红花多糖对人肝癌SMMC-7721细胞Bcl-2与Bax基因转录及蛋白表达的影响

Effects of Safflower Polysaccharide on Gene Transcription and Protein Express on of Bcl-2 and Bax in Human Hepatocarcinoma Cell Line SMMC-7721

目的:研究红花多糖(SPS)体外抑制人肝癌细胞株SMMC-7721增殖、诱导凋亡及对凋亡调控基因Bax,Bcl-2基因转录和蛋白表达的影响,探讨SPS诱导SMMC-7721细胞凋亡的机制。方法:不同剂量的SPS(0,0.02,0.04,0.08,0.16,0.32,0.64,1.28 g.L-1)分别作用于体外培养的SMMC-7721细胞,采用四甲基偶氮唑盐比色法(MTT)检测细胞的抑制率;用Ca2+依赖性磷脂结合蛋白(AnnexinⅤ)-异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染法观察细胞凋亡的形态学改变;通过罗丹明123(Rho123)荧光显微镜检测线粒体膜电位(Δψm);以实时荧光定量PCR技术(real-time PCR)和蛋白免疫印迹(Westernblotting)法检测细胞凋亡相关因子Bcl-2及Bax mRNA水平和蛋白水平的表达情况。结果:SPS对体外培养的人肝癌SMMC-7721细胞具有抑制增殖作用,且具有明显的时间和剂量相关性;荧光显微镜下SPS作用的细胞呈现典型的凋亡细胞形态;肝癌细胞内Rho123荧光强度明显减弱。SPS作用后细胞内Bcl-2蛋白和mRNA表达水平均降低,Bax蛋白和mRNA的表达水平均升高,二者的变化趋势均呈一定的时间依赖关系;而且,Bcl-2/Bax比值随SPS时间的延长显著降低且变化趋势均呈一定的时间依赖关系。结论:SPS能够显著抑制人肝癌细胞SMMC-7721增殖,诱导SMMC-7721细胞凋亡,其作用机制可能与上调Bax的表达及下调Bcl-2的表达和降低线粒体膜电位有关。

Objective：To explore how safflower polysaccharide（SPS） induce apoptosis of human liver cancer cell line SMMC-7721 through observing the influence of SPS on proliferation,apoptosis and expression of apoptosis gene and protein Bax and Bcl-2 of SMMC-7721.Method： SMMC-7721 cells were treated with different concentrations（0,0.02,0.04,0.08,0.16,0.32,0.64,1.28 g·L-1） of SPS.The cells growth inhibitory rate was measured by methyl thiazolyl tetrazolium（MTT） assay.The morphological changes of apoptosis were observed by AnnexinⅤ-FITC/PI staining.The mitochondrial membrane potential（Δψm） was assayed by fluorescent microscopy using Rhodamine 123（Rho123）.Quantitative real-time RT-PCR and Western blot analysis were used to detect the mRNA and protein expressions of Bcl-2 and Bax.Result： SPS could inhibit the proliferation of SMMC-7721 cells in a dose and time dependent manner.After treated with SPS,SMMC-7721 cells presented typical apoptosis under fluorescence microscope.Fluorescence intensity of Rho123 were significantly decreased in SMMC-7721 cells.The mRNA and protein expression of Bcl-2 expression were down-regulated while those of Bax were up-regulated and both changes had good time-dependent tendency.The ratio of Bcl-2/Bax decreased significantly in a time-dependent manner.Conclusion： SPS could inhibit the proliferation of SMMC-7721 and enhance apoptosis of SMMC-7721 and its mechanism maybe relate with its up-regulation of Bax expression as well as down-regulation of Bcl-2 expression and decline of Δψm.