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目的研究齐墩果酸对人肝癌细胞株Hep3B增殖和凋亡的影响及其与细胞内钙离子浓度{[Ca2+]i}关系。方法将浓度分别为40、80、100μg/ml齐墩果酸作用于肝癌Hep3B细胞24 h以后,DAPI染色,荧光显微镜观察对照组和处理组细胞形态变化;以11组不同浓度齐墩果酸(5～400μg/ml)作用Hep3B细胞24 h后,四甲基偶氮唑蓝(MTT)法检测Hep3B细胞增殖情况;分别以不同浓度齐墩果酸(80、100、150μg/ml)作用Hep3B细胞24 h后,流式细胞仪检测细胞周期变化、细胞凋亡率、细胞内钙离子浓度;对钙离子荧光强度与细胞凋亡率进行相关性分析。结果各处理组Hep3B细胞出现凋亡;不同浓度齐墩果酸能够抑制肝癌细胞Hep3B增殖,且在5～100μg/ml范围内呈剂量依赖性,药物作用细胞24、48 h的IC50分别为86.04、93.29μg/ml;各处理组细胞周期在G2/M期产生阻滞、[Ca2+]i较对照组显著增加(P<0.05),细胞凋亡率和[Ca2+]i增加与药物浓度呈依赖关系;钙离子荧光强度与细胞凋亡率之间存在线形相关性(P<0.05)。结论齐墩果酸能够抑制肝癌Hep3B细胞株增殖,可能通过上调[Ca2+]i诱导其凋亡。
Objective To study the effect of oleanolic acid on proliferation and apoptosis of human hepatocellular carcinoma cell line Hep3B and its relationship with intracellular calcium concentration {[Ca2 +] i}. Methods Hep3B cells were treated with oleanolic acid at concentrations of 40, 80 and 100 μg / ml for 24 h, respectively. DAPI staining was used to observe the morphological changes of cells in control and treatment groups. Eleven different concentrations of oleanolic acid Hep3B cells were treated with different concentrations of oleanolic acid (80, 100, 150μg / ml) for 24 hours, and the proliferation of Hep3B cells was detected by MTT assay. After 24 h, the changes of cell cycle, apoptosis rate and intracellular calcium concentration were detected by flow cytometry. Correlation analysis between calcium fluorescence intensity and apoptosis rate was carried out. Results Apoptosis of Hep3B cells was observed in all treatment groups. Oleanolic acid inhibited the proliferation of Hep3B cells in a dose-dependent manner in the range of 5-100 μg / ml. The IC50 values of drug-treated cells for 24 and 48 h were 86.04, 93.29μg / ml. The cell cycle arrest in G2 / M phase in each treatment group was significantly increased compared with the control group (P <0.05). The apoptosis rate and the increase of [Ca2 +] i were dependent on the drug concentration There was a linear correlation between calcium fluorescence intensity and apoptosis rate (P <0.05). Conclusion Oleanolic acid can inhibit the proliferation of Hep3B hepatocarcinoma cells and induce its apoptosis by up-regulating [Ca2 +] i.