型异质性和能量代偿调控机制研究
发布时间：2018-08-11 08:56
【摘要】：研究背景线粒体病(mitochondrial diseases)一组由线粒体DNA (mtDNA)或核DNA(nDNA)缺陷导致的线粒体结构和功能障碍、ATP合成不足所致的多系统疾病,包括：原发性线粒体病,由编码线粒体功能相关蛋白的基因突变引起的；继发性线粒体病,由药物、环境、感染等引起。该病于1959年被首次报道,此后几十年越来越多的疾病发现与线粒体功能异常有关,这其中不仅包括基因突变导致线粒体病,也包括一些常见疾病,例如糖尿病、肿瘤、肥胖、阿尔兹海默病、帕金森病、亨廷顿病和原发性癫痫等。Holt于1988年首次在线粒体肌病患者中发现mtDNA缺失,28年来,通过对线粒体患者及其家系的研究,几百种致病性核基因和线粒体基因被发现,从而证实了核基因和线粒体基因突变是原发性线粒体病的重要遗传因素。如mtDNA点突变可以导致线粒体脑肌病伴高乳酸血症、卒中样发作(MELAS),mtDNA单一大片段缺失引起眼外肌麻痹(CPEO),核基因Twinkle的致病突变可以导致家族性的眼外肌麻痹等。随着测序技术的发展,尽管越来越多的致病病基因被发现和报道,但是依然有很多未解的问题：①线粒体病基因型和表型多样性,临床复杂多样,受累器官不同,各年龄均可发病,这对于线粒体的诊断是个难题。②同一种致病性突变可以导致截然不同的临床表现,特殊的线粒体病亚型可以表现为特异性组织损害；③线粒体病的治疗,目前对于预防发作和延缓进展的治疗还是非常有限。自从成纤维细胞生长因子21在2005年被发现以来,由于其在机体能量代谢调控方面的广泛作用,引起了国内外研究团队的广泛兴趣。FGF21作为成纤维细胞生长因子(FGF)家族中特殊的一员,具有许多内分泌功能。FGF21和其他成纤维细胞因子家族不同,FGF21可以被分泌到血液中,作用于全身各个系统。人类的FGF21由187个氨基酸组成,主要由肝脏分泌,调节机体的葡萄糖和脂肪的代谢。研究发现：正常情况下,血液中的FGF21的浓度很低,在饥饿和酮体饮食的情况下,血中FGF21的浓度明显升高。此外,二型糖尿病、冠心病、肝功能异常等疾病都可以导致血FGF21轻度的升高。近期的研究发现,在线粒体病的小鼠模型以及线粒体病患者中,血清FGF21水平升高。虽然FGF21因子的作用在许多病理过程中被报道和研究,但是它们在线粒体病中的作用却一直未被阐明,此外关于FGF21如何调节细胞线粒体的功能的研究也很少。线粒体是细胞内三大物质代谢的主要场所,因此线粒体的功能缺陷可以导致糖、脂肪酸和氨基酸代谢的异常,不仅表现为血乳酸、丙酮酸和丙氨酸的异常,也可以引起尿液中物质代谢中间产物的升高,如2-氧戊二酸盐、琥珀酸盐、延胡索酸盐、苹果酸盐等。线粒体功能异常情况下,机体通过正反馈和负反馈机制对物质和能量代谢状态进行重新调控,最大限度地对线粒体的功能缺陷进行代偿,保证细胞相对正常的生理状态。这种能量代谢的调控机制被称为线粒体能量代谢重构。线粒体功能缺陷引起的能量代谢重构主要依赖于以线粒体为中心的生物能学体系。细胞内ATP、acetyl-CoA、NAD+、SAM的水平可直接影响DNA的甲基化、组蛋白的乙酰化以及蛋白的磷酸化和乙酰化水平,并通过上述因子重新调控细胞的能量代谢过程。为此,本课题围绕线粒体病基因型—临床表型异质性和能量代偿调控机制进行了以下几方面的工作：第一部分线粒体病中FGF21介导的能量代谢重构代偿线粒体功能缺陷的机制研究研究目的本部分围绕线粒体病中FGF21细胞因子如何重构整个机体的能量代谢过程,采用分子干预和体外实验研究的手段,明确FGF21在以肌肉损害为主的线粒体病患者肌肉标本中是否升高,阐明FGF21对机体能量代谢的影响,探索线粒体病基因型—临床表现关系复杂多变的机制。材料方法1)利用SYBR GREEN实时定量PCR方法检测分析线粒体病患者肌肉组织内FGF21 mRNA的表达,蛋白免疫电泳的方法分析线粒体病肌肉组织中FGF21蛋白的表达。2)利用线粒体呼吸链抑制剂,如鱼藤酮、寡霉素,以及解偶联剂FCCP诱导成肌细胞线粒体的功能障碍3)利用SYBR GREEN实时定量PCR方法检测分析FGF21诱导前后,肌细胞物质代谢相关基因的表达情况4)采用mtDNA数量、细胞内ATP含量、柠檬酸合酶活性以及线粒体ROS水平等方法评估成肌细胞的线粒体功能5)海马细胞能量分析仪测量细胞氧耗量和细胞外酸化率,获得细胞基础呼吸率、ATP产生量、质子漏、最大呼吸率、细胞基础糖酵解率以及最大糖酵解能力等指标。6)通过分析成肌细胞最大呼吸率评估细胞抵抗ROS的能力。7)蛋白免疫组化分析FGF21处理后,成肌细胞内mTOR-PGC1 α等信号通路的变化。8)雷帕霉素抑制mTOR蛋白激酶的活性,LY294002抑制PI3K-Akt通路。9)shRNA减少YY1和PGC1 α转录因子的表达,明确它们在FGF21促进能量代谢方面作用的分子机制。实验结果线粒体脑肌病患者肌肉组织内FGF21表达明显升高,线粒体呼吸链抑制剂诱导的线粒体损伤能够上调肌细胞FGF21的表达；FGF21因子能够促进肌细胞线粒体代谢相关基因(CPT1A, CPT2, PPARy,IDH3A, GLUT1)的表达翻译,增加成肌细胞mtDNA的数量,增强线粒体柠檬酸合酶的活性,增加细胞内ATP的含量,增加成肌细胞的氧气消耗率和促进细胞的糖酵解过程。FGF21促进成肌细胞能量代谢过程是通过激活细胞内mTOR-YY 1-PGC1 α信号通路实现的,雷帕霉素、shRNA降低PGCl α的表达能够减弱FGF21的这种促进作用。伴有线粒体功能障碍的一种组织可以通过分泌FGF21细胞因子重构整个机体的能量代谢过程。血清中升高的FGF21细胞因子,一方面作用于肝脏导致酮体的产生；另一方面作用于脂肪组织,导致脂肪的分解；这些酮体和脂肪酸可以被肌肉、脑等其他器官组织摄取和利用。实验结论①线粒体脑肌病患者肌肉组织内FGF21表达明显升高；②线粒体氧化呼吸功能异常可以引起肌细胞内FGF21细胞因子水平的升高,肌肉细胞通过分泌FGF21因子来代偿线粒体能量代谢的异常；③肌肉组织通过分泌FGF21细胞因子,一方面作用于肌肉组织本身,激活细胞内的mTOR-YY1-PGC1 α信号通路增强线粒体的功能和氧化呼吸效率,另一方面作用于肝脏和脂肪组织为肌肉供能；④伴有线粒体功能异常的肌肉组织可以通过分泌FGF21因子调控机体的能量代谢过程。第二部分线粒体基因C15620A多态位点影响G11778A突变临床表型的研究研究目的线粒体基因G11778A突变是Leber遗传性视神经病(LHON)的常见致病突变位点,而G11778A突变引起Leigh综合征(LS)确罕有人报道。我们报道一例携带有线粒体G11778A突变的LS患者的临床和病理特点,并探讨线粒体基因多态性位点C15620A对致病突变G11778A临床表型的影响。材料方法采集一例临床表现为LS的3岁汉族儿童相关病史、体格检查以及其他辅助检查资料,完善血、脑脊液乳酸、血尿有机酸、血脂酰肉碱和视神经检查,并行肌肉活检和线粒体基因全长序列分析,以明确致病突变位点。分离培养患儿的皮肤成纤维细胞,对其进行线粒体呼吸链酶活性分析、细胞线粒体膜电位及细胞ATP含量检测分析,以明确患儿的线粒体功能。实验结果该患儿自幼发育较同龄儿慢,一岁八个月会走,两岁会说话,在两岁十个月的时候开始出现意向性震颤、右眼内斜视和走路不稳。神经系统查体发现右眼内斜视,走路不稳和共济失调,眼底检查未见明显异常。颅脑MRI显示双侧延髓、脑桥、中脑及小脑对称性的长T1长T2信号,FLARI呈高信号。空腹血乳酸6.0mmol/L(正常值1.55mmol/L);脑脊液乳酸3. lmmol/L。肌肉切片酶组织化学染色提示部分肌纤维肌膜下琥珀酸脱氢酶活性增强。线粒体基因分析发现G11778A致病突变,其在肌肉组织突变率为91.6%,外周血的突变率为57%；同时并合并一个新的C15620A非致病突变,其在肌肉组织突变率分别为90%；患儿母亲的外周血中也发现了这两种突变,突变率分别是57%和79%。同时,患儿皮肤成纤维细胞呼吸链复合体Ⅰ活性、复合体Ⅲ酶活性和线粒体膜电位分别降低了67%、15%和50%。 C15620 A非致病突变可能通过降低复合体Ⅲ酶的活性而影响GI1778A致病突变的临床表型。实验结论线粒体基因GI1778A致病突变可以引起Leigh综合征。线粒体基因C15620A多态位点可能过降低复合体Ⅲ酶的活性而影响致病性GI1778A突变的临床表型,表现为Leigh综合征。第三部分Twinkle基因突变导致的家族性眼外肌麻痹的两例家系报道研究目的总结家族性眼外肌麻痹患者的肌肉病理、基因和临床表现的特点,分析Twinkle基因突变引起眼外肌麻痹的肌肉病理表现和临床特点。材料方法收集整理齐鲁医院神经肌肉研究室2005年以来的两例家族性眼外肌麻痹家系患者的临床及其他辅助检查资料,完善血乳酸、血脂酰肉碱及尿有机酸等检查,总结肌肉活检病理表现,Twinkle基因测序分析明确致病位点。总结文献资料,概括归纳Twinkle基因致病性突变的各种类型及其表型。实验结果这两个家系中的患者临床表现相对较轻,仅表现为轻-中度的眼睑下垂、轻度的眼外肌麻痹和进行性的肌无力。肌肉病理主要表现为少量的RRFs和COX酶活性缺失纤维。Twinkle基因测序分析发现了两个已报道的致病性突变,分别是c.1423GC,p.A475P和c.1061GC,p.R354P。对两个家系的先证者肌肉mtDNA分析发现,它们都存在多发缺失突变。实验结论Twinkle基因是家族性眼外肌麻痹的常见致病基因,p.A475P和p.R354P突变是常见的致病突变位点,它引起的临床表现相对较轻,肌肉病理主要特点是少量散在分布的破碎样红纤维(RRFs)和COX酶活性缺失纤维。
[Abstract]:Background Mitochondrial diseases are a group of multisystem diseases caused by mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) deficiencies and ATP deficiency, including primary mitochondrial diseases, caused by mutations in genes encoding mitochondrial function-related proteins; secondary mitochondrial diseases, caused by deficiencies in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). Drugs, environment, infection, etc. The disease was first reported in 1959. Since then, more and more diseases have been found to be associated with mitochondrial dysfunction, including not only genetic mutations causing mitochondrial diseases, but also common diseases such as diabetes, cancer, obesity, Alzheimer's disease, Parkinson's disease, Huntington's disease and primary disease. In 1988, Holt first found mtDNA deletion in patients with mitochondrial myopathy. In the past 28 years, hundreds of pathogenic nuclear and mitochondrial genes have been found in patients with mitochondrial myopathy and their families, which confirms that nuclear and mitochondrial gene mutations are important genetic factors in primary mitochondrial diseases. Mitochondrial encephalomyopathy with hyperlactinemia, stroke-like seizures (MELAS), extraocular muscle paralysis (CPEO) caused by deletion of a single large fragment of mtDNA, and familial extraocular muscle paralysis caused by mutations in the nuclear gene Twinkle. With the development of sequencing technology, although more and more pathogenic genes have been found and reported, there are still some. Many unsolved problems are as follows: 1. Mitochondrial disease is characterized by genotypic and phenotypic diversity, clinical complexity, organ involvement, and age. This is a difficult problem for the diagnosis of mitochondrial disease. 2. The same pathogenic mutation can lead to distinct clinical manifestations, and special mitochondrial disease subtypes can manifest specific tissue damage. Since the discovery of fibroblast growth factor 21 in 2005, because of its extensive role in regulating energy metabolism in the body, it has aroused widespread interest of research teams at home and abroad. Different from other fibroblast factor families, FGF21 can be secreted into the blood and acts on various systems throughout the body. Human FGF21 is composed of 187 amino acids, mainly secreted by the liver, which regulates glucose and fat metabolism. In addition, type 2 diabetes mellitus, coronary heart disease, liver dysfunction and other diseases can lead to a slight increase in serum levels of FGF21. Recent studies have found that in mice with mitochondrial disease and in patients with mitochondrial disease, serum levels of FGF21 water Although the role of fibroblast growth factor 21 in many pathological processes has been reported and studied, their role in mitochondrial diseases has not been elucidated. In addition, few studies have been done on how fibroblast growth factor 21 regulates the function of cellular mitochondria. It can lead to abnormal metabolism of sugar, fatty acids and amino acids, not only abnormal blood lactic acid, pyruvate and alanine, but also the increase of metabolic intermediates in urine, such as 2-oxoglutarate, succinate, fumarate, malate, etc. in the case of abnormal mitochondrial function, the body through positive and negative feedback The regulation mechanism of energy metabolism is called mitochondrial energy metabolism remodeling. The energy metabolism remodeling caused by mitochondrial dysfunction mainly depends on the mitochondrial function. Cellular ATP, acetyl-CoA, NAD+, and SAM levels directly affect DNA methylation, histone acetylation, and protein phosphorylation and acetylation, and through these factors re-regulate the energy metabolism process of cells. Quantitative compensatory regulation mechanisms have been studied in the following aspects: Part I: Mechanism of energy metabolism remodeling mediated by FGF21 in mitochondrial diseases to compensate for mitochondrial dysfunction To investigate whether FGF21 is elevated in the muscle samples of patients with mitochondrial disease, to clarify the effect of FGF21 on energy metabolism, and to explore the mechanism of complex and changeable relationship between mitochondrial genotype and clinical manifestation. Materials and Methods 1) To detect and analyze the muscle groups of patients with mitochondrial disease by SYBR GREEN real-time quantitative PCR. Expression of fibroblast growth factor 21 mRNA in tissues and expression of fibroblast growth factor 21 protein in mitochondrial disease muscle tissues were analyzed by protein immunoelectrophoresis. 2) Myoblast mitochondrial dysfunction induced by mitochondrial respiratory chain inhibitors such as rotenone, oligomycin and uncoupling agent FCCP was detected by SYBR GREEN real-time quantitative PCR before and after induction of fibroblast growth factor 21. Myoblast mitochondrial function was assessed by mtDNA quantity, intracellular ATP content, citrate synthase activity and mitochondrial ROS level. 5) Oxygen consumption and extracellular acidification were measured by hippocampal energy analyzer to obtain basal cell respiration rate, ATP production and proton leakage. Maximum respiration rate, basal glycolysis rate and maximal glycolysis ability were measured by analyzing maximal respiration rate of myoblasts. 7) Protein immunohistochemical analysis of the changes of signal pathways such as mTOR-PGC1alpha in myoblasts after treatment with FGF21. 8) Rapamycin inhibited the activity of mTOR protein kinase, LY294002 Inhibiting PI3K-Akt pathway.9) shRNA reduced the expression of YY1 and PGC1alpha transcription factors and clarified the molecular mechanism of their role in promoting energy metabolism by FGF21. Results The expression of FGF21 in muscle tissue of patients with mitochondrial encephalomyopathy was significantly increased, and mitochondrial injury induced by mitochondrial respiratory chain inhibitors could up-regulate the expression of FGF21 in muscle cells. Factor FGF21 can promote the expression and translation of mitochondrial metabolism-related genes (CPT1A, CPT2, PPARy, IDH3A, GLUT1) in myoblasts, increase the number of mtDNA, enhance the activity of mitochondrial citrate synthase, increase the content of ATP, increase the oxygen consumption rate of myoblasts and promote the glycolysis of myoblasts. Cellular energy metabolism is achieved by activating the mTOR-YY-1-PGC1alpha signaling pathway in cells. Reducing the expression of PGCl alpha by rapamycin and shRNA attenuates this stimulating effect of FGF21. A tissue with mitochondrial dysfunction can reconstruct the whole body's energy metabolism process by secreting cytokines from fibroblast growth factor 21. GF21 cytokines, on the one hand, induce ketone production in the liver; on the other hand, induce fat decomposition; these ketones and fatty acids can be absorbed and utilized by muscles, brain and other organs and tissues. Dysfunction of chemical respiration can induce the elevation of FGF21 cytokines in myocytes. Muscle cells compensate for the abnormality of mitochondrial energy metabolism by secreting FGF21 cytokines. Muscle tissue acts on the muscle tissue itself by secreting FGF21 cytokines and activates the mTOR-YY1-PGC1alpha signaling pathway to enhance mitochondria. Function and oxidative respiration efficiency, on the other hand, act on liver and adipose tissue to provide energy for muscle; (4) Muscle tissues with mitochondrial dysfunction can regulate energy metabolism by secreting factor FGF21. (2) Mitochondrial gene C15620A polymorphism affects the clinical phenotype of G11778A mutation. Mitochondrial G11778A mutation is a common pathogenic mutation in Leber's hereditary optic neuropathy (LHON), but G11778A mutation is rarely reported to cause Leigh syndrome (LS). We report the clinical and pathological characteristics of a LS patient with mitochondrial G11778A mutation and investigate the relationship between the mitochondrial gene polymorphism site C15620A and the pathogenic mutation G111. Materials and Methods A 3-year-old Han Chinese child with clinical manifestations of LS was collected for related medical history, physical examination and other auxiliary examinations. Blood, cerebrospinal fluid lactic acid, uric acid, serum lipid acyl carnitine and optic nerve examination were improved. Muscle biopsy and mitochondrial gene full-length sequence analysis were performed to identify the mutation site. Spot. Cultured skin fibroblasts were isolated and cultured, and their mitochondrial respiratory chain enzyme activity, mitochondrial membrane potential and cell ATP content were analyzed to clarify the mitochondrial function of the children. The results showed that the development of the children was slower than that of the children of the same age, walking at one year and eight months, talking at two years and ten months old. Intentional tremor, right intraocular strabismus, and instability of walking began to appear. Neurological examination revealed right intraocular strabismus, walking instability and ataxia. Fundus examination showed no obvious abnormalities. Craniocerebral MRI showed symmetrical long T1 and T2 signals in bilateral medulla oblongata, pons, midbrain and cerebellum. FLARI showed high signal. Fasting blood lactate 6.0 mmol/L (normal value 1.55 mmol/L). Cerebrospinal fluid lactate 3.lmmol/L.Muscle biopsy enzyme histochemical staining revealed an increased activity of succinate dehydrogenase in some myofibrils.Mitochondrial gene analysis revealed that G11778A was a pathogenic mutation with a mutation rate of 91.6% in muscle tissue and 57% in peripheral blood. Both mutations were found in the peripheral blood of the mothers of the children, 57% and 79% respectively. Meanwhile, the activity of respiratory chain complex I, complex III and mitochondrial membrane potential of the skin fibroblasts decreased by 67%, 15% and 50% respectively. Mitochondrial gene GI1778A mutation may cause Leigh syndrome. Mitochondrial gene C15620A polymorphism may affect the clinical phenotype of pathogenic GI1778A mutation by decreasing the activity of complex III enzyme. Part III Twinkle gene expression is Leigh syndrome. Objective To summarize the characteristics of muscle pathology, gene and clinical manifestations in patients with familial extraocular muscle paralysis caused by mutation of Twinkle gene, and to analyze the pathological and clinical features of extraocular muscle paralysis caused by mutation of Twinkle gene. Two families with familial extraocular muscular paralysis were studied for clinical and other auxiliary examinations. Blood lactate, serum lipid carnitine and urine organic acid were improved. Muscle biopsy pathological findings were summarized. Twinkle gene sequencing was used to identify the pathogenic site. The results showed that the clinical manifestations of these two families were relatively mild, with only mild to moderate blepharoptosis, mild extraocular muscle palsy and progressive myasthenia. Multiple deletion mutations were found in the muscles of the probands of the two families. Conclusion Twinkle gene is a common pathogenic gene of familial extraocular muscle paralysis. Mutations of P. A475P and P. R354P are common pathogenic mutations. The clinical manifestations caused by these mutations are relatively mild. The main pathological feature of meat is a small amount of fragmented red fibers (RRFs) and COX-deficient fibers.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R746
[Abstract]:Background Mitochondrial diseases are a group of multisystem diseases caused by mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) deficiencies and ATP deficiency, including primary mitochondrial diseases, caused by mutations in genes encoding mitochondrial function-related proteins; secondary mitochondrial diseases, caused by deficiencies in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). Drugs, environment, infection, etc. The disease was first reported in 1959. Since then, more and more diseases have been found to be associated with mitochondrial dysfunction, including not only genetic mutations causing mitochondrial diseases, but also common diseases such as diabetes, cancer, obesity, Alzheimer's disease, Parkinson's disease, Huntington's disease and primary disease. In 1988, Holt first found mtDNA deletion in patients with mitochondrial myopathy. In the past 28 years, hundreds of pathogenic nuclear and mitochondrial genes have been found in patients with mitochondrial myopathy and their families, which confirms that nuclear and mitochondrial gene mutations are important genetic factors in primary mitochondrial diseases. Mitochondrial encephalomyopathy with hyperlactinemia, stroke-like seizures (MELAS), extraocular muscle paralysis (CPEO) caused by deletion of a single large fragment of mtDNA, and familial extraocular muscle paralysis caused by mutations in the nuclear gene Twinkle. With the development of sequencing technology, although more and more pathogenic genes have been found and reported, there are still some. Many unsolved problems are as follows: 1. Mitochondrial disease is characterized by genotypic and phenotypic diversity, clinical complexity, organ involvement, and age. This is a difficult problem for the diagnosis of mitochondrial disease. 2. The same pathogenic mutation can lead to distinct clinical manifestations, and special mitochondrial disease subtypes can manifest specific tissue damage. Since the discovery of fibroblast growth factor 21 in 2005, because of its extensive role in regulating energy metabolism in the body, it has aroused widespread interest of research teams at home and abroad. Different from other fibroblast factor families, FGF21 can be secreted into the blood and acts on various systems throughout the body. Human FGF21 is composed of 187 amino acids, mainly secreted by the liver, which regulates glucose and fat metabolism. In addition, type 2 diabetes mellitus, coronary heart disease, liver dysfunction and other diseases can lead to a slight increase in serum levels of FGF21. Recent studies have found that in mice with mitochondrial disease and in patients with mitochondrial disease, serum levels of FGF21 water Although the role of fibroblast growth factor 21 in many pathological processes has been reported and studied, their role in mitochondrial diseases has not been elucidated. In addition, few studies have been done on how fibroblast growth factor 21 regulates the function of cellular mitochondria. It can lead to abnormal metabolism of sugar, fatty acids and amino acids, not only abnormal blood lactic acid, pyruvate and alanine, but also the increase of metabolic intermediates in urine, such as 2-oxoglutarate, succinate, fumarate, malate, etc. in the case of abnormal mitochondrial function, the body through positive and negative feedback The regulation mechanism of energy metabolism is called mitochondrial energy metabolism remodeling. The energy metabolism remodeling caused by mitochondrial dysfunction mainly depends on the mitochondrial function. Cellular ATP, acetyl-CoA, NAD+, and SAM levels directly affect DNA methylation, histone acetylation, and protein phosphorylation and acetylation, and through these factors re-regulate the energy metabolism process of cells. Quantitative compensatory regulation mechanisms have been studied in the following aspects: Part I: Mechanism of energy metabolism remodeling mediated by FGF21 in mitochondrial diseases to compensate for mitochondrial dysfunction To investigate whether FGF21 is elevated in the muscle samples of patients with mitochondrial disease, to clarify the effect of FGF21 on energy metabolism, and to explore the mechanism of complex and changeable relationship between mitochondrial genotype and clinical manifestation. Materials and Methods 1) To detect and analyze the muscle groups of patients with mitochondrial disease by SYBR GREEN real-time quantitative PCR. Expression of fibroblast growth factor 21 mRNA in tissues and expression of fibroblast growth factor 21 protein in mitochondrial disease muscle tissues were analyzed by protein immunoelectrophoresis. 2) Myoblast mitochondrial dysfunction induced by mitochondrial respiratory chain inhibitors such as rotenone, oligomycin and uncoupling agent FCCP was detected by SYBR GREEN real-time quantitative PCR before and after induction of fibroblast growth factor 21. Myoblast mitochondrial function was assessed by mtDNA quantity, intracellular ATP content, citrate synthase activity and mitochondrial ROS level. 5) Oxygen consumption and extracellular acidification were measured by hippocampal energy analyzer to obtain basal cell respiration rate, ATP production and proton leakage. Maximum respiration rate, basal glycolysis rate and maximal glycolysis ability were measured by analyzing maximal respiration rate of myoblasts. 7) Protein immunohistochemical analysis of the changes of signal pathways such as mTOR-PGC1alpha in myoblasts after treatment with FGF21. 8) Rapamycin inhibited the activity of mTOR protein kinase, LY294002 Inhibiting PI3K-Akt pathway.9) shRNA reduced the expression of YY1 and PGC1alpha transcription factors and clarified the molecular mechanism of their role in promoting energy metabolism by FGF21. Results The expression of FGF21 in muscle tissue of patients with mitochondrial encephalomyopathy was significantly increased, and mitochondrial injury induced by mitochondrial respiratory chain inhibitors could up-regulate the expression of FGF21 in muscle cells. Factor FGF21 can promote the expression and translation of mitochondrial metabolism-related genes (CPT1A, CPT2, PPARy, IDH3A, GLUT1) in myoblasts, increase the number of mtDNA, enhance the activity of mitochondrial citrate synthase, increase the content of ATP, increase the oxygen consumption rate of myoblasts and promote the glycolysis of myoblasts. Cellular energy metabolism is achieved by activating the mTOR-YY-1-PGC1alpha signaling pathway in cells. Reducing the expression of PGCl alpha by rapamycin and shRNA attenuates this stimulating effect of FGF21. A tissue with mitochondrial dysfunction can reconstruct the whole body's energy metabolism process by secreting cytokines from fibroblast growth factor 21. GF21 cytokines, on the one hand, induce ketone production in the liver; on the other hand, induce fat decomposition; these ketones and fatty acids can be absorbed and utilized by muscles, brain and other organs and tissues. Dysfunction of chemical respiration can induce the elevation of FGF21 cytokines in myocytes. Muscle cells compensate for the abnormality of mitochondrial energy metabolism by secreting FGF21 cytokines. Muscle tissue acts on the muscle tissue itself by secreting FGF21 cytokines and activates the mTOR-YY1-PGC1alpha signaling pathway to enhance mitochondria. Function and oxidative respiration efficiency, on the other hand, act on liver and adipose tissue to provide energy for muscle; (4) Muscle tissues with mitochondrial dysfunction can regulate energy metabolism by secreting factor FGF21. (2) Mitochondrial gene C15620A polymorphism affects the clinical phenotype of G11778A mutation. Mitochondrial G11778A mutation is a common pathogenic mutation in Leber's hereditary optic neuropathy (LHON), but G11778A mutation is rarely reported to cause Leigh syndrome (LS). We report the clinical and pathological characteristics of a LS patient with mitochondrial G11778A mutation and investigate the relationship between the mitochondrial gene polymorphism site C15620A and the pathogenic mutation G111. Materials and Methods A 3-year-old Han Chinese child with clinical manifestations of LS was collected for related medical history, physical examination and other auxiliary examinations. Blood, cerebrospinal fluid lactic acid, uric acid, serum lipid acyl carnitine and optic nerve examination were improved. Muscle biopsy and mitochondrial gene full-length sequence analysis were performed to identify the mutation site. Spot. Cultured skin fibroblasts were isolated and cultured, and their mitochondrial respiratory chain enzyme activity, mitochondrial membrane potential and cell ATP content were analyzed to clarify the mitochondrial function of the children. The results showed that the development of the children was slower than that of the children of the same age, walking at one year and eight months, talking at two years and ten months old. Intentional tremor, right intraocular strabismus, and instability of walking began to appear. Neurological examination revealed right intraocular strabismus, walking instability and ataxia. Fundus examination showed no obvious abnormalities. Craniocerebral MRI showed symmetrical long T1 and T2 signals in bilateral medulla oblongata, pons, midbrain and cerebellum. FLARI showed high signal. Fasting blood lactate 6.0 mmol/L (normal value 1.55 mmol/L). Cerebrospinal fluid lactate 3.lmmol/L.Muscle biopsy enzyme histochemical staining revealed an increased activity of succinate dehydrogenase in some myofibrils.Mitochondrial gene analysis revealed that G11778A was a pathogenic mutation with a mutation rate of 91.6% in muscle tissue and 57% in peripheral blood. Both mutations were found in the peripheral blood of the mothers of the children, 57% and 79% respectively. Meanwhile, the activity of respiratory chain complex I, complex III and mitochondrial membrane potential of the skin fibroblasts decreased by 67%, 15% and 50% respectively. Mitochondrial gene GI1778A mutation may cause Leigh syndrome. Mitochondrial gene C15620A polymorphism may affect the clinical phenotype of pathogenic GI1778A mutation by decreasing the activity of complex III enzyme. Part III Twinkle gene expression is Leigh syndrome. Objective To summarize the characteristics of muscle pathology, gene and clinical manifestations in patients with familial extraocular muscle paralysis caused by mutation of Twinkle gene, and to analyze the pathological and clinical features of extraocular muscle paralysis caused by mutation of Twinkle gene. Two families with familial extraocular muscular paralysis were studied for clinical and other auxiliary examinations. Blood lactate, serum lipid carnitine and urine organic acid were improved. Muscle biopsy pathological findings were summarized. Twinkle gene sequencing was used to identify the pathogenic site. The results showed that the clinical manifestations of these two families were relatively mild, with only mild to moderate blepharoptosis, mild extraocular muscle palsy and progressive myasthenia. Multiple deletion mutations were found in the muscles of the probands of the two families. Conclusion Twinkle gene is a common pathogenic gene of familial extraocular muscle paralysis. Mutations of P. A475P and P. R354P are common pathogenic mutations. The clinical manifestations caused by these mutations are relatively mild. The main pathological feature of meat is a small amount of fragmented red fibers (RRFs) and COX-deficient fibers.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R746
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