外源性线粒体DNA在细胞内的重建（简报）

To create human mitochondrial DNA (mtDNA)-transferred cells as cell model of mitochondria defects-related diseases, platelet of normal young and old subjects were isolated as donor of mtDNA, then fused with mtDNA-depleted cells (rho0 cells) under induction of PEG1500. Auxtrophy test, cytochrome c oxidase activity assay and PCR amplification of mtDNA were done to confirm the transferring of mtDNA. Cell clones were visible in the medium 10 to 15 days after fusion, which grew well in medium lacking uridine, had positive COX activity and contained objective fragment of mtDNA by PCR, opposite to rho0 cells. Transferring of mtDNA to rho0 cells was identified, and mtDNA-transferred cell models were successfully created.     

常见结直肠癌无线粒体DNA细胞株的建立

目的:流行病学研究表明结直肠癌细胞线粒体DNA(mt DNA)拷贝数变异与患者预后具有显著相关性,但因缺乏相关体外细胞模型导致其细胞生物学效应及具体分子机制尚不明确。因此研究建立结直肠癌ρ0细胞株(无mt DNA)的方对该研究域具有重大意义。方法:在含有丙酮酸、尿苷及不同浓度溴化乙锭的培养基中培养大肠癌细胞株SW480、HCT 116、HCT-8,以不加溴化乙锭的细胞为对照,利用实时定量PCR技术监测不同处理组mt DNA拷贝数的变化,并观察结直肠癌ρ0细胞线粒体形态数目及细胞形态的变化。结果:SW480细胞用50 ng/m L EB处理25代后,再以100 ng/m L EB处理14代可成功构建SW480ρ0细胞株;HCT116细胞加用EB 100 ng/m L培养32代,继续用EB 200 ng/m L培养10代,再用EB 500 ng/m L培养3代,可成功获得HCT116ρ0细胞株;HCT-8细胞用200 ng/m L EB培养24代,可成功获得HCT-8ρ0细胞株。同时,ρ0细胞较亲本细胞变大变长,其线粒体的个数增多,线粒体形态变大变长。结论:利用EB处理法可成功构建大肠癌ρ0细胞株,但不同肠癌细胞方法不尽相同。mt DNA拷贝数的降低可显著影响大肠癌细胞的形态。     

线粒体DNA病

线粒体ＤＮＡ病王学敏杨雨善谢惠君郑惠民（第二军医大学,上海２００４３３）关键词线粒体病线粒体ＤＮＡ近年确认了线粒体ＤＮＡ（ｍｔＤＮＡ）突变会引起人类疾病,临床症状有盲、聋、痴呆、运动障碍、肌肉衰弱、心力衰竭、糖尿病、肾功能不全、肝病等。ｍｔＤＮＡ呈裸...     

高等植物的线粒体DNA

高等植物的线粒体含有结构复杂的庞大基因组。线粒体ＤＮＡ有较为特殊的重组行为和来源,也可向细胞核转移,在植物系统发育中有重要作用。     

线粒体DNA编码细胞色素氧化酶亚基基因的进展

细胞色素氧化酶由13个亚基组成,其中构成级联反应核心的最大3个亚基(COXⅠ,COXⅡ和COXⅢ)由mtDNA编码,其余10个亚基(COⅣ,Ⅴa,Ⅴb,Ⅵa,Ⅵb,Ⅵc,Ⅶa,Ⅶb,Ⅶc,和Ⅷ)均由nDNA编码。COXⅠ亚基与hemea、hemea3、CuB结合,直接参与质子泵过程;COXⅡ亚基与CuA结合,位于线粒体包质面与细胞色素C进行反应;COXⅠП亚基参与氧化还原连接的质子易位过程;其余10个亚基的功能尚不明确。COX是线粒体组装所必需的基因,其表达调控与nDNA和mtDNA相互作用有关。     

线粒体DNA与DNA甲基化的关系

线粒体DNA（mitochondrial DNA,mtDNA）遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响。mtDNA的损伤与衰老、肿瘤等疾病的发生有关。DNA甲基化是调节基因表达的重要方式之一。mtDNA基因的表达受核DNA（nuclear DNA,nDNA）的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病。本文就近年来mtDNA与DNA甲基化的关系作一综述。     

线粒体DNA与DNA甲基化

线粒体是除细胞核之外唯一携带遗传物质的细胞器,其线粒体DNA（mitochondrial DNA,mtDNA）控制着线粒体一些最基本的性质,对细胞功能有着重要影响.DNA甲基化是调节基因表达的重要方式之一.研究表明mtDNA存在CpG位点的低甲基化,并且mtDNA基因的表达受核DNA（nuclear DNA,nDNA）及线粒体自身DNA甲基化的调控,mtDNA和nDNA协同作用参与机体代谢调节和疾病发生发展过程.就近年来mtDNA与DNA甲基化的关系作一综述.     

水稻线粒体DNA的电镜观察

从黑暗条件下培养的岗比亚水稻芽中提取线粒体DNA(mtDNA),电泳时发现除mtDNA主带外,还存在一些小分子DNA带,其中1.1和1.6kb(0.34和0.53μm)的两条DNA带含量最高。用电镜观察所提取的mtDNA时,发现有十多种大小不同的环型分子,其中0.34μm,0.53μm的两种环型分子出现频率最高,这两种DNA分子的大小与电泳时看到的两条明显DNA小分子带基本上相吻合,此外在mtDNA中,还观察到套索结构和D-环。     

高等植物线粒体DNA的制备方法

自从六十年代发现线粒体DNA(mtDNA)以来,mtDNA在遗传上的功能引起了广泛的重视。由于线粒体具有自已的基因组,能够自我复制,又能编码一些酶,比如生物氧化链上的一部分酶的亚基就是由线粒体基因编码的,可以推测生物的某些性状的表达可能与mt-DNA有关;另外由于实现线粒体基因组的复制与表达所需的许多酶又是由核基因编码的(如DNA聚合酶,RNA聚合酶、DNA连接酶等),可以推测     

转线粒体小鼠的研究进展

线粒体转移技术的发展和成熟使得我们业已成功的建立了转线粒体小鼠动物模型。目前常用的方法主要有:一是直接应用显微注射技术将活的线粒体转入小鼠胚胎;二是通过脱核胞质体与胚胎干细胞融合,再将胚胎干细胞显微注入小鼠囊胚,从而形成嵌和鼠;三是将脱核胞质体与小鼠胚胎直接融合而产生的转线粒体小鼠。随着越来越多线粒体相关疾病的发现,各种不同线粒体疾病的转线粒体小鼠的开发具有十分重要的应用价值和广阔的研究前景。     

SPHEROSOMES AND MITOCHONDRIA IN THE LIVING FUNGAL CELL

Study of living hyphae of Fusarium oxysporum Schlect., Fomes annosus (Fries) Cooke, Ceratocystis fagacearum (Bretz) Hunt, Basidiobolus ranarum Eidam, and Mycotypha microspora Fenner with phase contrast revealed that these fungi have spherosomes similar to those in vascular plants. The spherosomes are conspicuous in the hyphal tip, suggesting some function other than fat synthesis. It may be that the Woronin bodies reported by other workers are spherosomes. Mitochondria in these fungi are highly pleomorphic and exhibit saltatory movement. They often interact with nuclei in a manner suggesting close membrane contact.     

外源胆固醇对水稻根端线粒体ATP酶活力的影响

外源胆固醇无论是通过根系吸收或是直接与离体线粒体一起温育的方式,在试验浓度范围内均能提高水稻根端线粒体ATP酶的活力,同时观察到外源胆固醇能明显降低ATP酶表现活化能(Apparent activation energy,AEa)在Arrhenius图上的折点温度。其中通过根系吸收进入线粒体膜内后,其线粒体ATP酶AEa的两个折点温度由对照的27.7℃和15.5℃分别降低到24.5℃和12.7℃;直接与离体线粒体一起温育的两个折点温度分别降低到18.8℃和9.6℃。试验结果证明,适量的外源胆固醇不仅对水稻根端线粒体ATP酶活力具有明显的促进作用,而且对降低线粒体膜脂的相变温度也有明显的调节作用。     

OXIDATIVE PHOSPHORYLATION AND ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA IN THE INTACT ASCITES TUMOR CELL

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.     

STUDIES ON THE BIOCHEMISTRY OF MITOCHONDRIA AND CELL MORPHOLOGY IN THE NEONATAL SWINE HEPATOCYTE

Mitochondrial preparations isolated from neonatal swine hepatocytes show a marked increase in oxidative and concomitant phosphorylative capacity between birth and 2 days postpartum. There are no changes in the coupling parameters (respiratory control ratio and adenosine diphosphate/O ratio) with age. Changes in sedimentation properties in a sucrose gradient suggest qualitative changes in the mitochondria. Some of the lipid measurements (increased phospholipid) might be interpreted as supportive of this suggestion, although most could also be regarded as indicative of quantitative changes (increased number of mitochondria). Electron microscopy of isolated mitochondria and of the hepatocyte demonstrated an increased number of mitochondria but no change in shape, size, or structure as the pig developed. An increase in a number of cytoplasmic components (Golgi apparatus and endoplasmic reticulum) and a decrease in glycogen were also observed. The functional changes in mitochondria seem to occur within a short period of time (6–12 hr postpartum).     

THE OXIDATION OF EXOGENOUS AND ENDOGENOUS CYTOCHROME C IN MITOCHONDRIA : A Biochemical and Ultrastructural Study

The effect of the nonionic detergent Lubrol on the oxidation of endogenous and exogenous cytochrome c by cytochrome oxidase in intact and fragmented mitochondria was studied. Mitochondria and mitochondrial fragments from liver, kidney, heart, and skeletal muscle have been used. Negatively stained preparations of intact mitochondria showed the particles of Fernández-Morán on the matrix side of their inner membrane system: under these conditions, the oxidation rate of externally added cytochrome c was very high, and it was stimulated very poorly by Lubrol. Mechanical fragmentation of liver mitochondria yielded vesicles with a smooth external profile: also under these conditions, the oxidation of externally added cytochrome c was very high, and poorly stimulated by Lubrol. The oxidation of endogenous cytochrome c was also unaffected by Lubrol. On the other hand, fragmentation of heart and skeletal muscle mitochondria yielded vesicles having numerous particles of Fernández-Morán on their external profiles. Under these conditions, the oxidation of exogenous cytochrome c was low and was markedly stimulated by Lubrol. On the contrary, no activation of the oxidation of endogenous cytochrome c was induced by the detergent. The results indicate a difference in the permeability properties of the two faces of the inner mitochondrial membrane: a permeability barrier for cytochrome c is suggested to exist at the inner face.     

A DEMONSTRATION OF SEVERAL DEOXYRIBONUCLEASE ACTIVITIES IN MAMMALIAN CELL MITOCHONDRIA

Extracts of purified mitochondria from adult rabbit liver and kidney have been prepared by lysis with Triton X-100. Such extracts contain deoxyribonuclease activity demonstrable at alkaline pH. Studies utilizing the effects of substrate variation, differing ionic strength, nucleoside di- and triphosphates, and SH-group inhibitors reveal the existence of at least five distinguishable deoxyribonuclease activities in these extracts. Assay of lysosomal and mitochondrial enzyme markers indicates no significant lysosomal contamination of the mitochondrial extracts. Further studies also suggest that the alkaline deoxyribonuclease activity is specifically located in or in association with mitochondria.     

牵牛花受精前后卵细胞质体和线粒体及其细胞质DNA的变化

应用透射电镜研究了圆叶牵牛（Ｐｈａｒｂｉｔｉｓｐｕｒｐｕｒｅａ （Ｌ．） Ｖｏｙｇｈｔ）和大花牵牛（Ｐ． ｌｉｍ ｂａｔａ Ｌｉｎｄｌ．）卵细胞在受精前和受精后质体和线粒体的变化。观察到卵细胞在受精前高度液泡化,在核周及卵细胞周围的薄层细胞质中有质体和线粒体。质体含１～２ 个较大淀粉粒。线粒体多呈环形或杯状。卵细胞受精后,细胞质增多,质体的数量也明显增加。质体基质变得更加浓厚,并且普遍含有嗜锇的球体。合子中线粒体丰富,但缺乏卵细胞中那种环形或杯状线粒体,多呈圆形。细胞质ＤＮＡ检测的结果表明,卵细胞质ＤＮＡ荧光有大小和形状不同的两类,它们在细胞中随机分布。一类较大,呈环状,可能是线粒体中的ＤＮＡ显示的荧光;另一类小的点状荧光,可能大多是质体ＤＮＡ荧光,前者比后者多。卵细胞受精后,细胞质类核发生变化,表现在数量上明显地比受精前少。研究揭示了牵牛花合子中细胞质ＤＮＡ 减少的现象,说明了母系质体ＤＮＡ 在受精后可能被降解,提供了母系质体不传递到后代的可能机制。     

THE ORGANIC-INORGANIC RELATIONSHIP IN CALCIFIED MITOCHONDRIA

Experimentally induced calcification within mitochondria has been studied electron rnicroscopically. Cells investigated comprise hepatic cells damaged by CCl₄ intoxication, myocardial cells damaged by prolonged dihydrotachysterol (DHT) administration, and cells from skeletal muscle (gastrocnemius) damaged by DHT sensibilization and local injury. Cells from a human bowel carcinoma were studied too. Two types of intramitochondrial inorganic inclusion have been found. The first consists of clusters of apatite-like, needle-shaped crystals (crystalline aggregates), the second of clusters of very fine granules (granular aggregates). The former have been found mainly in mitochondria in apparently normal myocardial and muscular cells, the latter in mitochondria of degenerated hepatic, neoplastic, and myocardial cells. Crystalline aggregates are closely related to the membranes of cristae at first, but they later spread to occupy the whole mitochondrial matrix. Granular aggregates are initially found in the mitochondrial matrix near, but perhaps not touching, cristae; by growing they come into close contact with cristal membranes. Both types of aggregate show intrinsic electron opacity, which disappears after formic acid decalcification. Only the crystalline aggregates give an electron diffraction pattern of crystallinity. Uranium and lead staining of decalcified sections shows that both types of aggregate are intimately connected with an organic substrate. The substrate of crystalline aggregates consists of very thin, elongated structures shaped like the inorganic crystals. The substrate of granular aggregates consists of amorphous material gathered in clusters, with the same roundish shape and intercristal position as the inorganic granules. Both types of substrate are stained by phosphotungstic acid at low pH and by silver nitrate-methenamine after periodic acid oxidation. These results show that the organic content of the substrates includes glycoproteins; they have been confirmed by the periodic acid-Schiff (PAS) method under the optical microscope. These findings have been discussed in relation to the recent discovery of organic Ca²⁺-binding sites in mitochondria and to the general problems of soft tissue calcification.