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为了探讨酪氨酸蛋白激酶SYK和CBL家族泛素连接酶在蟾蜍灵诱导HL-60细胞凋亡过程中的作用机制,采用台盼蓝染色检测细胞活力,采用流式细胞术检测细胞凋亡,采用Western blot和免疫沉降技术检测CBL、CBL-b表达和SYK的磷酸化。结果显示,蟾蜍灵以时间和剂量依赖方式抑制HL-60细胞增殖,24、48和72小时抑制细胞活力的IC50浓度分别约为26.3,7.8和2.0 nmol/L。高剂量蟾蜍灵从8小时即开始诱导HL-60细胞凋亡;与此同时蟾蜍灵快速活化SYK,并以时间依赖的方式下调CBL和CBL-b表达。结论:SYK活化及CBL家族蛋白表达下调可能参与蟾蜍灵对HL-60细胞的增殖抑制和凋亡诱导作用。
In order to investigate the mechanism of tyrosine kinase AKK and CBL family ubiquitin ligase in bufalin induced HL-60 cell apoptosis, trypan blue staining was used to detect cell viability, flow cytometry was used to detect apoptosis, Western blot and immunoprecipitation were used to detect CBL, CBL-b expression and SYK phosphorylation. The results showed that bufalin inhibited the proliferation of HL-60 cells in a time-and dose-dependent manner, with IC50 concentrations of 26.3, 7.8 and 2.0 nmol / L at 24, 48 and 72 hours respectively. High dose bufalin induced apoptosis of HL-60 cells from 8 hours on; at the same time, bufalin rapidly activated SYK and down-regulated the expression of CBL and CBL-b in a time-dependent manner. CONCLUSION: Down-regulation of SYK activation and CBL family protein may be involved in the inhibitory effect of bufalin on HL-60 cell proliferation and apoptosis induction.