沙利度胺对人肝癌细胞株SMMC-7721体外生长的抑制作用

Inhibitory effect of thalidomide on growth of human hepatoma cell line SMMC-7721 cells

目的研究沙利度胺对人肝癌细胞株SMMC-7721体外生长的抑制作用及其可能的机制。方法将不同浓度的沙利度胺作用于人肝癌细胞株SMMC-7721，采用四甲基偶氮唑蓝（MTr）法检测沙利度胺对SMMC-7721细胞的增殖抑制作用。将SMMC07721细胞培养至对数生长期，采用DNA琼脂糖凝胶电泳、荧光显微镜观察、流式细胞仪检测等方法观察沙利度胺处理后SMMC-7721细胞的凋亡梯度、形态学变化和凋亡率，并对凋亡调控蛋白caspase-3的表达进行测定。采用酶联免疫吸附（ELISA）法测定不同浓度的沙利度胺处理后SMMC-7721细胞表达血管内皮生长因子（VEGF）的变化。结果沙利度胺的浓度从3．125μg／ml增至200μg／ml时，其对SMMC-7721细胞的增殖抑制率从11．7％增至34．2％；当沙利度胺的浓度〉25μg／ml时，其对SMMC07721细胞的增殖抑制作用明显强于空白对照组（P〈0．05）。200μg／ml的沙利度胺处理SMMC-7721细胞24h后，行琼脂糖凝胶电泳，可见到DNA梯形条带；48h后梯形条带更明显，并且在荧光显微镜下可见SMMC-7721细胞出现核固缩和核裂解现象。200μg／ml的沙利度胺处理SMMC-7721细胞12、24、48和72h时，碘化丙啶（e1）法检测SMMC-7721细胞的凋亡率分别为3．1％±0．5％、8．4％±1．3％、19．4％±3．5％和25．8％±2．1％，24h起的凋亡率均明显高于空白对照组SMMC-7721细胞48h的自然凋亡率（1．6％±0．6％，均P〈O．05）。50、100和200μg／ml的沙利度胺处理SMMC-7721细胞48h时，AnnexinV—FITC／PI双标法检测SMMC-7721细胞的凋亡率分别为8．7％±1．2％、16．8％±2．5％和25．4％±4．5％，均明显高于空白对照组SMMC-7721细胞48h的自然凋亡率（2．1％±0．5％，均P〈0．05）。随着沙利度胺浓度的增加，表达caspase-3蛋白的SMMC-7721细胞数量不断增加，而SMMC-7721细胞中VEGF的含量却逐渐下降。结论沙利度胺可能通过诱导肝癌细胞的凋亡、抑制肿瘤血管的生成而发挥双重抗肿瘤生长的作用。

Objective The aim of this study was to investigate the effect of thalidomide on the growth of human hepatoma cell line SMMC-7721 cells in vitro, and to explore the curative possibility of hepatocellular carcinoma with thalidomide. Methods SMMC-7721 cells were treated with Thalidomide at different concentrations. The cell growth and proliferation was assessed by MTT assay. DNA ladder, apoptosis rate and changes of cell nuclei were studied by agarose electrophresis, fluorescence microscopy and flow cytometry, respectively. The expression of caspase-3 was analyzed with flow cytometry. The VEGF content of SMMC-7721 ceils in culture medium was tested by ELSIA. Results When the concentration of Thalidomide solution was increased from 3. 125 μg/ml to 200 μg/ml, the cell growth was inhibited by from 11.7% to 34.2%. Compared with the control group, the thalidomide solution at a concentration of 25,50, 100 and 200 μg/ml solution significantly inhibited the proliferation of SMMC-7721 cells （ P 〈 0.05 ）. A ladder pattern of DNA fragments appeared after SMMC-7721 cells exposed to 200 μg/ml thalidomide for 24 h, especially for 48 h. Fluorescence microscopy revealed that the cell nuclei were condensed and fragmented after the cells were exposed to 200 μg/ml thalidomide for 48 h. In cells treated with 200 μg/ml thalidomide for 12,24,48 and 72 h,the apoptotic rate was 3.1% ＋0.5% ,8.4% ± 1.3% ,19.4% ±3.5% and 25.8% ±2.1%, respectively, significantly higher than that in the negative control group 1.6% ±0.6%. The cells treated with thalidomide at a concentration of 50, 100, 200 μg/ml for 48 h, the apoptotic rate was 8.7% ±1.2%, 16. 8% ±2.5% and 25.4% ±4.5%, respectively, increasing in a dose-dependent manner, also significantly than that in the cells of control group 2.1% ±0.5% , （all were P 〈0.05）. The caspase-3 positivity of SMMC-7721 cells treated with thalidomide was increasing along with the increase of treatment time or drug concentration, but not in the control cells. The VEGF content in SMMC-7721 cells was lowering when thalidomide was used in an increasing concentration. Conclusion Under the conditions used in this study, thalidomide can inhibit the proliferation of SMMC-7721 cells in vitro. Induction of apoptosis and inhibition of angiogenesis may be possibly two mechanisms for its anticancer action.