| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
|
|---|
| Research Abstract |
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
|
