异补骨脂素对L02细胞增殖、周期和凋亡的影响

Effects of isopsoralen on proliferation, cell cycle and apoptosis in L02 cells

目的研究异补骨脂素对人正常肝细胞(L02)增殖、细胞周期和凋亡的影响,并探讨其相关作用机制。方法以异补骨脂素为研究对象,L02细胞为体外模型,采用MTT试验检测不同浓度异补骨脂素(5、10、20、40、80和100μg/ml)对L02细胞增殖的影响,流式细胞仪检测不同浓度异补骨脂素体外作用24 h对L02细胞周期分布和凋亡率的影响,通过蛋白印迹法(Western blot)检测细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白E1(Cyclin E1)、细胞周期素依赖性激酶2(CDK2)、细胞周期素依赖性激酶4(CDK4)、B淋巴细胞瘤-2基因(B-cell lymphoma 2,Bcl-2)和Bcl-2相关X蛋白(Bcl-2-like protein4/Bcl-2-associated X protein,Bax)的表达,探讨异补骨脂素毒性的作用机制。结果 MTT试验表明,异补骨脂素对L02细胞生长有明显抑制作用,且抑制作用呈现时间-剂量依赖性;流式细胞仪检测结果显示,各组细胞经异补骨脂素(20、40和80μg/ml)作用24 h后,细胞总凋亡率分别为5.59%,7.8%和20.91%(P<0.001),40和80μg/ml异补骨脂素组L02细胞处于G1期的细胞数增多,S期的细胞减少,说明异补骨脂素能将L02细胞的细胞周期阻滞在G1期;Western blot蛋白质印迹结果显示,同对照组相比,不同浓度异补骨脂素处理L02细胞24 h后,Cyclin D1蛋白表达水平明显下降(P<0.05),而Bax蛋白表达水平明显升高,差异有统计学意义(P<0.05)。结论异补骨脂素可能通过调控周期蛋白及细胞凋亡相关蛋白抑制L02细胞增殖,并诱导细胞凋亡。

Objective To investigate the effects of isopsoralen on the proliferation, cell cycle and apoptosis in human normal hepatocytes cells(L02) and explore its mechanism. Methods With isopsoralen as the research object and L02 cells as the model in vitro, the effect of different concentrations of isopsoralen on the proliferation of L02 cells was evaluated by MTT. The effect of the drugs on cell cycle was detected by PI/RNase staining, the apoptosis after different concentrations of drug treatment was detected by Annexin V:PE/7-AAD double staining, and flow cytometry was used to analyze the cell cycle and apoptosis finally. The expressions of cell cycle and apoptosis-related proteins CDK2, CDK4, Cyclin D1, Cyclin E1, Bcl-2 and Bax were measured by Western blot. Results MTT result showed that the inhibition effect of isopsoralen was in time-and dose-dependent manner on the growth of L02 cells, the flow cytometry demonstrated that isopsoralen increased the apoptotic rate of L02 cells in 80 μg/ml. In the administration group, the number of L02 cells in G1 phase increased and the number of cells in S phase decreased, suggesting that isopsoralen could arrest the cell cycle of L02 cells in G1 phase(P<0.01 or P<0.001). Western blot result showed that the expression of Cyclin D1 decreased and Bax increased at a certain concentration of isopsoralen. Conclusion These result suggest that isopsoralen could inhibit L02 cells proliferation and induce cell apoptosis via regulation the expression of cell cycle protein and cell apoptotic related protein.