Study On Mitochondrial DNA Damage Caused By Carbon-ion Irradiation And The Related Methodology Development

Purpose: This thesis aims to investigate mitochondrial DNA damage caused bycarbon-ion irradiation and the related methodology development. The investigationconsists of quantitative detection of mitochondrial DNA damage and mutationcumulant after carbon-ion irradiation, and tests of two materials（silicon carbonquantum dots and mitochondrial ROS inducer）which will be used in mitochondrialdamage research.Materials and Methods: MCF-7cells were irradiated by12C6+ion beam or X-ray,Real-time PCR was used to detect instant damage of mitochondrial DNA codingregion5464-7287and4977large fragments deletion. Clone survival rate of MCF-7cells after irradiation was measured. Direct sequencing method was used to quantifyD310point mutations. Test of silicon carbon quantum dots (SiCDs) includes MTT test,contents of SOD，MDA and ROS in cells and zebrafish, and the qualification of SiCDsact as biological dye. Study of mitochondrial ROS inducer（Mitochondros）includescell MTT test and detection of fluorescence intensity.Results: Mitochondrial DNA coding region5464-7287was seriously damagedby carbon-ion irradiation. Vitamin C, a typical antioxidant, is protective for the codingregion if added half an hour before irradiation. When measured by10%survivalfraction (D10), relative biological effectiveness of carbon-ion irradiation on MCF-7cell is3.1(1.5Gy carbon-ion equals to4.65Gy X-ray). D310SNP detection found that X-ray caused mutations in D310area, while decrease of normal sequence C7wasless than that were caused by carbon-ion irradiation (58%vs.74%), single-base insertC8and single-base deletion C6abundance caused by carbon-ion irradiation wereobviously higher, which were18%and24%, respectively. Mitochondrial DNA4977deletion after carbon-ion and X-ray irradiation performed consistently, it peaked at24hours after radiation, and quickly dropped to the background level in the following48and72hours. It showed no obviously differences of carbon-ion and X-ray irradiationin mitochondrial DNA4977deletion. After24h incubation，low concentrations ofSiCDs didn’t affect survival of cells and zebrafish, SOD and MDA content in cells andzebrafish decreased. SiCDs in living cells and in vivo imaging showed strongfluorescent signals in405,488and555nm, only cytoplasmic region showedfluorescence. Cell MTT test results of Mitocondros showed that middle and lowconcentrations of Mitocondros didn’t affect survival of HepG2cells, Mitocondrosexcitated at428nm and emitted at600nm. Its intracellular fluorescent signals showedno difference after24h incubation, except the minimum concentration at0.1ng/ml.Conclusion: Mitochondrial DNA coding region5464-7287is sensitive tocarbon-ion irradiation, Vitamine C eliminated ROS thus protected mitocondral DNA,this implies damage in the coding regions was mediated by oxidative damage.Carbon-ion irradiation caused more D310mutations than X-ray.The existence of mitochondrial DNA4977deletions induced severe oxidative stress,which makes mitochondrial DNA4977deletions a harmful mutations, and cannot becoexist in live cells. SiCDs is an qualified fluorochrome in living cells: lowconcentrations of SiCDs have no toxicity to cells and zebrafish, it didn’t cause celldamage or oxidative stress, besides, it showed strong fluorescent signals and located inthe cytoplasm. However, its targeted mitochondria-specificity needs to be improved.Mitocondros cytotoxicity results show that middle and low concentrations ofMitocondros are not toxic and can be observed constinuously， which hints that it is qualified to be a ROS inducer. Limited amount of Mitocondros can be accommodatedin mitochondria, and0.1ug/ml, the concentration of highest fluorescence intensityvalues, will be used in the following-up experiments.