线粒体脱氧核糖核酸缺失与病毒性心肌疾病患者心功能损伤的关系

目的探讨不同心功能状态病毒性心肌疾病患者外周淋巴细胞线粒体脱氧核糖核酸(mtI)NA)缺失率,为进一步明确mtDNA损伤在病毒性心肌疾病发病中的作用提供分子依据方法将83例病毒性心肌疾病患者分为病毒性心肌炎组(VMC组,n=50)和扩张型心肌病组(DCM组,n=33),23例健康献血者为对照组.用定量多聚酶链反应(PCR)法检测患者外周淋巴细胞mtDNA4977碱基对(mtDNA4977)和mtDNA7436碱基对(mtDNA7436)缺失率,并分析mtDNA缺失程度与心脏扩大及心功能状态的关系.结果VMC组和DCM组外周淋巴细胞mtDNA缺失率显著高于对照组(P<0.001);mtDNA缺失率的高低与心功能损害呈一致性改变,NYHA心功能I级与IV级者mtDNA缺失率分别比对照组高12.4倍和110.3倍;mtDNA缺失率与心功能分级、心胸比率及心腔内径呈正相关,与左心室射血分数(LVEF)呈负相关;mtDNA4977和mtDNA7436缺失率与LVEF的相关系数分别为-0.681和(0.675(P均<0.05).结论mtDNA缺失突变与病毒性心肌疾病患者心功能的损伤密切相关,可能在该病发生与发展中起一定作用.     

病毒性心肌炎患者心肌细胞线粒体DNA缺失的定量分析

为探讨病毒性心肌炎 (VMC)患者心肌细胞线粒体 DNA(mt NDA)缺失突变情况及意义 ,用定量 PCR法检测 2 0例 VMC患者心肌细胞及其外周血淋巴细胞 mt DNA4 977碱基对 (mt DNA4 977)和 mt DNA74 36 碱基对 (mt D-NA74 36 )缺失率。取 10例健康意外死亡者心肌和 2 0例献血员外周血淋巴细胞作正常对照。结果显示 ,正常对照者和 VMC患者心肌细胞均存在 m t DNA4 977及 mt DNA74 36缺失 ,合计缺失率分别为 0 .176 %、0 .384 % ,二者差异显著 ,P<0 .0 5 ;VMC患者外周淋巴细胞 mt DNA缺失程度与心肌细胞呈一致性改变 ,且有良好的相关性 (r=0 .92 0 ,P<0 .0 0 1)。提示 mt DNA缺失可能是 VMC发病过程中重要的心肌损伤机制 ;外周淋巴细胞在研究心肌细胞 mt DNA缺失中的作用值得进一步探讨     

风湿性心脏病患者心肌、骨骼肌细胞mtDNA突变的相关性研究

目的探讨风湿性心脏病(风心病)患者心肌细胞与骨骼肌细胞线粒体DNA(mtDNA)突变的相关性.方法随机选取56例风湿性二尖瓣狭窄为主患者(下称实验组,冠心病除外),另选10例意外死亡的成人做为正常对照组.术中切取后乳头肌作为心室肌标本,胸大肌作为骨骼肌标本.定量每例患者心肌及骨骼肌mtDNA4977缺失率,分析其相关性及与心功能的关系.结果实验组心肌mtDNA4977缺失率为1.18%～13.75‰,对照组为0～0.06‰,两者有显著性差异(P＜0.01).随着心功能恶化,实验组心肌mtDNA4977缺失率增加.按NYHA心功能分级,Ⅱ级(B组)、Ⅲ级(C组)、Ⅳ级(D组)心肌mtDNA4977缺失率分别为3.79‰、6.32‰和9.28‰,两两比较,P＜0.05.实验组心肌、骨骼肌mtDNA4977缺失率呈正相关(γ=0.75,P＜0.01).结论风心病患者的心肌mtDNA4977缺失率与其心功能损害程度密切相关;其骨骼肌mtDNA4977缺失率与心肌mtDNA4977缺失率呈正相关.     

心肌mtDNA4977缺失与扩张型心肌病猝死的关系

目的探讨扩张型心肌病(DCM)猝死者心肌线粒体DNA(mtDNA)4977缺失情况及其与猝死的关系.方法从近7年尸检案例中挑选11例DCM猝死和14例对照组病例及心肌组织蜡块,按常规方法提取心肌mtDNA,用PCR、琼脂糖紫外凝胶成像技术确定扩增产物激光密度,初步定量检测mtDNA4977缺失率.结果DCM猝死11例中,未成年人2例;成人9例,均为男性,年龄22～49(平均38)岁.对照组14例中,未成年人1例;成人13例,其中男11例,女2例,年龄19～47(平均37.23)岁,死因为机械性损伤和窒息各2例,电击死2例,中毒2例,非心肌病猝死6例.DCM11例(占100%)、对照组2例(占14.28%)检见不同程度的mtDNA4977缺失;两组病例mtDNA4977缺失率均值分别为0.92%和0.09%,差异有统计学意义.结论DCM猝死者心肌可检见mtDNA49 77缺失;其心肌mtDNA4977缺失突变与DCM猝死有关.     

抗人心肌线粒体抗体在病毒性心肌炎和扩张型心肌病中的检测及其意义

目的检测病毒性心肌炎(VMC)和扩张型心肌病(DCM)患者血清中是否有抗人心肌线粒体抗体存在及其意义。 方法以人心肌线粒体作抗原,应用免疫点印迹在29例VMC(VMC组)、24例DCM(DCM组)、33例其它心脏病(OCD组)及20例健康献血者(HBD组)进行抗人心肌线粒体抗体检测,用免疫印迹方法检测其抗原分子量。 结果VMC组(12例,41.4%)DCM组(10例,41.7%)抗人心肌线粒体抗体阳性明显高于OCD组(2例,6.1%)及HBD(0%);②抗体阳性的VMC和DCM患者中有43.8%的心肌肌钙蛋白T升高,抗体阴性者为12.0%(P＜0.05)有显著性差异;③人心肌线粒体特异性抗原的分子量为30KD。 结论①在VMC和DCM患者血清中存在抗人心肌线粒体抗体,说明部分VMC及DCM与自身免疫有关,该抗体是引起心肌损伤的因素之一,对该抗体的检测可作为VMC和DCM的辅助诊断手段之一;②该抗体的特异性抗原可能为人心肌线粒体腺苷酸转位酶。     

心力衰竭患者心肌线粒体脱氧核糖核酸损伤与衰竭心肌线粒体酶活性的关系

目的 :为探讨心肌线粒体脱氧核糖核酸 (mt DNA)获得性损伤对衰竭心肌线粒体酶活性改变的影响 ,确立 mt DNA损伤在衰竭心肌发病中的作用。　　方法 :对 2 8例慢性心力衰竭患者活检心肌 ,用定量聚合酶链式反应 (PCR)及生化方法 ,观察衰竭心肌中常发缺失型突变 mt DNA4977和 mt DNA7436 缺失率与线粒体呼吸酶活性和心脏功能受损之间的关系。　　结果 :慢性心力衰竭患者活检心肌中的 mt DNA存在不同程度的缺失突变损伤 ,两种缺失类型合计的缺失率在0 .10 2 %～ 1.745 %之间 ;mt DNA缺失率与心肌生化水平上的线粒体呼吸酶活性改变相关性良好 ;同时与整体水平上的心脏泵功能受损呈一致性关系。　　结论 :心肌 mt DNA损伤与心肌线粒体呼吸功能下降和心脏功能受损程度密切相关 ,提示 mt DNA损伤可能参与了心力衰竭的形成与发展。     

老年耳聋的线粒体DNA4977缺失研究

目的探讨mtDNA4977缺失与老年耳聋的关系。方法提取老年耳聋组(20例)与老年听力正常组(20例)的外周血DNA,采用聚合酶链反应(polymerase chainreaction,PCR)及巢式PCR技术,扩增正常及缺失区mtDNA片段。结果在6例老年耳聋中检测到了mtDNA4977缺失,而听力正常的老年组中未检测到mtDNA4977缺失。结论mtDNA4977缺失是老年耳聋的发病原因之一。     

病毒性心肌炎小鼠心肌与骨骼肌细胞线粒体损伤及其相关性

目的探讨病毒性心肌炎（VMC）小鼠心肌与骨骼肌细胞线粒体损伤（线粒体膜磷脂脱失和线粒体DNA3867缺失）程度及二者的相关性。方法50只BALB/c小鼠随机分为2组,实验组（40只）腹腔注射内含柯萨奇B3病毒（Coxsackievirus B3,CVB3,TCID50=108）的Eagle液制备VMC小鼠模型,另10只为对照组。分别于病毒感染后3、11和24 d行心肌和骨骼肌细胞线粒体膜磷脂脱失程度和mtDNA3867缺失率的测定,并用Spearman法对其进行相关分析。结果实验组小鼠在病毒感染后3 d,可见心肌和骨骼肌细胞mtDNA3867缺失率显著高于对照组（P<0.05）,而线粒体膜磷脂脱失程度与对照组相比无显著性差异;病毒感染后11 d,心肌和骨骼肌细胞mtDNA3867缺失性损伤达高峰（P<0.05）,线粒体膜磷脂脱失程度亦显著高于对照组（P<0.05）;病毒感染后24 d,心肌和骨骼肌细胞线粒体膜磷脂脱失和mtDNA3867缺失程度与感染后11 d组比较无显著性差异,但与对照组和病毒感染后3 d组比较,仍有显著性差异（P<0.05）。线粒体的上述损伤性改变在心肌和骨骼肌细胞呈一致性同步变化,且具有良好相关性（P<0.05）。结论CVB3可显著损伤心肌和骨骼肌线粒体DNA和膜磷脂,两者损伤具有相关性,提示骨骼肌有望成为反映心肌细胞线粒体损伤的外周细胞“窗口”。     

扩张型心肌病患儿线粒体DNA缺失的研究

采用PCR法检测了15例扩张型心肌病（DCM）、13例急性心肌炎（病程＜3个月）患儿和10例正常儿童外周血淋巴细胞中线粒体DNA（mtDNA）的缺失情况,以研究mtDNA突变在DCM发病中的作用。结果显示,被检者线粒体均存在5Kb和7.4Kb的mtDNA缺失,但DCM患儿的缺失率显著高于心肌炎患儿和正常儿童（缺失率分别为7.97±3．51％,2.5±1．64％和2.28±1．76％,P＜0．05）。提示mtDNA缺失与部分DCM的发病有关。     

病毒性心肌炎患儿血清肌酸激酶同工酶及心肌肌钙蛋白Ⅰ的动态变化

目的通过连续观测急性病毒性心肌炎(VMC)患儿血清肌酸激酶同工酶(CK-MB)和心肌肌钙蛋白(cTnI)与外周血淋巴细胞等的变化,探讨VMC心肌细胞受损的机制。方法对32例急性期和12例迁延期及慢性期VMC患儿血清cTnI、CK-MB,外周血T淋巴细胞亚群、B淋巴细胞、自然杀伤细胞,肠道病毒RNA,柯萨奇病毒(CBV)特异性抗体IgM(CBV-IgM),淋巴细胞增殖活性(PI),补体C     

Multiple skeletal muscle mitochondrial DNA deletions in patients with unilateral peripheral arterial disease

Peripheral arterial disease (PAD) is associated with metabolic derangements and accumulation of the common 4977 bp mitochondrial DNA (mtDNA) deletion mutation. The current study was undertaken to test the hypothesis that PAD is associated with multiple mtDNA deletions. Gastrocnemius biopsies were obtained from nine patients with unilateral PAD. DNA extracted from the biopsies was analyzed for mtDNA deletions using a primer-shift PCR strategy. Multiple primers and strict, prospective criteria were used to identify deletions. PAD was associated with multiple mtDNA deletions (average of 8.2 distinct deletions in muscle from the hemodynamically affected limb). mtDNA injury was present in both the worse- and less-affected limbs of the unilateral PAD patients, and the estimated degree of mtDNA injury was strongly correlated in the two limbs on an intra-subject basis. The 4977 bp deletion was frequently identified, but was not always the deletion of highest frequency in individual samples. The estimated relative frequency of the 4977 bp deletion was correlated with the overall mtDNA injury in the biopsies. In summary, PAD is associated with mtDNA injury as reflected by multiple deletion mutations. As the mutations are not limited to the ischemic limb in unilateral patients, they are unlikely to contribute to the pathophysiology of claudication.     

Oxidative damage to mitochondrial DNA and its relationship to diabetic complications

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Oxidative stress is known to increase the conversion of deoxyguanosine (dG) to 8-hydroxydeoxyguanosine (8-OHdG) in DNA, which is linked to increased mitochondrial DNA (mtDNA) deletions. We investigated mtDNA deletions and 8-OHdG in the muscle DNA of non-insulin-dependent diabetes mellitus (NIDDM) patients. mtDNA deletion of 4977 bp (delta mtDNA4977) and the content of 8-OHdG in the muscle DNA of the NIDDM patients were much higher than those of the control subjects. There was a significant correlation between delta mtDNA4977 and the 8-OHdG content (P < 0.0001). Both delta mtDNA4977 and the 8-OHdG content were also correlated with the duration of diabetes. Delta mtDNA4977 and the 8-OHdG content in muscle DNA increased in proportion to the severity of diabetic nephropathy and retinopathy. This is the first report that an increase in delta mtDNA4977 and 8-OHdG is proportional to the severity of diabetic complications. Oxidative mtDNA damage is speculated to contribute to the pathogenesis of diabetic complications though a defect in mitochondrial oxidative phosphorylation or other mechanisms. 8-OHdG and delta mtDNA4977 are useful markers to evaluate oxidative mtDNA damage in the diabetic patients.     

The frequency of 4977 base pair deletion of mitochondrial DNA in various types of liver disease and in normal liver

Using a polymerase chain reaction (PCR) method, we tested for the hepatic mitochondrial DNA (mtDNA) deletion in 40 hepatic tumors (28 hepatocellular carcinomas [HCcs], 9 other malignant tumors, and 3 benign tumors) and in the livers of 71 patients, including 16 pediatric patients with end-stage liver disease who underwent living related donor liver transplantation and 16 liver donors. A 4977 base pair (bp) deletion of mtDNA was detected in 36 of 55 specimens of non-tumor portions of adult liver (65.5%). However, none of the specimens obtained from cirrhotic livers of the 16 pediatric patients younger than 13 years of age had the 4977 bp deletion. The frequency of mtDNA deletion was significantly decreased compared with normal liver in HCCs (7 of 28) and other malignant liver tumors (2 of 9). The frequency of this deletion was unrelated to the presence of liver cirrhosis, patient's gender, hepatitis B virus surface antigen status, and hepatitis C virus antibody status.     

Oxidative stress and its impact on mitochondrial DNA in pulmonary tuberculosis patients- a pilot study

BackgroundPulmonary tuberculosis (PTB) remains a major cause of morbidity and mortality all around the world. Recent studies have pointed out increased oxidative stress and also DNA damage in peripheral blood in PTB. Till date, to the best of our knowledge, no study has so far been conducted to show the mitochondrial DNA (mtDNA) deletions mapping in PTB patients. Therefore we performed the present study with the aim to investigate oxidative stress parameters along with mtDNA damage in newly diagnosed untreated PTB patients.Material and methodsThis is a prospective study carried out in Mahatma Gandhi Institute of Medical Sciences, Sevagram,Wardha, Maharashtra during september 2017 to september 2018.Thirty newly diagnosed untreated PTB patients and thirty age matched healthy controls were enrolled in the present study. Analysis of Oxidative stress parameters such as nitric oxide (NO) and malondialdehyde (MDA) were done by calorimetric methods. Assessment of mitochondrial DNA damage was carried out by mtDNA deletions mapping using primer shift long range polymerase chain reaction technique.ResultsThere was significant increase in levels of oxidative stress parameters, nitric oxide and malondialdehyde, in PTB patients compared to controls (p < 0.01). Generally there are two common deletion sites of “13 bp direct repeats” (ACCTCCCTCACCA) in mtDNA. One at the junction sites from bp 8470 to 8482 bp and another from bp 13447 to 13460 bp which make mtDNA more prone for 4977bp deletion. Out of thirty cases of PTB, two cases showed mtDNA damage in the form of mtDNA deletion of 4977bp. There was no mtDNA deletion in any control which can be attributed to continuous generation of oxidative stress.ConclusionThis pilot study has been able to demonstrate that compared to controls, in newly diagnosed pulmonary tuberculosis patients some mtDNA damage did occur and was probably due to continuous generation of oxidative stress in tuberculous patients. However, sample size is too small to draw any conclusions but definitely a more comprehensive study, by recruiting more number of pulmonary tuberculosis patients is warranted to establish correlation between oxidative stress and mtDNA damage in PTB.     

Detection of the 4977 bp deletion of mitochondrial DNA in different human blood cells

As recently reported, it is possible to detect and quantify the amount of the deleted human mitochondrial DNA (mtDNA) in whole blood, platelets and peripheral blood mononuclear cells using real-time PCR. The aim of this study was to identify the cell types in human blood carrying the 4977 bp deleted mtDNA and their accumulation with regard to donor age. Whole blood from 10 healthy donors (five individuals aged from 19 to 22 years, five aged from 57 to 61 years) was separated in various cell populations such as granulocytes, B cells/monocytes and T cells. Purity of the cell isolates was determined by flow cytometry. Total DNA was extracted and 250 ng DNA of each cell type was subjected to PCR using fluorescent-labelled primer pairs. The specific PCR product of the 4977 bp deletion was quantified using an automated detection system. The accumulation of the 4977 bp deletion was more pronounced in T lymphocytes and granulocytes in comparison to B lymphocytes/monocytes. The amount of the 4977 bp deletion in whole blood varied from 0 to 0.00018%, in T lymphocytes from 0.00009 to 0.00160%, in granulocytes from 0 to 0.00162% and in the B lymphocyte/monocyte fraction from 0 to 0.00025%. The higher amount of the deletion in T lymphocytes may be due to a subset of lymphocytes with a longer lifespan thus facilitating the accumulation of mitochondrial damage. The higher amount in granulocytes could have the explanation in the higher release of free radicals for prevention of infectious diseases, because free radicals are supposed to damage the macromolecules of this cell type. The 10 donors displayed differences in the pattern of the accumulation with regard to the different cell types, but no age-dependent accumulation was observed. Differences of the accumulation pattern may be due to actual individual living behaviour or environmental factors.     

Atrial fibrillation is associated with accumulation of aging-related common type mitochondrial DNA deletion mutation in human atrial tissue

STUDY OBJECTIVE: Accumulation of somatic mutations of mitochondrial DNA (mtDNA) contributes to the aging process and progressive organ dysfunction. We investigated the mitochondrial DNA with 4977-base-pair mtDNA deletion mutation (mtDNA(4977)) in human atrial tissue and correlated the amount of mtDNA(4977) to clinical atrial fibrillation (AF). METHODS AND RESULTS: Atrial tissue from the right atrial appendage was obtained in 88 patients during open-heart surgery (22 children/adolescents and 66 adults). The amount of mtDNA(4977) was measured using a nested polymerase chain reaction protocol and normalized to wild-type mtDNA. We found that the mtDNA(4977) was absent in all 22 pediatric/adolescent patients. In the adult group, the relative amount of mtDNA(4977) was significantly higher in patients with AF than in patients without AF (0.55 +/- 0.26 vs 0.35 +/- 0.29, p < 0.007) [mean +/- SD]. The amount of mtDNA(4977) was also positively associated with age (r = 0.29, p < 0.01). Left and right atrial pressures, left atrial dimension, hypertension, and cardiac diagnosis did not influence the amount of mtDNA(4977) significantly. Further multivariate analysis showed that both aging and AF contributed independently to the accumulation of mtDNA(4977). CONCLUSION: AF is associated with an increase of mtDNA(4977). This change is similar to the aging process of atrial tissue and might contribute to atrial dysfunction in AF.     

克山病患者心肌肠道病毒VP1结构蛋白检测

目的 探讨肠道病毒感染与克山病发病的关系。方法 应用抗柯萨奇B5病毒(Coxsackie B5 virus，CVB5)VP1蛋白单克隆抗体，采用免疫组化方法，检测黑龙江、山东、云南省克山病病区急型、亚急型、慢型、潜在型克山病死亡病例心肌标本83例，风心病10例，冠心病10例，心肌炎21例，扩张型心肌病29例，非病区非正常死亡的正常人10例，病区发病季节非克山病死亡病例13例。结果 克山病83例心肌标本中有74例VP1结构蛋白阳性，阳性率89．2％；风心病、冠心病、非正常死亡健康人VP1阳性检出率均为10％；心肌炎、扩张型心肌病VP1阳性检出率分别为66．7％和70％；病区发病季节非克山病死亡病人VP1阳性检出率为38．5％。结论 来自3省份的各型克山病死亡病例心肌标本中均能检出肠道病毒VP1结构蛋白，克山病的发生与肠道病毒感染高度相关。