保肝蒙药Ⅰ号对人肝癌Huh-7细胞的作用及机制研究

Study on the role and mechanism of Hepatoprotective Mongolian Ⅰ on hepatocellular carcinoma Huh-7 cells

目的探讨保肝蒙药Ⅰ号对人肝癌Huh-7细胞增殖的抑制作用及机制,为临床应用与推广提供科学的理论及实验数据。方法体外培养人肝癌Huh-7细胞,实验分为四组:对照组(生理盐水)、保肝蒙药Ⅰ号组、5-氟尿嘧啶(5-Fu)组、保肝蒙药Ⅰ号联合5-Fu组。分别采用MTT法、流式细胞术和Q-PCR技术观察药物干涉后对肝癌细胞增殖的抑制作用,对肝癌细胞凋亡、细胞周期阻滞和相关基因表达的影响。结果 5-Fu组、保肝蒙药Ⅰ号(4 mg/m L)组和保肝蒙药Ⅰ号联合5-Fu组对Huh-7肝癌细胞增殖抑制率分别达到67.70%、73.67%和75.35%。保肝蒙药Ⅰ号组单独作用于Huh-7肝癌细胞以及保肝蒙药Ⅰ号联合5-Fu组联合作用,细胞的凋亡率均显示增加(P<0.05)。与对照组比较,保肝蒙药Ⅰ号组和5-Fu组分别作用于Huh-7肝癌细胞后显示细胞G_0/G_1期比率明显增加(P<0.05);然而,保肝蒙药Ⅰ号联合5-Fu组作用于Huh-7肝癌细胞后显示细胞S期比率明显增加(P<0.05)。保肝蒙药Ⅰ号组对凋亡相关基因Caspase-3、Caspase-8和Caspase-9的表达具有调控作用,能上调细胞色素C的表达。结论保肝蒙药Ⅰ号对人肝癌Huh-7细胞增殖有显著的抑制作用,其机制与细胞周期阻滞及影响肿瘤细胞凋亡相关基因的表达相关。研究成果将对阐明临床应用的保肝蒙药Ⅰ号治疗肝癌的机制及推广应用有重要意义。

Objective To explore the role and mechanism of Hepatoproteetive Mongolia I on hepatocellular carcinoma Huh-7 cells and to provide the scientific theory and experimental data for the clinical application of Hepatoproteetive Mongolia I . Methods Hepatocellular carcinoma Huh-7 cells were cultured in vitro, and the experiment was divided into four groups： control group （normal saline）, 5-Fluorouracil （5-Fu） group, Hepatoprotective Mongolia I group and Hepatoproteetive Mongolia I combined with 5-Fu group. Cell proliferation was measured by MTF in Huh-7 cells. Apoptosis and cell cycle were measured by flow cytometry. Gene expression was examined by real-time fluorescence PCR. Results The proliferation inhibition rate of 5-Fu group, Hepatoprotective Mongolia I （4 mg/mL） grqup and Hepatoprotective Mongolia I combined with 5-Fu group on the Huh-7 liver cancer cell was 67.70%, 73.67% and 75.35% respectively. Hepatoprotective Mongolia I group and Hepatoprotective Mongolia I combined with 5-Fu group acted on the Huh-7 cell showed that the apoptosis rate was significantly increased （P 〈 0.05）. Compared with control group, Hepatoprotective Mongolia I group and 5-Fu group acted on the count of Huh-7 cells in GdGl phase was significantly increased （P 〈 0.01）, while Hepatoprotective Mongolia I combined with 5-Fu group in S phase was significantly increased （P 〈 0.01）. Q- PCR data also showed that apoptosis genes, such as Caspase-3, Caspase＇8 and Caspase-9 had regulatory role upon Hepatoprotective Mongolia I group and expression of cytochrome C was increased. Conclusion Hepatoprotective Mongolia I shows significant anti-proliferative effects on the Huh-7 cells and its inhibition mechanism involves cell cycle arrest and related gene expression, which affects tumor cell apoptosis. The research results have important significance to the application and mechanism of liver cancer treated by Hepatoprotective Mongolia I in explanation for clinical application.