摘要[目的]观察探讨小檗胺(BBM)对体外培养的视网膜母细胞瘤(RB) HXO-RB44细胞增生和凋亡的影响及其机制.[方法]体外培养RB细胞,取对数生长期细胞分为BBM处理组和空白对照组.BBM处理组分别加入2、4、8、16、32 mg/L BBM,作用24、48、72 h后,四氮唑(MTT)比色法检测细胞增生活性;4、8、16 mg/L BBM作用24 h后,流式细胞仪检测细胞早期细胞凋亡率,酶联免疫吸附试验(ELISA)检测细胞内B细胞淋巴瘤/白血病-2(bcl-2)、Bax蛋白的表达,比色法检测半胱氨酸蛋白酶-3 (Caspase-3)的活性.[结果]BBM以时间-剂量依赖方式显著抑制RB细胞的增生(24 h:F=70.547,48 h:F=603.438,72 h:F=577.521;P＜0.01).作用24、48、72 h,半数抑制浓度分别为25.26、10.94、6.25 mg/L.空白对照组和不同浓度BBM处理组细胞坏死率分别为(1.25±0.45)％、(4.10±2.95)％、(4.39±0.21)％、(10.54±4.38)％,与空白对照组比较,差异有统计学意义(F=6.527,P＜0.05)；晚期凋亡和坏死率分别为(2.13±0.71)％、(5.45±2.31)％、(9.86±3.18)％、(11.10±1.70)％,与空白对照组比较,差异有统计学意义(F=10.845,P＜0.05):早期凋亡率分别为(0.51±0.26)％、(2.68±0.35)％、(5.97±0.50)％、(11.22±1.17)％,与空白对照组比较,差异有统计学意义(F=144.976,P＜0.01).BBM剂量依赖性地减少bc1-2的表达,增加Bax的表达,降低bcl-2/Bax比值,增强Caspase-3活性,差异均有统计学意义(bcl-2:F=835.726,Bax:F=111.963,Caspase-3:F=298.058;P＜0.01).[结论]BBM体外能抑制RB细胞增生并诱导细胞凋亡或坏死.其机制可能与下调bcl-2的表达,上调Bax的表达,并增强Cspase-3的活性有关.

abstracts[Objective] To investigate the effect of berbamine (BBM) on the proliferation and apoptosis of retinoblastoma (RB) HXO-RB44 cells and its possible mechanism in vitro.[Methods] RB cells in logarithmic growth phase were divided into BBM treated group and control group.RB cells in BBM treated group were cultured with different concentrations of BBM (2,4,8,16 and 32 mg/L) for 24,48 and 72 hours,respectively.The proliferation was assayed by methyl Thiazolyl tetrazolium (MTT).RB cells were cultured with different concentrations of BBM (4,8 and 16 mg/L) for 24 hours.The early apoptotic rates were detected by flow cytometry; the expression of bcl-2 and Bax were measured by enzyme-linked immunosorbent assay (ELISA) and the activity of Caspase-3 was detected by colorimetric assay.[Results] BBM could obviously inhibit the proliferation of RB cells in a time-and dose-dependent manner (24 hours:F=70.547,P＜0.01; 48 hours:F=603.438,P＜0.01; 72 hours:F=577.521,P＜0.01).The 1C50value at 24,48 and 72 hours were 25.26,10.94 and 6.25 mg/L,respectively.Necrosis rates of control group andBBM treated group were (1.25±0.45)％,(4.10±2.95)％,(4.39±0.21)％ and (10.54±4.38) ％ respectively; the difference between two groups was statistically significant (F =6.527,P＜0.05).Apoptotic and necrosis rates in advanced stage of control group and BBM treated group were (2.13±0.71)％,(5.45 ± 2.31)％,(9.86 ± 3.18)％ and ( 11.10 ± 1.70)％,respectively.The difference between two groups was statistically significant (F =10.845,P＜0.05).Early apoptotic rates of control group andBBM treated group were (0.51±0.26)％,(2.68±0.35)％,(5.97±0.50)％ and (11.22±1.17) ％,respectively.The difference between two groups was statistically significant (F=144.976,P＜0.01).In addition,BBM dose-dependently reduced bcl-2 level and increased Bax expression,causing the reduction of the bcl-2/Bax protein ratio as well as increased the Caspase-3 activity in RB cells remarkably (bcl2:F=835.726,P＜0.01; bax:F=111.963,P＜0.01; Caspase-3:F=298.058,P＜0.01).[Conclusion]s BBM can inhibit the proliferation and induce apoptosis or necrosis of RB cells in vitro,downregulating the expression of bcl-2,up-regulating the expression of Bax.Along with increased Caspase-3activity these may be the apoptotic mechanisms.