小檗胺诱导体外培养的视网膜母细胞瘤HXO-RB44细胞凋亡的研究

Apoptosis induced by berbamine in retinoblastoma HXO-RB44 cells

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bcl-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。

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 IC50 value at 24,48 and 72 hours were 25.26, 10.94 and 6.25 mg/L, respectively. Necrosis rates of control group and BBM 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 and BBM 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 （bcl-2： F=835.726,P〈0.01; bax： F=111.963, P〈0.01;Caspase-3：F=298.058,P〈0.01）.Conclusions BBM can inhibit the proliferation and induce apoptosis or necrosis of RB cells in vitro, down regulating the expression of bcl-2, up regulating the expression of Bax. Along with increased Caspase-3 activity these may be the apoptotic mechanisms.