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

这项研究旨在为线粒体疾病患者建立治疗方法，这些患者的组织中有正常和突变的线粒体DNA。为了研究从Melas患者获得的培养的成纤维细胞中突变的线粒体DNA复制的修饰因子，我们通过缺乏线粒体DNA和患者缺乏缺乏核心的患者的HELA细胞而形成了细胞抗酸剂克隆。获得两个气缸克隆，这些克隆的突变线粒体DNA（A3243G）的速率分别为11％（克隆A）和42％（克隆B）。将这两个Cybrid克隆在1）下培养72小时的氧气浓度，例如70、150和500 mmHg的PO 2、2）不同变化的葡萄糖浓度由0、0.01、0.01、0.05、0.1、0.2和0.5％，0.5％，0.5％，3）供应proconosolon或胰岛素或胰岛素或胰岛素或胰岛素。调节培养后突变的线粒体DNA速率的变化由单细胞PCR确定，然后是…更多的PAI RFLP。使用Mann-Whitney U检验对结果进行了统计评估。显着性设置为p <0.05。分辨率：1）在三种条件的PO2培养物下，克隆A没有显着差异。但是，克隆B在500 mmHg的PO2以下聚集后突变率显着降低。在高氧浓度培养物中克隆B中细胞活力的降低，在每个培养条件下相似地研究了细胞活力测试。这表明，线粒体DNA突变率高的细胞易受功能性细胞损伤，导致选择性细胞死亡受到通常在线粒体中通常产生的活性氧时的选择性细胞死亡。但是，克隆B在0％和0.01％的葡萄糖浓度中显示出显着降低的突变率。细胞活力测试显示，在培养基中，克隆B的活力降低了0％和0.01％的葡萄糖浓度。这表明突变速率相对较高的细胞高度依赖于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中突变率降低。 3）培养基中补充胃溶性和胰岛素补充剂在克隆A和B中的突变率没有显着变化。较少的