山绿茶中RA对HepG2肿瘤细胞抑制作用的实验研究

Experimental Study of the Inhibiting Effects of Rotundioic Acid (RA) from Ilex Hainanensis on Tumor Cell HepG2

目的探讨中药山绿茶中乌苏烷三萜类化合物3β,19α-二羟基乌苏-12-烯-23,28-二羧酸(Rotundioic acid,RA)对肝癌细胞株HepG2细胞周期、凋亡的影响及其作用机制。方法人肝癌细胞株HepG2以RA(15,30,60,90mg.ml-1)作用不同的时间后,分别使用四甲基偶氮唑蓝比色法(MTT),吖啶橙(AO)荧光染色法,免疫细胞化学SP法,流式细胞术等实验方法检测HepG2细胞的增殖抑制情况、细胞周期和凋亡的形态学变化,以及细胞内半胱天冬酶-3(Caspase-3)、半胱天冬酶-9(Caspase-9)两个重要的细胞凋亡相关蛋白表达的变化。结果 RA对HepG2细胞有增殖抑制作用,其作用随着药物浓度和作用时间的增加而增强。RA作用24 h后HepG2细胞周期的主要特点是大量细胞累积于G1期,进入S期的细胞减少,RA能够诱导HepG2细胞凋亡,RA(30,60,90 mg.ml-1)经过24 h作用后,细胞凋亡率为8.19%,8.67%,9.81%,细胞内Caspase-3蛋白的表达随着药物浓度增加逐渐增加;Caspase-9的表达随着药物浓度增加无明显变化。结论 RA能够抑制HepG2细胞的增殖,并使细胞大量累积于G1期,进入S期的细胞减少;Caspase-3蛋白的表达增加来诱导细胞凋亡。

Objective To investigate the proliferation inhibition and apoptosis in human hepatic cancer cell strain HepG2 induced by Rotundioic acid（RA） and its mechanism.Methods HepG2 were treated by RA with different concentration and time.MTT assay,around totally orange（AO） fluorescence staining method,flow cytometry（FCM）,SP method of immunocyte chemistry were used to determine the inhibiting effect on HepG2 cells,the cell cycle and apoptosis morphological changes and the expressions of Caspase-3 and Caspase-9.Results RA inhibited the proliferation of HepG2 cells in dosage and time dependent manner.The HepG2 cells that were treated by RA for 24 hours.RA induced a G1 cell cycle arrest and reduced the cell population in S phase and the apoptosis rate were 8.19%,8.67%,9.81%,respectively.With the increasing of the medicine density,the expression of Caspase-3 was up-regulated,while the expression of Caspase-9 unchaned in the apoptosis.Conclusion RA can inhibit proliferation of the HepG2 cells and induce a G1 cell cycle arrest and reduce the cell population in S phase.It can also induce apoptosis of human prostatic carcinoma through up-regulating the expressions of Caspase-3.