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Objective: To investigate Clematis saponins induced NB4 cell apoptosis and the possible mechanisms and the PML-RARa mRNA for leukemia treatment and to explore the possibility, to provide a theoretical basis for the development of new anti-tumor drugs. Methods: According to the literature extract Weilingxian saponins 0.01% DMSO as a negative control, 1.0umol / l of arsenic trioxide as a positive control, various concentrations of Clematis saponins experimental group. 0.01% DMSO, 1.0umol / l of arsenic trioxide and different concentrations of Clematis saponin were added to cultured NB4 cell culture fluid, so that the saponins in the reaction system in a final concentration of 10ug/ml, 20ug/ml, 40ug/ml 80ug/ml, 160ug/ml, each group of five parallel control, and repeat three experiments. Remove NB4 cells cultured for 24, 48 and 72 hours after adding MTT solution, measuring the value of A, to calculate the rate of cell growth inhibition and IC50 values, and to identify the optimal concentrations. NB4 cells were added with 30ug/ml Clematis saponin, 0.01% DMSO, cultured in the culture system of the 1.0umol / l of arsenic trioxide, were cultured for 48 and 72 hours (after the groups of three parallel control, and repeating the test three times ), Wright's-Giemsa staining NB4 cells in each group, and Morphological changes were observed under an optical microscope and photographed. NB4 cells were added with 30ug/ml Clematis saponin, 0.01% DMSO, 1.0umol / l of arsenic trioxide that are cultured in the culture system, were cultured for 24, 48 hours after (groups of three parallel-group, and repeating the test three times), take 1 × 105 cells, adding the 500ul a Binding Buffer suspended cells, joined 5ul Annexin V-FITC and mix 5ul Propidium iodide mixing, at room temperature, protected from light, reaction 20min apoptosis rate detected by flow cytometry. NB4 cells were added the containing saponins of 30ug/ml Weilingxian, 0.01% DMSO culture system culture were cultured for 24, 48 hours (groups of three parallel control, and repeat the test three times), take the 1 × 105 cells fixed with 75% ethanol, and the RNA-containing enzyme PI dye to detect cell cycle distribution. NB4 cells were added to the culture containing 30ug/ml Clematis saponin, 0.01% DMSO in the culture system, were cultured for 48 and 72 hours (after the groups of three parallel control, and repeating the test three times), 1 × 106 cells extracted RNA using Real-time PCR method to detect PML-RARa mRNA in NB4 cells. Results: different concentrations of Clematis of saponins role cells inhibited cell growth inhibition rate gradually increased with the extension of drug concentration and time of intervention, a pairwise comparison P-lt; 0.05, the difference was statistically significant, but the inhibition rate of less than arsenic trioxide group. Calculate the IC50 value for each time period saponins: intervention group of 24 hours: 247.91ug/ml; the interventions the 48 hours group: 30ug/ml; intervention group of 72 hours 4.00ug/ml a select concentration for 30ug/ml saponins as the optimal concentration. Clematis saponins NB4 cells by morphological examination found the cell size smaller nucleus highly concentrated gathered, even fracture the formation of apoptotic cytoplasm loose, vacuolization, the same changes and arsenic trioxide. Clematis saponins NB4 cells by flow cytometry found, apoptosis rate increase with intervention prolonged time, the same change before experiments, between groups p lt; 0.05, the difference was statistically significant, but the The apoptosis rate of less than arsenic trioxide group. The Clematis saponins role NB4 cells by flow cytometry find most of the cell cycle arrest at the G2 phase, and saponins intervention group than 48 hours, and statistical analysis P lt; 0.05, the difference was statistically significant. NB4 cells of the Clematis after the saponin role of PML-RARa mRNA expression differences before and after the drug effect was not statistically significant (P gt; 0.05), and the difference in change over time and no statistical significance (P gt; 0.05), Table Hemingway Ling Xian saponins not affect PML-RARa mRNA expression. Conclusion: Clematis saponins inhibit cell growth in a time-and concentration-dependent manner. Clematis saponins can induce apoptosis was significantly time-dependent, but the apoptosis rate of less than arsenic trioxide group. Clematis saponins inhibited cell growth and induced apoptosis optimum concentration and optimal duration of action is 30ug/ml and 48 hours respectively. Clematis saponins induce apoptosis mechanism may be through the cell cycle arrest, arrest at G: period, and then induce apoptosis. Clematis the saponin do not affect the expression of the PML-RARa mRNA.
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