Therapeutic research for mitochondrial disease

线粒体疾病的治疗研究

基本信息

批准号：
12670723
负责人：
HAGINOYA Kazuhiro
金额：
$ 2.18万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

This study aimed at establishing therapeutic approach to the patients with mitochondrial disease who have a heteroplasmy of normal and mutated mitochondrial DNA in their tissues. In order to investigate modifying factors on replication of mutated mitochondrial DNA in cultured fibroblasts obtained from patient with MELAS, we developed cybrid clone by fusion of Hela cell lacking mitochondrial DNA and patient's fibroblasts lacking nuclei. Two cybrid clone were obtained and the rate of mutated mitochondrial DNA(A3243G) of these clone was 11 % (clone A) and 42 % (clone B), respectively. These two cybrid clone were cultured for 72 hours under 1) variously changed oxygen concentration such as 70, 150, and 500 mmHg of pO 2, 2) variously changed glucose concentration consisted of 0, 0.01, 0.05, 0.1, 0.2, and 0.5 %, 3) conditioned medium supplied with predonisolon or insulin. Change in the rate of mutated mitochondrial DNA after conditioned culture was determined by single cell PCR followed by A … More paI RFLP. The results were statistically evaluated using Mann-Whitney U test. Significance was set at p<0.05.Results: 1) There was no significant difference in clone A under three conditioned pO2 cultures. However, clone B showed significant decrease of mutation rate after culturing under 500 mmHg of pO2. Cell viability test that was simaltaneously studied in each culture condition showed decrease in cell viability in clone B in high oxygen concentration culture. This suggests that cells with high mitochondrial DNA mutation rate were susceptible to functional cell damage leading to selective cell death when it is subjected to reactive oxygen species that is normally produced in the mitochondria.2) There was no significant change in the rate of mutated mitochondrial DNA in clone A under cultures with various concentration of glucose. However, clone B showed significant decrease of mutation rate in 0 % and 0.01 % of glucose concentration. Cell viability test showed decrease in viability in clone B under 0 % and 0.01 % of glucose concentration in the culture medium. This suggests that cells with relatively high mutation rate are highly dependent on glycolysis for ATP production rather than oxidative phosphorylation in the mitochondria. This caused selective cell death leading to decrease in mutation rate in cells cultured in those medium. 3) Predonisolon and insulin supplementation in the medium showed no significant changes in mutation rate in both clone A and B. Culture study supplied with other drugs that might affect on replication of mutated mitochondrial DNA is now under way. Less

本研究旨在建立对组织中存在正常线粒体 DNA 和突变线粒体 DNA 异质性的线粒体疾病患者的治疗方法。为了研究从 MELAS 患者获得的培养成纤维细胞中突变线粒体 DNA 复制的修饰因素，我们开发了一种方法。将缺乏线粒体DNA的Hela细胞与患者缺乏细胞核的成纤维细胞融合，获得两个cybrid克隆，并观察线粒体突变率。这些克隆的DNA(A3243G)分别为11％(克隆A)和42％(克隆B)。这两个细胞杂种克隆在1)不同变化的氧浓度(例如70、150和500mmHg)下培养72小时。 pO 2, 2) 不同变化的葡萄糖浓度包括 0, 0.01, 0.05, 0.1, 0.2 和 0.5 %, 3) 条件培养基在条件培养后通过单细胞 PCR 和 A … More paI RFLP 测定突变线粒体 DNA 的变化率。使用 Mann-Whitney 严格评估结果。 U检验显着性设定为p<0.05。结果：1)在三种条件pO2培养物中克隆A没有显着差异，但是克隆B显示突变显着减少。在 500 mmHg pO2 下培养后的细胞活力测试显示，在高氧浓度培养物中，克隆 B 的细胞活力下降，这表明线粒体 DNA 突变率高的细胞容易受到功能性细胞损伤。当受到线粒体中正常产生的活性氧的影响时，会导致选择性细胞死亡。2）在不同浓度的葡萄糖培养物中，克隆A中的线粒体DNA突变率没有显着变化，但克隆B的突变率没有显着变化。表现出显着的0%和0.01%葡萄糖浓度下突变率下降。细胞活力测试显示，在培养基中0%和0.01%葡萄糖浓度下，克隆B的活力下降，这表明突变率相对较高的细胞具有高度依赖性。 ATP 产生的糖酵解而不是线粒体中的氧化磷酸化，这导致选择性细胞死亡，导致在这些培养基中培养的细胞的突变率降低。 3) 在培养基中补充泼尼松和胰岛素。培养基显示克隆 A 和 B 的突变率没有显着变化。与可能影响突变线粒体 DNA 复制的其他药物一起进行的培养研究正在进行中。