葛根总黄酮体外诱导Kasumi-1细胞凋亡及其分子机制

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

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

Objective To explore the effects and the molecular mechanism of puerariae radix fla-vones（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 apoptoticcells 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, O. 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 AML1-ETO fusion gene.