松花粉对衰老成纤维细胞线粒体DNA缺失突变的影响

目的 通过研究松花粉对衰老细胞线粒体(mt) DNA4977缺失突变的影响,探讨松花粉抗衰老的作用机制.方法 将细胞分为青年组、衰老细胞组、含240 mg/dl松花粉的衰老细胞处理组,三组细胞分别用不同培养基培养后,抽提线粒体DNA,进行PCR检测mtDNA4977缺失突变,以线粒体DNA中保守序列PCR扩增结果作为内参,比较各组mtDNA4977缺失突变在总线粒体DNA中的比例;同时对各组细胞进行β-半乳糖苷酶染色;并测定各组细胞超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量.结果 松花粉能够改善衰老成纤维细胞的衰老变化,并能降低衰老细胞mtDNA4977的缺失突变,提高细胞SOD活性及降低其MDA含量(P＜0.05).结论 松花粉可能通过减轻成纤维细胞的氧化损伤,从而保护细胞mtDNA延缓成纤维细胞的衰老.     

衰老小鼠线粒体DNA缺失的研究

目的：探讨线粒体DNA(mtDNA)大片段缺失与衰老的关系。方法：用聚合酶链反应（PCR）检测不同年龄段小鼠心肌组织mtDNA片段缺失，同时用电镜观察其组织学改变。结果：用PCR检测到2只老年小鼠心肌mtDNA存在着3.87Kb片段缺失，电镜下老年小鼠心肌线粒体明显肥大肿胀，数目减少。结论：MtDNA片段缺失的发生随年龄增加而增加，可能中衰老的过程中起重要作用。     

康欣口服液对老年小鼠肾线粒体DNA缺失突变的影响

目的 研究康欣口服液对老年Balb/c小鼠肾线粒体DNA（mtDNA）缺失突变的影响.方法 老年Balb/c小鼠随机分为空白组和康欣口服液组,分别给予生理盐水和康欣口服液4个月.采用聚合酶链反应（PCR）技术和光密度扫描检测两组mtDNA的缺失突变情况.结果 与空白对照组比较,康欣口服液能显著减少老年Balb/c小鼠肾mtDNA的缺失（P＜0.001）.结论 康欣口服液可以抑制老年小鼠mtDNA的缺失突变,对mtDNA有保护作用.     

鸡胚低分子提取物对D-半乳糖拟衰老小鼠线粒体DNA缺失突变的影响

目的 探讨鸡胚低分子提取物对D-半乳糖诱导的衰老小鼠线粒体DNA(mtDNA)缺失突变的影响.方法 采用D-半乳糖诱导制备衰老小鼠模型并用鸡胚低分子提取物处理.用聚合酶链反应技术和琼脂糖凝胶电泳检测mtDNA缺失片段,密度扫描技术对扩增片段进行相对定量.缺失片段构建质粒后,直接测序进行鉴定.结果 全部小鼠的肝、大脑皮质与海马组织内均存在4239 bp的mtDNA缺失片段.衰老模型鼠不同组织mtDNA缺失的相对百分含量均明显高于正常对照组(均为P%0.01),而鸡胚低分子提取物可明显降低模型鼠肝、大脑皮质与海马组织内mtDNA缺失的比例(均为P＜0.05).肝组织中mtDNA缺失的比例较大脑皮质和海马组织更高.结论 4239 bp的mtDNA缺失片段普遍存在于小鼠中,鸡胚的低分子提取物可以明显减少D-半乳糖拟衰老小鼠4239 bp的mtDNA缺失的发生.     

抗衰1号对老年小鼠肝线粒体DNA缺失的影响

目的研究抗衰1号对老年Balb/c小鼠肝线粒体DNA(mtDNA)缺失突变的影响.方法老年Balb/c小鼠随机分为老年空白组和抗衰1号组,分别给予生理盐水和抗衰1号4个月.采用聚合酶链反应(PCR)技术和光密度扫描检测两组mtDNA的缺失情况.结果与老年空白对照组比较,抗衰1号能显著减少老年Balb/c小鼠mtDNA的缺失(P＜0.001). 结论抗衰1号可以抑制老年小鼠mtDNA的缺失,提示补肾健脾活血通腑法组方能减少mtDNA的氧化损伤,对mtDNA有保护作用,从而从分子水平提供了本法延缓衰老的可能机理.     

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。     

维生素C对D-半乳糖致亚急性衰老小鼠模型的影响

目的从mtDNA缺失改变，线粒体呼吸酶活力和氧化应激水平的角度研究维生素C（Vc）对D-半乳糖诱导亚急性衰老小鼠模型的影响。方法建立衰老小鼠模型，PCR法检测实验小鼠mtDNA固定片段的缺失情况，氧电极法检测肝线粒体呼吸酶琥珀酸脱氢酶（SUCOX）和NADH氧化酶（NADHOX）活力，荧光法检测肝和脑MDA含量和GPX活性，化学法检测SOD的活性。结果模型组和Vc保护组小鼠都出现mtDNA固定片段缺失，但Vc保护组荧光强度明显低于模型组。与模型组相比，对照组线粒体呼吸酶琥珀酸脱氢酶和NADH氧化酶活性显著降低，Vc保护组线粒体呼吸酶琥珀酸脱氢酶和NADH氧化酶活性显著升高；对照组和Vc保护组小鼠肝脑MDA含量和SOD活性降低。结论Vc可以减轻D-半乳糖诱导亚急性衰老动物模型的氧化损伤，保护mtDNA和增强线粒体氧化呼吸酶活性。     

康欣口服液对老年小鼠脑线粒体DNA缺失突变的影响

目的 研究补肾健脾养血活血法对Balb/c小鼠脑线粒体DNA(mtDNA)缺失突变的影响.方法 近交系Balb/c老年小鼠(14个月)分别灌胃给予生理盐水(老年空白对照组),康欣口服液(老年康欣口服液组),连续4个月后处死,提取脑组织的mtDNA,采用PCR技术分别扩增野生型和缺失型的mtDNA 片段,运用凝胶成像仪进行光密度扫描,比较两组缺失型mtDNA/野生型mtDNA的光密度比值.结果 近交系老年Balb/c小鼠脑mtDNA中存在明显的片段缺失,与老年空白对照组相比,康欣口服液能显著降低老年Balb/c小鼠mtDNA的缺失率(P＜0.001).结论 补肾健脾养血活血法可以抑制老年小鼠脑mtDNA的缺失.     

病毒性心肌炎与扩张型心肌病心肌线粒体DNA缺失的定量分析

目的对比研究病毒性心肌炎(VMC)与扩张型心肌病(DCM)患者活检心肌组织线粒体DNA(mtDNA)缺失突变情况及其与外周淋巴细胞mtDNA缺失程度的相关性.方法用定量PCR法检测20例VMC患者、12例DCM患者心肌细胞及其外周血淋巴细胞mtDNA4977碱基对(mtDNA4977)和mtDNA7436碱基对(mtDNA)缺失率.取12例健康意外死亡者心肌和23例献血员外周血淋巴细胞作正常对照.结果正常对照者、VMC和DCM患者心肌细胞均存在mtDNA4977及mtDNA7436缺失,合计缺失率分别为0.175%、0.385%和3.004%;外周淋巴细胞mtDNA缺失程度与心肌细胞呈一致性改变,且有良好的相关性(r=0.960,P＜0.001).结论mtDNA缺失可能是VMC发病及其向DCM演变的一个重要心肌损伤机制;外周淋巴细胞在研究心肌细胞mtDNA缺失中的作用值得进一步探讨.     

8-甲氧补骨脂素及长波紫外线诱导培养皮肤成纤维细胞线粒体DNA突变的研究

目的 探讨线粒体DNA4977bp缺失突变与皮肤成纤维细胞光老化之间的关系。方法 采用8－甲氧补骨脂素及长波紫外线（8－MOP／UVA）共同作用诱导培养皮肤成纤维细胞光老化，一步法提取线粒体DNA（mtDNA），三引物PCR的方法检测皮肤成纤维细胞线粒体DNA4977bp缺失突变，密度扫描半定量分析。结果 8－MOP／UVA作用下，培养皮肤成纤维细胞迅速出现细胞老化的形态学改变；28d时，8－MOP／UVA组、UVA组4977bp缺失突变的发生率及占总mtDNA中的比较分别为100％、11％和0.253、0.053；8－MOP组、正常对照组均为阴性，8－MOP／UVA组珉春他各组比较差异均有显著性（P＜0.01）。结论 线粒体DNA4977bp缺失突变累积与皮肤光老化密切相关，可能作为衡量皮肤老化程度的分子生物学标志。     

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.     

冠心病患者线粒体细胞色素C氧化酶亚基Ⅱ基因片段的重排

采用玻璃粉微量吸附法获取DNA直接作为PCR反应的模板,利用巢式PCR技术,检测冠心病患者血细胞内编码细胞色素C氧化酶亚基II的线粒体DNA扩增片段多态性,探讨线粒体DNA的片段缺失与动脉粥样硬化发生发展的关系。研究发现在全部冠心病患者编码细胞色素C氧化酶亚基II的基因中出现了大约100bp的缺失片段,其中1例冠心病患者中还出现了大约450bp核基因组和600bp的线粒体DNA扩增片段,而在正常对照老人中没有出现片状的重排现象,表明冠心病患者中存在该基因片段的缺失和模板量减少的现象,该基因可能参与了冠状动脉弱样硬化的形成与发展,编码呼吸链复合物一些亚基线粒体DNA片段的重排可能是动脉粥样硬化进行性发展的恶化因素。     

Detection of chromosome 20q deletions in bone marrow metaphases but not peripheral blood granulocytes in patients with myeloproliferative disorders or myelodysplastic syndromes

Myeloproliferative disorders and myelodysplastic syndromes arise in multipotent progenitors and may be associated with chromosomal deletions that can be detected in peripheral blood granulocytes. We present here seven patients with myeloproliferative disorders or myelodysplastic syndromes in whom a deletion of the long arm of chromosome 20 was detectable by G-banding and/or fluorescence in situ hybridization in most or all bone marrow metaphases. However, in each case, microsatellite polymerase chain reaction (PCR) using 15 primer pairs spanning the common deleted region on 20q showed that the deletion was absent from most peripheral blood granulocytes. The human androgen receptor clonality assay was used to show that the vast majority of peripheral blood granulocytes were clonal in all four female patients. This represents the first demonstration that the 20q deletion can arise as a second event in patients with pre-existing clonal granulopoiesis. Microsatellite PCR analysis of whole bone marrow from two patients was consistent with cytogenetic studies, a result that suggests that cytogenetic analysis was not merely selecting for a minor subclone of cells carrying the deletion. Furthermore, in one patient, the deletion was present in both erythroid and granulocyte/monocyte colonies. This implies that the absence of the deletion in most peripheral blood granulocytes did not reflect lineage restriction of the progenitors carrying the deletion but may instead result from other selective influences such as preferential retention/destruction within the bone marrow of granulocytes carrying the deletion.     

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     

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets

AIM/HYPOTHESIS: Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. METHODS: We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. RESULTS: There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. CONCLUSION/INTERPRETATION: Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.     

Mitochondrial DNA deletions and the aging heart

Mitochondrial DNA (mtDNA) mutations appear to be associated with a wide spectrum of human disorders and proposed to be a potential contributor of aging. However, in an age-dependent increase of the common 4977 bp deletion of human mtDNA still many unanswered questions remain. Comparing mtDNA copy levels in different tissues revealed that cardiac muscle had the highest, while the cortex cerebelli showed the lowest copy number of mtDNA in every donor. Intriguingly, mtDNA copy number showed no changes during aging. In heart tissue, the amount of 4977 bp mtDNA deletion increased in an age-dependent manner showing significant differences at the age of 40 years and older (p<0.005). In vitro studies analyzing human normal cells transfected with telomerase (BJ-T) revealed that oxidative stress (OS)--a well accepted promoter of aging--induced 4977 bp deletion and point mutations as demonstrated by real-time PCR and DHPLC analysis. Interestingly, OS induced apoptosis only in transformed human fibroblasts by activation of the intrinsic (mitochondrial-mediated) signalling pathway as indicated by morphological damage of mitochondria, DNA laddering and increase of the Bax/Bcl-2 ratio. In conclusion, in heart tissue, the amount of the 4977 bp deletion increased in an age-dependent manner and it was more detectable after the 4th decade of life, although there was some scatter in the data. Since, apoptosis was induced by the mitochondria-mediated pathway only in transformed cells, the role for apoptosis in normal tissue of the aging heart remains unclear.     

Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys

While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging.     

Hematologic improvement of Pearson's syndrome confirmed by mitochondrial DNA analysis]

We report on an 8-month-old girl with Pearson's syndrome who presented with transfusion-dependent pancytopenia, exocrine pancreatic dysfunction, and lactic acidosis. Bone marrow findings were consistent with sideroblastic anemia and marked vacuolization of myeloid and erythroid precursors. Southern blot analysis of mitochondrial DNA (mtDNA) revealed a 4.5 kb deletion in peripheral blood cells. Gradual hematologic improvement was observed thereafter, and the patient was relieved of the need for blood transfusions. We were able to confirm a decrease of the mtDNA deletion in lymphocytes as well as in lymphoblastoid cell lines cultured from peripheral lymphocytes as the patient made steady hematologic progress.