鱼腥草注射液诱导人肝癌程序性死亡的机制研究

The mechanism research of houttuynia injection induced inhuman apoptosis of liver cancer

目的探究鱼腥草注射液诱导人肝癌细胞程序性死亡和程序性死亡相关基因表达的p38MAPK信号转导通路，阐明肝癌治疗的分子机理。方法将5、10、20mg／L鱼腥草注射液分别培养人肝癌SMMC-7721细胞48h后，通过免疫组织染色观察细胞程序性死亡的情况；通过琼脂糖凝胶电泳分析和观察程序性死亡细胞特征性DNA区带；流式细胞仪法（Flow cytometry，FCM）检测细胞程序性死亡和细胞周期；荧光定量PCR检测Fas和Caspase-9基因mRNA的表达水平，并比较阻断p38MAPK信号通路后鱼腥草注射液对肝癌细胞凋亡和Fas和Caspase-9基因mRNA的表达。结果鱼腥草注射液作用48h后，荧光显微镜下观察到经Hoechst染色的典型程序性死亡细胞。琼脂糖凝胶电泳可见程序性死亡细胞DNA呈规律性的梯状条带。5、10、20mg／L浓度鱼腥草注射液作用人肝癌细胞后的细胞程序性死亡率分别为12．83％±1．51％，17．86％±2．70％和29．24％±7．58％，与对照组6．30％±2．08％比较均有显著性差异（P〈0．01），阻断p38MAPK信号通路后，程序性死亡率和G0／G1期细胞比例明显降低（P〈0．01）。10mg／L鱼腥草注射液作用人肝癌细胞48h后FasmRNA和Caspase-9mRNA的表达明显上升，阻断p38MAPK信号通路后，鱼腥草注射液作用人肝癌细胞的FasmRNA和Caspase-3mRNA的表达明显下降。结论鱼腥草注射液能诱导人肝癌细胞株SMMC-7721程序性死亡，阻滞肝癌细胞于G0/G1期，通过p38MAPK信号转导通路上调Fas、Caspase-9mRNA的表达可能是其诱导肝癌细胞程序性死亡的重要机制。

Objective To explore induced apoptosis of liver cancer induced by Houttuynia injection and the related gene expression of P38MAPK signal transduction pathway, clarifies the molecular mechanism of the treatment of liver cancer. Methods Forty eight hours after 5, 10 and 20 mg/L Houttuynia injection respectively to develop liver cancer SMMC-7721 cells , programmed cell death were observed by immunohistochemical staining; By agarose gel electrophoresis, analyzed and observed programmed cell death characteristic DNA zone; Flow cytometry instrument method（Flow cytometry, FCM）detected programmed cell death and cell cycle; Fluorescence quantitative PCR detected Fas and Caspase-9 gene mRNA expression level, and compared the blocking p38MAPK signaling pathway after Houttuynia injection on liver cancer cell apoptosis and the expression of Fas and Caspase-9 gene mRNA. Results Forty eight hours after Houttuynia injection, with fluorescence microscope Hoechst staining of typical programmed cell death was observed. DNA agarose gel electrophoresis was programmed cell death regularity of ladder shaped banding. 5, 10 and 20 mg/L concentration heartleaf houttuynia herb injection of human liver cancer cells after the programmed cell death rate of 12.83% plus or minus 1.51%, 17.86% plus or minus 2.70%, and 29.24% plus or minus 7.58%, compared with the control group 6.30% plus or minus 2.08% had significant difference （P 〈 0.01）, after blocking p38MAPK signaling pathways, pr^edural mortality and G0/G1 phase cell percentage significantly lower （P〈0.01）. 10 mg/L heartleaf houttuynia herb injection effect of human liver cancer cells after 48 h Fas mRNA and Caspase-9 mRNA expression of the obvious rise, blocking p38MAPK signaling pathway, heartleaf houttuynia herb injection effect of human liver cancer cells FasmRNA and Caspase 3 mRNA expression decreased obviously. Conclusion Houttuynia injection can induce liver cancer cell apoptosis of SMMC-7721, and block liver cancer cells in G0/G1 phase, through P38MAPK signal transduction pathways to raise Fas, Caspase-9 mrna expression may be its important mechanism to induction of programmed cell death of liver cancer.