葛根总黄酮体外诱导Kasumi-1细胞凋亡及其分子机制

目的 探讨葛根总黄酮(puerariae radix flavones,PRF)对人急性髓系白血病细胞系Kasumi-1细胞凋亡的影响,并对其可能的分子机制进行初步研究.方法 以不同浓度PRF作用Kasumi-1细胞48 h,瑞氏染色及Hoechst 33258荧光染色观察细胞凋亡变化,FITC-Annexin V/PI双染法检测细胞凋亡率,实时定量PCR技术检测AMLl -ETO融合基因表达,Western blot法检测Bcl-2、Bim及Caspase相关酶的变化.结果 PRF能有效诱导Kasumi-1细胞凋亡.以50、200及500 μg/ml的PRF分别处理细胞,其早期凋亡率依次为(14.1±0.8)％、(17.7±1.3)％及(32.4±1.4)％,较空白对照组[(7.8±0.7)％]明显增加(P＜0.05),作用呈浓度依赖性;抗凋亡蛋白Bcl-2相对表达量依次为0.85±0.05、0.62±0.07及0.43±0.05,呈下调趋势(P＜0.01);而促凋亡蛋白Bim相对表达量分别为0.21±0.06、0.39±0.04及0.75 +0.05,Caspase-3蛋白相对表达量分别为0.92±0.04、1.21±0.07及1.33±0.04,Caspase-9蛋白相对表达量分别为0.35±0.05、0.53±0.03及0.69±0.07,均呈上调趋势(P＜0.01).Caspase-8蛋白表达量及AML1 -ETO融合基因表达量则无明显改变.结论 一定浓度PRF诱导Kasumi-1细胞凋亡可能与下调细胞Bcl-2、上调Bim及活化Caspase相关酶有关,与AML1 -ETO融合基因无明显相关性.

Apoptosis of Kasumi-1 cells induced by puerariae radix flavones and its molecular mechanism

Objective To explore the effects and the molecular mechanism of puerariae radix flavones(PRF) on acute myeloid leukemia cell line Kasumi-1 cells in vitro.Methods Kasumi-1 cells treated by PRF for 48 hours were observed with Wright' s and Hoechst 33258 dying.The apoptotic cells were analyzed by flow cytometry with AnnexinV/PI staining.The expression levels of bcl-2,Bim and Caspase-3/-8/-9 protein were assayed by Western blot and the AML1-ETO fusion gene was detected by real-time polymerase chain reaction.Results PRF could induce Kasumi-1 cells to apoptosis effectively.The proportion of apoptotic cells in 50,200 and 500 μg/ml PRF treatment groups were ( 14.1 +0.8)％,( 17.7 + 1.3)％ and (32.4 ± 1.4) ％,respectively,and significantly higher than that of control [ ( 7.8 + 0.7 ) ％ ].The relative expression levels of the anti-apoptotic Bcl-2 protein were 0.85 ± 0.05,0.62 ± 0.07 and 0.43 ± 0.05 ; the apoptotic Bim protein were 0.21 ± 0.06,0.39 + 0.04 and 0.75 ± 0.05 ; the caspase-3 and caspase-9 were 0.92 ± 0.04,1.21 ± 0.07,1.33 ± 0.04 and 0.35 ± 0.05,0.53 ± 0.03,0.69 ± 0.07,respectively.Compared to the blank control group,all these changes were significant ( P ＜ 0.01 ).Nevertheless,nearly no changes could be observed on the expression level of AML1-ETO fusion gene and caspase-8 protein.Conclusion Apoptosis of Kasumi-1 cells induced by PRF might correlate to the down-regulation of Bcl-2 protein expression and the activation of caspase-3 and caspase-8 protein in the cells.It seemed that all these effects had no relationship with the AMLI-ETO fusion gene.