Relationship Between DNA Damage In Peripheral Blood Lymphocytes Of Bronchial Asthma And Oxidative Stress

Objective:Examine the DNA damage in peripheral blood lymphocytes and plasma MPO and MDA of the Patients with bronchial asthma, to explore the correlation of DNA damage and Oxidative Stress so provide new idea for the prevention and cure of bronchial asthma. Method:1. patients with asthma as study objects,20 patients with severe asthma as the severe group,9 patients with mild asthma as mild group and 9 cases of healthy persons as normal group. Blood, collected from the cubital veins,to examine the DNA damage in lymphocytes and the content of MPO,MDA.2. To detect DNA damage in the lymphocytes by Comet Assay:An amount of 2 ml heparinized blood mixing with the HanK's liquid 2ml was carefully layered over 2ml Lympoprep and centrifuged for 15 min at 1500 rpm. The interface band containing lymphocytes was collected by 15 min centrifugation at 1500 rpm, collect 20μl lymphocyte cell resulting pellets, mixed with 75μl 0.7% low-melting-point agarose, subsequently,75μl of this mixture was layered onto slides that had previously been coated with 1.0% hot normal melting point agarose (NMA) and covered with a coverslip at 4℃for at least 10 min to allow the agarose to solidify. After removing the coverslips,the slides were submersed in freshly prepared cold (4℃) lyzing solution for at least 1 h. Take out the slides and washed with 0.9% NS twice, Slides were then immersed in freshly prepared alkaline electrophoresis buffer, kept in room temperature for unwinding(20 min) and then electrophoresed (25 V/300 mA,20 min). All of the above steps were conducted without direct light in order to prevent additional DNA damage.After electrophoresis, take out the slides and washed with 0.9% NS twice, the slides were stained with ethidium bromide (70μl/slide) for 10min in dark place and analyzed using a fluorescence microscope (excitation wavelength 515-560nm). observe nuclear DNA and migration DNA within 200 field of vision,50 cells are photographed in each subject, save the file, utilize TriTek CometScoreTM to do the image analysis, with tail length and tail moment as criteria, observe the damaged degree of DNA.3. Determine the plasma MPO content:Plasma MPO levels were determined using ELISA.Inject MPO to the plate coated MPO monoclonal antibody in advance, incubation; after washed,add HRP marked MPO antibody.Incubation and wash again, remobe unconjugated enzyme, then add substrate A,B, produce blue, convert to final yellow by the effects of acid.Different shades of colors correlated positively with the MPO concentration of the sample. Detected OD in wavelengh 450nm, base on the OD of standard and sample, calculate MPO content of sample.4. Determine the plasma MDA content:the MDA in lipid peroxidation degradation product can condensate with thiobarbituric acid, produce the red creature, has maximal absorption peak at he wavelength of 532nm, determination of absorbance by chromatometry, calculate the plasma MDA content by calculational method. Results:1.The observation of DNA damage in the lymphocyte cells.οDifference of TL:severe group are more remarkably than mild group and normal group, have statistical sense(p<0.05); mild group are significantly higher than normal group (p<0.05).②Difference of TM:severe group are more remarkably than mild group and normal group, have statistical sense (p<0.05); no significant changes of mild group and normal group, not have statistical sense (p>0.05).2. Difference of the plasma MPO content:The plasma MPO content of severe group are obviously higher than mild group and normal group, (p<0.05); mild group are higher than normal group, have statistical sense (p<0.05).3. Difference of the plasma MDA content:The plasma MDA content of severe group are obviously higher than mild group and normal group (p<0.05); mild group are higher than normal group (p<0.05).4. Correlative analysis reveal:TL and TM of severe group are correlated positively (p<0.01);TL and MPO content of severe group are correlated positively (p<0.01); TM and MPO content of severe group are correlated positively (p<0.01); TL and MDA content of severe group are correlated positively (p<0.01); TM and MDA content of severe group are correlated positively (p<0.01); MPO content and MDA content of severe group are correlated positively (p<0.01). Conclusion:1. There exists obvious DNA damage in lymphocytes during a Acute exacerbation of bronchial asthma, the more severe the disease, the more obvious damage.2.The plasma MDA and MPO as Oxidative Stress indicators, which content increase evidently at acute stage of asthma, obviously in severe asthma.3.The content of the oxidative stress indicators MDA and MPO in peripheral blood from asthmatic patients are correlated positively with the DNA damage in peripheral blood lymphocytes.4. Oxidative Stress enter the DNA damage in peripheral blood lymphocytes in bronchial asthma.