马钱子对人肝癌细胞生长的影响及其作用机制

目的 探讨马钱子对人肝癌细胞生长影响及作用机制.方法 体外培养人肝癌细胞SMMC-7721,分别加入5％、10％、20％的马钱子药物血清,噻唑蓝(MTT)比色法测定对人肝癌细胞SMMC-7721的生长抑制作用.20％马钱子药物血清作用肝癌细胞SMMC-77210、24、48 h后,分别收集细胞,提取蛋白和总RNA,应用Westem blot和荧光定量聚合酶链反应(PCR)检测Fas、Cyclin D1、PCNA蛋白和mRNA表达.结果 20％马钱子药物血清对人肝癌细胞72 h的生长抑制率为(54.94±8.19)％,与5％、10％浓度抑制率(19.928±7.653)％、(29.020 ±6.100)％比较差异有统计学意义(F=18.23,P＜0.01).药物血清作用24、48 h后,肝癌细胞SMMC-7721 Fas蛋白表达分别为(0.302±0.009)、(0.399±0.021),与对照组(0.226±0.022)比较,差异有统计学意义(F=68.25,P＜0.05)；药物血清作用48 h后,肝癌细胞SMMC-7721 PCNA蛋白表达为(0.7457±0.0386),与对照组、24 h(0.8529±0.0099、0.9016±0.0139)比较,差异有统计学意义(P＜0.05)；药物血清作用24、48 h后,肝癌细胞SMMC-7721 Cyclin D1 mRNA表达分别为0.045 680 ±0.038 130、0.026 714±0.019934,与对照组(0.145246±0.048543)比较,差异有统计学意义(F=8.671,P＜0.05)；药物血清作用24、48 h后,肝癌细胞SMMC-7721 Cyclin D1蛋白表达,PCNA、Fas mRNA表达变化差异无统计学意义(F =68.25,P＞0.05).结论 马钱子具有抑制人肝癌细胞增殖作用,其抑制效应与药物血清浓度成正相关；通过上调肝癌细胞Fas蛋白和下调PCNA蛋白及Cyclin D1 mRNA表达,是马钱子抑制肝癌细胞增殖的机制之一.

Effect of Nux Vomica on growth of hepatocellular carcinoma cells and action mechanism

Objective To investigate the inhibitory effects of Nux Vomica on human hepatocellular carcinoma (HCC) cells and the action mechanism.Methods Human HCC SMMC-7721 cells were cultured and the serum from Nux Vomica at the concentrations of 5％,10％,20％ was added into cell culture fluid respectively.The inhibitory effect of Nux vomica on SMMC-7721 cells was measured by using methyl thiazol tetrazolium (MTT) assay.After treatment of SMMC-7721 cells with the serum from 20％ Nux Vomica for 0,24 and 48 h,the cells were collected,and the total protein and RNA were extracted.The protein and mRNA expression of Fas,Cyclin D1 and PCNA was detected by using Western blotting and fluorescence quantitative polymerase chain reaction (PCR) method.Results The proliferation of SMMC-7721 cells was obviously inhibited by the serum from Nux Vomica in a concentration-dependent manner.The inhibition ratio was (54.94 ±8.19)％ with the serum from 20％ Nux Vomica and there was statistically significant difference in comparison with the serum from 5％ and 10％ Nux Vomica [ ( 19.928 ±7.653)％,(29.020 ± 6.100) ％ ] ( F =18.23,P ＜ 0.01 ).The relative expression of Fas protein was respectively (0.302 ±0.009) and (0.399 ±0.021 ) at 24 and 48 h respectively,which was significantly higher than in control group ( 0.226 ± 0.022) ( F =68.25,P ＜ 0.05 ).The relative expression of PCNA protein was 0.7457 ±0.0386 at 48 h,which was significantly lower than in control group and 24 h group (0.8529 ±0.0099,0.9016 ±0.0139) ( P ＜ 0.05 ).The Cyclin D1 mRNA expression was 0.045680 ± 0.038130 and 0.026714 ± 0.019934 at 24 and 48 h respectively,which was significantly difference from that in control group (0.145 246±0.048 543,F=8.671,P ＜0.05).There was no significant difference in the cyclinD1 protein expression ( F =68.25,P ＞ 0.05 ).Conclusion The proliferation of HCC cells can be inhibited with Nux Vomica in a dose-dependent manner.The high expression of Fas,low expression of PCNA as well as low expression of Cyclin D1 mRNA in HCC cells is closely correlated to the inhibitive effect on tumor cell proliferation.