线粒体dna在α粒子致人支气管上皮细胞体外恶性转化中作用-卫生毒理学专业论文.docx

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2018-05-05 发布于上海
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线粒体dna在α粒子致人支气管上皮细胞体外恶性转化中作用-卫生毒理学专业论文.docx

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线粒体dna在α粒子致人支气管上皮细胞体外恶性转化中作用-卫生毒理学专业论文

MitochondrialDNA-dependentsignalingpathwayis involvedintheprocess ofαparticles-induced malignanttransformationinvitroAbstractObjective：ToexploretheroleofmitochondrialDNA(mtDNA)intheprocess of malignanttransformationinducedbyα-particles,andinthemechanismsofdisabled apoptosis.Methods:(1)Humanbronchialepithelialcells(HBE)wereculturedinmedium containing50ng/mLethidiumbromide(EtBr)for9daysandconsecutivelywith12.5 ng/mLEtBrforother30daystoestablishmtDNA-depletedHBE(HBEρ-).ThemtDNA copynumbersweredeterminedbyreal-timePCR.TheEtBr-treatedHBEwerethen scoredformtDNAdepletionbylivecellimagingafterstainingwithPicogreenand MitoTrackerRed.Also,HBEρ-wasculturedin“ρ0testmedium”toverifyits auxotrophiccharacteristicforuridineandpyruvate.Weassessedthegrowthkineticsof HBEρ+andHBEρ-byrecordingcellsgrowthcurve.Cytochromecoxidase(COX) activitywasmeasuredwithbiochemicalkit.Flowcytometry(FCM)wasusedtodetect mitochondrial membrane potential (ΔΨm) and intracellular reactive oxygenspecies(ROS).(2)HBEρ+ andρ-cellswereexposedtodifferentdoses(0、0.3、0.6Gy)ofα-particles,andweresub-culturedtopassage20and40.Anchorage-independentcell proliferationwasdeterminedbythesoftagarassayandwound-healingassaywas performedtoassessinvasiveness/metastaticpotential.Theplatingefficiencyassaywas performedtodetectthesurvivingfraction.Spontaneousapoptosiswasmeasuredwith theuseofJC-1,AnnexinV-FITC/PIorPI-stainingflowcytometryseparately. Calculationofthedoublingtimeswasbasedoncellsgrowthcurve.Distributionofthe cellcyclewasanalyzedbyflowcytometryafterbeingstainedwithPI.(3)The expression of apoptosis-related molecules Bax, Bcl-2, cyt-c, caspase-9, caspase-8,英文摘要线粒体DNA在α粒子致人支气管上皮细胞体外恶性转化中的作用caspase-3inirradiatedρ+andρ-atpassage20and40wasdetectedbywesternblotting analysis.ROSproductionwasdeterminedbyfluorescentstainingwithH2DCFDAand mtDNAcontentwas analyzedbyreal-timePCR.Results:(1)Real-timePCRandmtDNAstainningshowedthatthecontentof mtDNAinEtBr-treatedHBEcellswas24%ofthewide-typeHBE.Moreover,the EtBr-treatedcellscouldn’tsurv