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目的研究肌醇六磷酸(IP6)对人肝癌细胞株HepG2细胞周期的影响并探讨其作用机制。方法以体外培养人肝癌细胞株HepG2为研究对象,应用流式细胞仪检测不同浓度IP6作用24h后对HepG2周期的影响;以免疫细胞化学法检测IP6对细胞周期相关蛋白cyclinD1、Rb、P27表达的影响;RT-PCR法检测IP6对HepG2细胞cyclinD1、CDK4 mRNA表达的影响。结果经IP6作用处理的HepG2细胞的细胞周期发生G1期阻滞。免疫细胞化学结果显示:与对照组比,各IP6浓度组均能抑制cyclinD1蛋白的表达(F=225.02,q=15.20-25.35,P<0.05),上调Rb(F=63.31,q=2.77-13.06,P<0.05)、P27蛋白的表达(F=254.75,q=4.71-25.71,P<0.05);RT-PCR结果显示,与对照组相比,各IP6浓度组均能抑制cyclinD1(F=672.34,q=16.41-41.99,P<0.05)和CDK4 mRNA(F=108.35,q=5.32-16.27,P<0.05)的表达。结论 IP6对HepG2细胞生长具有明显的抑制作用。其机制可能是IP6降低cyclinD1、CDK4水平,上调Rb、P27蛋白的水平而作用于G1-S限制点,使HepG2细胞周期发生G1期阻滞,从而起到抑制细胞增殖的作用。
Objective To study the effect of phytic acid (IP6) on the cell cycle of human hepatocellular carcinoma cell line HepG2 and to explore its mechanism. Methods Human hepatocellular carcinoma cell line HepG2 was cultured in vitro. The effect of different concentrations of IP6 on the HepG2 cycle was detected by flow cytometry. The expression of cyclinD1, Rb and P27 was detected by immunocytochemistry The effect of IP6 on the expression of cyclinD1 and CDK4 mRNA in HepG2 cells was detected by RT-PCR. Results The cell cycle of HepG2 cells treated with IP6 showed G1 arrest. The results of immunocytochemistry showed that compared with the control group, the expression of cyclinD1 protein was inhibited in all the IP6 groups (F = 225.02, q = 15.20-25.35, P <0.05) and Rb increased (F = 63.31, q = (F = 254.75, q = 4.71-25.71, P <0.05). Compared with the control group, the expression of P27 protein in each IP6 group was lower than that in the control group (F = 672.34, P <0.05) , q = 16.41-41.99, P <0.05) and CDK4 mRNA (F = 108.35, q = 5.32-16.27, P <0.05). Conclusion IP6 has a significant inhibitory effect on the growth of HepG2 cells. The mechanism may be that IP6 can reduce the level of cyclinD1 and CDK4, upregulate the levels of Rb and P27 and act on the G1-S restriction point to block G1 phase of HepG2 cell cycle, thereby inhibiting cell proliferation.