葡萄柚黄酮抑制白血病细胞株增殖的体外实验研究

The Mechanism of Pure Total Flavonoids from Citrus Paradisi Macfad for Inhibiting in vitro Proliferation of Leukemia Cell Strain

目的研究葡萄柚黄酮组分(PTFC)对Kasumi-1、K562细胞株的生长抑制作用及诱导凋亡情况,从而为白血病的临床治疗提供实验基础。方法不同浓度的PTFC(0、0.125、0.25、0.5、1、2 mg/mL)作用于Kasumi-1、K562细胞株,采用MTT法检测PTFC对Kasumi-1、K562细胞株的生长抑制作用;AnnexinⅤ/PI流式细胞术检测PTFC诱导Kasumi-1、K562细胞株的凋亡情况;Hoechst 33258染色法观察PTFC诱导白血病细胞凋亡的形态学改变;Western Blot法检测PTFC作用后Caspase-3、Caspase-8、Caspase-9、PARP蛋白表达变化。结果 0.125～2 mg/mL PTFC可明显抑制Kasumi-1、K562细胞株增殖,与空白对照组(PTFC 0 mg/mL)比较,细胞存活率明显降低(P<0.05),对Kasumi-1的IC_(50)(24、48 h)分别为1.99、0.89 mg/mL,对K562的IC_(50)(24、48 h)分别为1.23、1.03 mg/mL;通过AnnexinⅤ/PI流式细胞术检测,0.125～2 mg/mL PTFC作用24 h可诱导Kasumi-1、K562细胞凋亡,诱导Kasumi-1凋亡率分别为2.98%、3.2%、3.66%、8.6%、19.8%,K562的凋亡率分别为3.19%、3.19%、3.49%、7.25%、16.87%;Hoechst 33258染色法检测发现PTFC作用后Kasumi-1、K562细胞中出现明显凋亡小体;与空白对照组比较,Caspase-3、Caspase-9、PARP被激活(P<0.05)。结论 PTFC可抑制Kasumi-1、K562细胞增殖并诱导其凋亡,其凋亡相关机制与PARP、Caspase-3,Caspase-9蛋白表达变化相关。

Objective To observe the inhibition of pure total flavonoids from Citrus paradisi macfad(PTFC)on Kasumi-1 and K562 cell strains,and induction on their apoptosis,thus providing experimental evidence for clinical therapy of leukemia.Methods Leukemia Kasumi-1 cell strain and K562 cell strain were treated by PTFC(0,0.125,0.250,0.500,1.000,2.000 mg/mL,respectively).MTT colorimetric assay was used to detect the growth inhibition of PTFC on leukemia Kasumi-1 and K562 cell strains.Their apoptosis rates were analyzed by AnnexinⅤ/PI flow cytometry.The morphological changes of apoptotic cells were observed using Hoechst 33258 staining.The expressions of Caspase-3,Caspase-8,Caspase-9,and PARP were detected by Western Blot.Results PTFC in certain concentration range(0.125-2.000 mg/mL)markedly inhibited the proliferation of Kasumi-1 and K562 cell strains.Compared with the blank control group(PTFC 0 mg/mL),the cell survival rate was significantly reduced(P<0.05)in a dose-and time-dependent manner.The IC50(24 h and 48 h)for Kasumi-1 was 1.99 mg/mL and 0.89 mg/mL respectively,and the IC50 for K562(24 h and 48 h)was 1.23 mg/mL and 1.03 mg/mL respectively.Through AnnexinⅤ/PI flow cytometry detection,0.125-2.000 mg/mL PTFC induced apoptosis of Kasumi-1 and K562 cells at 24 h.The induced apoptosis rate of Kasumi-1 cell was 2.98%,3.20%,3.66%,8.60%,19.80%,respectively.The induced apoptosis rate of K562 cells was 3.19%,3.19%,3.49%,7.25%,and 16.87%,respectively.Hoechst 33258 staining showed typical apoptotic body occurred after treated by PTFC.Caspase-3,Caspase-9,and PARP were activated in Kasumi-1 and K562 cell strains,as compared with the blank control group(P<0.05).Conclusions PTFC significantly inhibited the proliferation of Kasumi-1 and K562 cell strains and induced their apoptosis.Its mechanism might be related to the activation of Caspase-3,Caspase-9,and PARP proteins.