苦碟子对恶性黑色素瘤细胞抑制及凋亡蛋白Bcl-2/Bax表达的影响

目的观察不同浓度中药苦碟子对恶性B16黑色素瘤细胞的抑制作用和凋亡蛋白Bcl-2/Bax表达的变化,探讨中药苦碟子抑制B16黑色素瘤细胞生长的可能机制。方法通过细胞培养和MTT实验,检测苦碟子对B16黑色素瘤细胞的生长抑制情况,Western blot测定凋亡蛋白Bcl-2/Bax表达,免疫荧光染色检测Bcl-2/Bax蛋白在不同浓度苦碟子处理的黑色素瘤细胞中的表达。透射电镜观察不同浓度梯度中药苦碟子诱导细胞凋亡,其细胞结构的特征性变化。结果随着苦碟子药物浓度的增加,黑色素瘤细胞活细胞数目逐渐减少,细胞存活率显著下降(<0.05),凋亡抑制蛋白Bcl-2表达水平呈现下降趋势,而促凋亡蛋白Bax呈现上升趋势(<0.05),免疫荧光染色显示Bcl-2表达逐渐减少,Bax表达逐渐增加;电镜下凋亡小体数目不断增加,核固缩逐渐明显,细胞间隙增大,线粒体数目减少。结论中药苦碟子有浓度依赖性抑制黑色素瘤细胞生长,促进细胞凋亡,凋亡蛋白Bcl-2/Bax变化呈线性关系。

Effect of Chinese drug kudiezi on the expression of Bcl-2/Bax protein and apoptosis of B16 malignant melanoma

Objective To study the effect of kudiezi on the expression of Bcl-2/Bax protein and apoptosis of B16 malignant melanoma to explore the possible mechanism that kudiezi inhibits the growth of B16 melanoma cell.Methods B16 melanoma cells were cultured and MTT was used to determine the growth of the cells,Western blot was performed to determine the apoptosis Bcl-2/Bax,immunofluorescence staining was performed to test the expression of Bcl-2/Bax protein in melanoma cells after treatment with various concentrations of kudiezi.The morphological characteristics of melanoma cell apoptosis were observed by transmission electron microscopy.Results Kudiezi decreased the number of B16 melanoma living cells and the cell survival rate significantly(<0.05),downregulated the expression level of anti-apoptotic protein Bcl-2,upregulated the expression level of pro-apoptotic protein Bax,increased the number of apoptotic bodies and evident karyopyknosis,decreased the number of mitochondria with dose-dependent manner.Conclusion Kudiezi inhibits malignant melanoma cell growth and increased apoptosis cells number with dose-dependent manner,apoptosis related protein Bcl-2/Bax showed a linear relationship.