魚腥草注射液誘導(dǎo)人肝癌程序性死亡的機(jī)制研究
魚腥草注射液誘導(dǎo)人肝癌程序性死亡的機(jī)制研究
曾紅波
【摘要】目的 探究魚腥草注射液誘導(dǎo)人肝癌細(xì)胞程序性死亡和程序性死亡相關(guān)基因表達(dá)的p38MAPK信號(hào)轉(zhuǎn)導(dǎo)通路,闡明肝癌治療的分子機(jī)理。方法 將5、10、20mg/L魚腥草注射液分別培養(yǎng)人肝癌SMMC-7721細(xì)胞48h后,通過免疫組織染色觀察細(xì)胞程序性死亡的情況；通過瓊脂糖凝膠電泳分析和觀察程序性死亡細(xì)胞特征性DNA區(qū)帶；流式細(xì)胞儀法(Flow cytometry, FCM)檢測細(xì)胞程序性死亡和細(xì)胞周期；熒光定量PCR檢測Fas和Caspase-9基因mRNA的表達(dá)水平,并比較阻斷p38MAPK信號(hào)通路后魚腥草注射液對(duì)肝癌細(xì)胞凋亡和Fas和Caspase-9基因mRNA的表達(dá)。結(jié)果 魚腥草注射液作用48h后,熒光顯微鏡下觀察到經(jīng)Hoechst染色的典型程序性死亡細(xì)胞。瓊脂糖凝膠電泳可見程序性死亡細(xì)胞DNA呈規(guī)律性的梯狀條帶。5、10、20mg/L濃度魚腥草注射液作用人肝癌細(xì)胞后的細(xì)胞程序性死亡率分別為12.83%±1.51%,17.86%±2.70%和29.24%±7.58%, 與對(duì)照組6.30%±2.08%比較均有顯著性差異(P<0.01),阻斷p38MAPK信號(hào)通路后,程序性死亡率和G0/G1期細(xì)胞比例明顯降低(P<0.01)。10 mg/L魚腥草注射液作用人肝癌細(xì)胞48h后Fas mRNA和Caspase-9 mRNA的表達(dá)明顯上升,阻斷p38MAPK信號(hào)通路后,魚腥草注射液作用人肝癌細(xì)胞的FasmRNA和Caspase-3mRNA的表達(dá)明顯下降。結(jié)論 魚腥草注射液能誘導(dǎo)肝人癌細(xì)胞株SMMC-7721程序性死亡, 阻滯肝癌細(xì)胞于G0/G1期，通過P38MAPK信號(hào)轉(zhuǎn)導(dǎo)通路上調(diào)Fas、Caspase-9mRNA的表達(dá)可能是其誘導(dǎo)肝癌細(xì)胞程序性死亡的重要機(jī)制。
【關(guān)鍵詞】p38MAPK信號(hào)；肝癌；程序性死亡
The mechanism research of Houttuynia injection induced inhuman apoptosis of liver cancer, Zeng Hongbo, The surgery department of The NO.2 People’s Hospital，Tian Men，Hubei 431700 China
【Abstract】 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, procedural 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.
【Key words 】 p38MAPK signal; Live Cancer；Apoptosis