鼠双微粒体2在小檗碱诱导急性淋巴细胞白血病EU-4细胞凋亡中的作用  

Role of murine double minute 2 in apoptosis of acute lymphoblastic leukemia EU-4 cells induced by berberine

作　　者：刘健[1] 张小玲[1] 胡群[1] 刘爱国[1] 刘双又[1] 张柳清[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030

出　　处：《中华实用儿科临床杂志》2014年第3期182-185,共4页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（81100357）

摘　　要：目的 探讨鼠双微粒体2（MDM2）在小檗碱诱导儿童急性淋巴细胞白血病EU-4细胞凋亡中的作用.方法 取对数生长期的EU-4细胞进行实验.终浓度为0、1、10、100 μmol/L小檗碱分别作用72 h后,流式细胞仪检测以上各组细胞凋亡情况,Western blot法检测以上各组细胞MDM2蛋白表达水平.100 μmol/L小檗碱分别作用0、24、48、72 h,采用实时荧光定量PCR法检测MDM2基因表达水平.使用终浓度为150 nmol/LMDM2小干扰RNA （siRNA）转染MDM2 siRNA组细胞,相同终浓度对照siRNA转染对照siRNA组细胞,48 h后采用流式细胞仪检测2组细胞凋亡情况.结果 经不同浓度小檗碱作用后,EU-4细胞凋亡率呈逐渐上升趋势,100 μmol/L小檗碱作用72 h后细胞凋亡率最高达（60.13±4.21）％,各组差异均有统计学意义（F=280.56,P＜0.05）;MDM2蛋白表达水平则随小檗碱浓度上升逐渐降低,差异有统计学意义（F=73.82,P＜0.01）.MDM2 mRNA表达水平随小檗碱作用时间延长逐渐降低,差异有统计学意义（F=45.37,P＜0.01）.对照siRNA组及MDM2 siRNA组细胞凋亡率为（11.09±1.63）％和（29.84±1.75）％,2组比较差异有统计学意义（t=-13.57,P＜0.01）.结论 小檗碱浓度和时间依赖地抑制MDM2的表达,可能是小檗碱诱导白血病细胞凋亡的机制之一.Objective To explore the role of murine double minute 2 （MDM2） in apoptosis of acute lymphoblastic leukemia EU-4 cells induced by berberine.Methods EU-4 cells at logarithm growth phase were chosen to carry out the experiments.EU-4 cells were incubated with different doses of berberine for 72 hours,and the final doses of berberine were 0,1,10 and 100 μmoL/L,respectively.Then the apoptosis rate of EU-4 cells were detected by flow cytometry,and the expression levels of MDM2 protein were tested by Western blot.The levels of MDM2 gene were examined by means of real-time PCR at 0,24,48 and 72 hours after incubation of EU-4 cells with 100 μmol/L berberine,respectively.EU-4 cells in MDM2 small interfering RNA（siRNA） group were transfected with 150 nmol/L MDM2 siRNA,and the control siRNA group were transfected with 150 nmol/L control siRNA.The apoptosis rates of EU-4 cells were detected 48 hours after transfection by flow cytometry.Results The apoptosis rates of EU-4 cells were increased in a dosedependent manner,and the highest apoptosis rate was up to（60.13 ± 4.21）%,which was incubated with 100 μmol/L berberine.Significant differences were found in all groups （F =280.56,P 〈 0.05）.Berberine treatment suppressed the protein expression of MDM2 in a dose-dependent manner（F =73.82,P 〈 0.01）.The mRNA expression of MDM2 was inhibited in a time-dependent manner by 100 μmol/L berberine（F =45.37,P 〈0.01）.The apoptosis rates of control siRNA group and MDM2 siRNA group were（11.09 ± 1.63） % and （29.84 ± 1.75） %,respectively.Significant difference was found between the 2 groups （t =-13.57,P 〈 0.01）.Conclusions Berberine can inhibit the expression of MDM2 in a dose-and time-dependent manner,which may be one of the mechanisms of apoptosis induced by berberine in leukemia cells.

关 键 词：小檗碱 白血病细胞 凋亡 鼠双微粒体2 

分 类 号：R733.71[医药卫生—肿瘤]