维生素K3通过上调细胞Fas的表达诱导肝癌SMMC-7721细胞凋亡的研究

Apoptosis of SMMC-7721 cells induced by vitamin K3 via up-regulating Fas expression on the surface of cells

目的研究维生素K3（VK3）对人肝癌细胞株SMMC-7721凋亡的影响并探讨其凋亡机制。方法用人肝癌细胞株SMMC-7721进行传代培养。CCK-8法检测VK3对SMMC-7721细胞的抑制作用。使用Hoechst33342荧光染色法观察凋亡细胞核的形态。细胞周期（PI法）、细胞凋亡（Annexin V／PI法）和细胞膜表面Fas表达量使用流式细胞仪进行检测。Fas mRNA的检测采用逆转录RT-PCR法。细胞培养上清液的可溶性Fas配体（sFasL）水平采用ELISA法检测。结果不同浓度VK3（2、5、10、20、25μmol／L）作用SMMC-7721细胞48h后，细胞生长抑制率分别为33．8％，50．1％，63．9％，78．5％和84．7％；细胞周期研究发现G0／G1期细胞增加，S期细胞减少；细胞的早期凋亡率分别为18．75％，25．80％，38．80％，29．92％和26．18％；Hoechst33342荧光染色法发现凋亡细胞核呈致密浓染；在VK3浓度低于10μmol／L，SMMC-7721细胞膜表面Fas平均荧光强度和细胞内Fas mRNA表达及培养上清液sFasL水平均逐渐升高，而在高浓度组其结果均又下降。结论VK3能抑制SMMC-7721细胞增殖并诱导其发生凋亡，而Fas及sFasL的表达水平与VK3诱导SMMC-7721细胞的凋亡密切相关。

Objective To investigate vitamin K3 （VK3） effect on apoptosis of human liver cancer cells and its mechanism. Methods The SMMC-7721 cells were cultured in the experiment. The inhibitory effects of VK3 on SMMC-7721 cells were tested by CCK-8. Morphological evaluation of apoptosis was performed by Hoechst33342 staining. The distribution of cell cycle and apoptosis, and the expression of Fas were assayed by flow cytometry. Fas mRNA expression were detected by RT-PCR. And the concentration of soluble Fas（sFasL） in the culture supernatant were measured by ELISA. Results The inhibitory rates of VK3 （ at concentrations of 2,5,10,20,25 μmol/L for 48 h） on SMMC-7721 growth were 33. 8 % ,50. 1%, 63.9% ,78.5% and 84.7%, respectively. Compared with the control group, the number of cells in the G0/ G1 phase increased, while that of S phase decreased. The apoptotic cell rates were 18.75%, 25.80%, 38. 80% ,29.92% and 26. 18%, respectively. The apoptosis cells were strongly stained by Hoechst33342. On exposure to VK3 at the concentration of （2,5,10 μmol/L） after 48 h, the mean fluorescence intensity of Fas on cell surface and the expression of fas mRNA and the concentration of FasL in the culture supernatant in SMMC-7721 were increased, but they all decreased at the high concentration of VK3. Conclusion VK3 can inhibit the proliferation of SMMC-7721 cells and induce apoptosis via up-regulating expression of Fas and sFasL.