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[摘要]
目的 研究苦参碱对原代人白血病细胞的体外作用及可能的分子机制。方法 取临床初诊的髓系白血病患者外周血, 分离得到原代白血病细胞体外培养。不同浓度苦参碱作用后, 观察细胞的形态学改变、CCK8法检测细胞增殖、Annexin V-FITC/PI分析细胞凋亡及流式检测细胞周期。结果 苦参碱作用24 h, 细胞增殖明显受抑, 0.5、0.8 mg/mL组的生长抑制率分别为54.98%和72.96%, 与对照组相比差异显著(P<0.01), 抑制效应有明显剂量相关性。苦参碱作用24 h的IC50为0.5 mg/mL。苦参碱作用24 h, 0.2、0.5、0.8 mg/mL组细胞早期凋亡率分别为11.8%、37.6%、54.7%, 明显高于对照组自发凋亡率(7.50%)(P<0.05)。苦参碱作用48 h后, 细胞周期阻滞于G0/G1→S期。结论 苦参碱可显著抑制体外培养的人原代白血病细胞增殖, 其机制与诱导细胞早期凋亡, 促进细胞周期G1→S期阻滞有关。
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[Abstract]
Objective To investigate the effects of matrine on the human primary 1eukemia cells and its possible molecular mechanisms. Methods The primary leukemia cells were isolated from the PBMC of the patients with myeloid leukemia and cultivated under the regular culture medium. After exposed to the matrine with different concentration, the morphological changes of leukemia cells were observed under the light biomicroscopy. The proliferation was determined by CCK-8 assay. Annexin V-FITC/PI affinity assay was used to analyze the apoptosis of leukemia cells induced by matrine. The cell cycles were analyzed by flow cytometry before and after matrine treatment. Results Compared to the vehicle cells, there was a dramatically proliferation suppression was observed in the cells after matrine treatment. The inhibitory rates of primary leukemia cells were 54.98% and 72.96% after treated with matrine for 24 h at the doses of 0.5 and 0.8 mg/mL, respectively (P < 0.01), which has a significant dose-dependent manner. The half maximal inhibitory concentration of matrine (IC50) was 0.5 mg/mL for 24 h treatment. Matrine could induce early cell apoptosis. The percentage of apoptotic cells were 11.8%, 37.6%, and 54.7% in the cells treated with 0.2, 0.5, or 0.8 mg/mL matrine for 24 h, respectively (with spontaneous apoptosis rate 7.50% for untreated cells) (P < 0.05). FCM method showed that cells at the G0/G1 phase decreased and cells at the S phase were declined obviously, suggested a blockage of cell cycles transition at the G1/S key checkpoint for 48 h. Conclusion Matrine manifests a significant inhibitory effects on the proliferation of human primary leukemia cells. Apoptosis induction and the arrest at G1 phase of cell cycle maybe contribute to its roles on the suppression of cell growth.
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[基金项目]
国家自然科学基金(81101647);南京医科大学博士后课题