| PrefaceThe oxidative stress reaction is exaggerated in chronic renal failure (GRF) patients. Hemodialysis (HD) may activate the complement system, and stimulate the oxi dative metabolism of neutrophil and monocyte. Reactive oxidative species (ROS) are produced by this mean. So HD may exaggerate the oxidative reaction in CRF patients. On the other hand, some antioxidative substances may lose during the HD process, for example vitamin C, E, glutathin, etc. The oxidative system and antioxidative system is imbalanced in HD patients. When treated by imbiocompatibile membrane, the blood - membrane contaction may lead to more ROS production. ROS mainly react with ribose; seize the hydrogen atom at C - 4, leading to unstable of DNA structure and resulting in DNA chain breakage. At present, the principal method to detect breakage of DNA chain is single cell gel electrophoresis technique - that is comet assay. Our experiment try to comprehend the degree of DNA damage of lymphocyte in MHD patients through the comet assay, and observe the effect of different membranes and vitamin C on it.Materials and Methods1. Main reagents and apparatus1% low and normal melting point agarose hoemothermia bath box; centrifu-ger; incubator; electrophresis apparutus; fluorescence microscope.2. Experimental methods2.1 Study objects-. Selected 20 patients from our blood purification center who were more than 20 years old, treated by MHD more than 6 months and hadstable clinical condition. Randomly divided the 20 patients into 2 groups. One was the HE group, which hemodialized by hemophan for 12 weeks; the other was PS group, which hemodialized by polysulfone for 12 weeks. 7 healthy people who were matched in age and sex with the patients were selected to be the normal control.2. 2 Test methodIsolate lymphocyte:took 50ul anticoagulated venous blood, added 1ml 1640 to diluted it,then added lymphocyte separating medium,4C ,4500r/min centrif-ugated for 3 minutes to abtain lymphocyte.Incubation;The lymphocyte was divided into 2 groups, added 1 ml 1640 in one group and 1ml 10ug/ml vitamin C into the other group. The two groups were incubated in 37C together for 1 hour.COMET assay: after centrifugating, added low melting point agarose to the cell suspension. Spread out one layer of normal melting point agarose, lymphocyte suspension, low melting point agarose on a slide . When the agarose coagulated , put the slide to split, unwind, electrophoresis, neutraliz, stain, then observed the result using fluorescence microscopeResult observation; For each patient two slides were prepared and images of at least 100 cells (50 cells from each of two slides) were evaluated.3. Statistical analysis: Experimental data were dealt with SPSS 11.5 statistical analysis software to describe and analyze the t test and analysis of variance, and do the correlated analysis of data. P >0. 05 was not accepted.Result1 The measured value of tail length of comet;before vitamin C treatment; the tail length of HE group is 0.970 +0.120um,the percentage of comet is 14. 8% ;the tail length of PS group is 0. 656 +0.187um,the percentage of comet is 11.5% The tail length of normal control is 0. 286 + 0. 194um, the percentage of comet is 5. 3% The tail length and the percentage of comet of the MHD patients are significantly higher than the normal control, and HE group is significantly higher than the PS group. After vitamin C treatment; the tail length of HEgroup is 0.788 +0. 260um,percentage of comet is 13. 5% ;the tail length of PS group is 0. 670 +0. 295um,percentage of comet is 11.6% There are no significant difference before and after vitamin C treatment.2 The correlation between DNA oxidative damage and serum creatinine and urea nitrogen: There is significantly positive correlation between the tail length and the concentration of serum creatinin and blood urea nitrogen;3 The correlation between DNA oxidative damage and other factors; the correlation between DNA oxidative damage and age, history of hemodialysis is...
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