Chowdhury S K, Drahota Z, Floryk D, Calda P, Houstek J
Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 4, Prague, Czech Republic.
Clin Chim Acta. 2000 Aug;298(1-2):157-73. doi: 10.1016/s0009-8981(00)00300-4.
Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9-22 independent cultures of amniocytes.
羊膜细胞代表了一类胎儿细胞群体，可用于对疑似线粒体氧化磷酸化（OXPHOS）缺陷的家庭进行产前诊断。在本文中，我们提出了一个复杂的方案，用于使用三种独立且互补的方法评估培养的羊膜细胞中线粒体OXPHOS酶的功能：（a）分光光度法，作为测定线粒体呼吸链酶（NADH泛醌氧化还原酶、琥珀酸和甘油磷酸细胞色素c还原酶、细胞色素c氧化酶和柠檬酸合酶）能力的工具；（b）极谱法，作为使用洋地黄皂苷通透的羊膜细胞原位评估线粒体OXPHOS酶功能的工具（鱼藤酮敏感的丙酮酸+苹果酸氧化、抗霉素A敏感的琥珀酸氧化、KCN敏感的细胞色素c氧化、ADP激活的底物氧化）；以及（c）细胞荧光法测定洋地黄皂苷通透的羊膜细胞中四甲基罗丹明甲酯（TMRM）荧光，作为在稳态条件下（琥珀酸存在时的状态4）测定线粒体膜电位的灵敏方法。这些方案还给出了使用9 - 22个独立的羊膜细胞培养物的参考对照值。
