| Objective:To study the apoptosis mechanism of Yiqi Yangyin Jiedu Fang (YQYYJDF) on human leukemia K562/A02cells.Methods:①Cell culture:K562、K562/A02cells were inoculated with RPMI1640medium containing10%fetal bovine serum, under the conditions of37degree Centigrade and5%CO2, then took the logarithm vegetal period cells,2or3days transfer generation one time, replacing the cell fluid;②Preparation of serum containing:12New Zealand white rabbits were randomly divided into experimental and control groups, each group have6rabbits. Experimental group was administered with with YQYYJDF consecutively for3days; control group was administered with distilled water. After two hours of the last lavage, collect the blood from the rabbit heart in sterile condition. Separation of the serum and mixed the serum of the same group,that is the drug-containing serum, then-20℃conservation.③The proliferation and cytotoxicity of K562, K562/A02cells at different concentrations of YQYYJDF contained serum were detected by MTT.The antiapopototic protein Bcl-2and tumor suppressor gene P53of K562/A02cells at different concentrations of YQYYJDF contained serum were measured by Flow cytometry (FCM).The apoptosis inhibitory protein Survivin expression of K562/A02cells at different concentrations of YQYYJDF contained serum were detected by immunohistochemical.Results:①Different concentrations of YQYYJDF contained serum could inhibit the proliferation of K562/A02with dose-dependence.There was statistically significant between groups (P<0.05).The IC10of YQYYJDF containing serum was16.7%.②YQYYJDF containing serum can significantly increase the apoptosis of K562, K562/A02cells.The lowest apoptosis rate of K562/A02cells in each concentration of serum containing at24h, and the highest apoptosis rate at72h. YQYYJDF containing serum joint daunorubicin has synergistic effect on the apoptosis rate of K562/A02cells.The apoptosis rate of high-dose,medium-dose,low-dose serum containing and containing serum joint daunomycin groups at48hours was39.19±1.39,36.43±1.09,32.70±1.48,45.01±1.74, respectively.There has a statistical differences between groups (P<0.05).③The fluorescence intensity of Bcl-2on K562/A02cell in high-dose, medium-dose, low-dose serum containing and containing serum joint daunomycin groups after48hours was431.67±0.86,453.69±0.40,516.66±0.40,218.48±1.53respectivc-ly.There has a statistical significance differences between groups(P<0.01).The fluorescence intensity of P53on K562/A02cell in high-dose, medium-dose, low-dose serum containing and containing serum joint daunomycin groups after48hours was234.33±12.22,192.33±4.04,172.00±6.00,292.33±7.02respectively. There has a statistical significance differences between groups (P<0.01).④The expression of protein Survivin on K562/A02cell in blank control group was strongly positive. The expression intensity of the low dose group was much higher than the medium-dose group and high-dose group. The expression intensity of the containing serum joint daunomycin group was weakest. The cell membrane and cytoplasm were brown-yellow. And the intensity of expression decreased with drug-dose increasing, There has a statistical significance differences between groups (P<0.01).Conclusion:YQYYJDF containing serum can induce apoptosis of leukemia K562/A02cells,and its possible mechanism was to enhanced cytotoxicity of K562/A02and reduced the expression of gene Bcl-2, inhibited the expression of protein Survivin and upregulated the expression of gene P53. |
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