| ObjectivesCigarette smoking is a seriously worldwide problem of public health, and has been proven to be contributable to carcinogensis of lung, larynx, throat, esophagus, kidney, pancreas, bladder, cervix and diseases of respiratory, digestive and cardiovascular systems. Cigarette smoke contains over 4000 constituents including reactive oxygen/nitrogen species and many pro - oxidants, which generate reactive substances during metabolizing. Many reports have suggested that cigarette smoking - related diseases could be induced by oxidative stress mechanism.In the present study, cigarette smoke bubbled - PBS was prepared to treat lymphocytes in vitro. Cell viability and oxidative stress - associated index were evaluated to explore the mechanism of oxidative damage. Pretreated cigarette smoke - administrated lymphocytes with vitamin C, glutathione or melatonin, and the interventional mechanisms of these antioxidants were also discussed. DNA damage of human lymphocytes treated with cigarette smoke was detected at different time, and DNA repair ability was discussed. The study was expected to offer evidence for researching mechanism of cigarette smoke - related damage, and preventing and remedying cigarette smoke - associated diseases.Methods1. Isolation of lymphocytes.Lymphocytes were isolated by Ficoll - Hypaque density gradient centrifuga-tion and washed twice with RPMI1640 medium. Cells were counted and checked for viability by trypan blue exclusion.2. Preparation of aqueous cigarette smoke solution (CSS)A cigarette holder was attached to one end of two serial glass tubes containing 5ml phosphate -buffered solution (PBS) respectively, and a 50 -ml glass syringe was attached to the other end of the serial tubes. Smoldering cigarette was puffed for 2s, once every min for a total of ten puffs, at a flow rate of 0.05 liters/min. Two cigarettes were burned at one time to form 0.2 cig/ml cigarette smoke solution. For every experiment CSS was freshly prepared.3. Cell pretreatment and administration1) 0,1 10-3cig/ml,2 10-3cig/ml,4 10-3cig/ml,8 10 -3cig/ml, 12 10-3cig/ml,16 10-3 cig/ml CSS were added to lymphocytes respectively to study the effect of cigarette smoke on cells. 2) Pretreated with vitamin C, gluta-thione or melatonin for 2h, cells were exposed to 8 10 -3cig/ml for another 2h to learn the impact of antioxidants. 3 ) Human lymphocytes had been exposed to 8 10-3cig/ml CSS for 0h, 0.5h, Ih, 2h, 3h, 4h, 5h and6h respectively, to learn DNA damage and its repair in normal cells.4. Evaluation of cell viabilityCells were plated in 96 - well plates at a density of 3 106 /ml. After being added 10% Alamar blue, cells were incubated at 31 C, 5% C02. After 4 hours, absorbance at 540nm and 620nm was detected by Labsystem Ascent plate reader, then Alamar blue reduction rate of cells was computed.5. Evaluation of intracellular ROSAdjusted the cell density to 1 106/ml, cells were pretreated with 2' ,7'-dichlorofluorescein diacetate (DCFH -DA) for Ih, cell -free groups were prepared in parallel. After 2h CSS - exposure, fluorescence density of dichlorofluo-rescein ( DCF) in cell suspension was detected, with the excitation wave 485nm, exit5nm, emission 530nm, exit 10nm.6. DNA damage evaluation by comet assayComet assay was conducted basically according to Singh et al. Briefly, 0.1ml 1% normal melting agarose (NMA) gel was used for the first layer, while 85 l 1% low melting agarose (LMA) gel together 3 106 cells (10 l cells suspension + 75 l LMA) , was used for the second layer. Finally, a third layer of 85 l LMA was added. Slides were immersed in ice - cold freshly pre-pared lysis solution to lyse the cells and to allow DNA unfolding. After 1.5 h at 4 C in the dark, slides were placed on a horizontal electrophoresis unit. The DNA was allowed to unwind for 0. 5 h, in electrophoresis running buffer solution. Electrophoresis was conducted for 20 min at 25 V and 300 mA in dark. Subsequently, the slides were removed gently and washed in a neutralization buffer.
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