| Over the past several years, extensive epidemiological surveys and anima1experiments and celluIar or molecuIar studies have been conducted to evaluatethe biological effects of extremely low frequency(ELF) magnetic fields,particularly on cancer development. However, the knowledge of the magneticfields on human health is still 1imited. The reason fOr this is that the aPpropriatecellular effects whether beneficiaI or harmful by ELF MF and the potentialmechanism is not stil1 cIarified. Because the cu1lular function change caused byMF re1ate to the change of gene transcription, there is the effective passport forpresuming the possible cullular effect to ensure the gene species occuringtranscription change induced by MF In l997, Binninger et al exposed the yeastSaccharomyces cervisiaer to 20 ll T 60 Hz MF for 24 hours and identifiedcharacteristically three EMF-responsive genes by using differential displayreverse transcreption PCR(DDRTPCR). The EMF-responsive genes in yeastprovide valuable information for highly developed organisms. However, there isquite different between the lowly and highly developed cells in both response toinsu1ts and genetic control mechanisms, especially in the cell-specific effects.Therefof, our lab ideniify and characterize the EMF-responsive genes in highlydeveloped cells(Daudi celI and discover l3 gene fragmenis. MF-l gene fragmentis an lowly expression fragment, absent only in MF exposed cu1tures, using DDtechnology. By using reverse Northem blot and Northern blot, we have futherproved that MF-l fragmellt is real DD fragment. After cloned, EST sequencedand Genebank blast, it was found that MF-l has a hundred percent homologousto cytochrome oxidase subunit l. But the present study is not to investigate therelation of ELF MF and cytochrome oxidase subunit l. we first filtrated andcloned the cytochrome oxide subunit l gene is the responsive gene to MF inDaudi cell Iines and discovered MF could lower this gene expression. If we canfurther prove this gene responsive universality and time characteristic in otherMF- sensitive cell lines, we will find a new breakhrough to reveal the relation ofMF and cell growth, EMF epigenetic toxicity nerval biology effect byresearched the function and distribution of cytochrome oxidase subunit l. Basedupon the above researches, the present study is tO prove this gene responsiveuniversality and particularity to use Northern blot observing cytochrome oxidasesubunit l gene expression changes with some MF-sensitive cell lines(HL-60,Jurkat, Ll2l0 and CHL).Materials and Methodsl.MFs exposure system:The ELF exposure system is composed of tWo Heimholtz coils which50Hz AC passed through during exposure and MF intensities distributedunifOrmIy with a variation of i 10% within the coils.2.Make 8ure thc exposure densities and exposure periods:Daudi, a human lymphoma cel1 line, was cultured routinely inRPMl-l640 medium coniaining 2 mM L-gIulamine and 25mM HEPES, andsupplemented with 15% fetal bovine serum, penicillin G(50U/ml),streptomycin sulfate (50 u g/ml) and Kanamycin sulfate(50U/ml). TheexPeriments consisted of nine MF exposure periods (20h, 24h, l6h, l2h, 8h,6h, 4h, lh and 20min) for MF flux of 0.8mT along with sham-exPosedcontrols. Each experimental run was repeated tWo times.Cells wereconcentrated(3000rPm for 5 min at 4oC) immediately fol1owing MFs exposureand lysed in Trizol. TOtal cellular RNA was isolated by Trizol method.Quantitative Northern blot hybridization were used to measure endogenousquantities of MF-l. GAPDH was used as an intCrnal standard tO determinesteady state transcript levels and to assure that dat8 are compatable both acrossdiffereat asmples within an experiment as wel1 as between awptalexperiments. Data was analyzed with an Ambis radioanalytic imager ThetranscriPtion of MF-l was normalized against that of GAPDH for each bolt,and the ration of data obtained from MF and sham exposed s... |
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