老瓜头生物总碱抑制小鼠黑色素瘤B16细胞增殖及促凋亡作用的研究

Inhibition of Proliferation and Promotion of Apoptosis of Mouse Melanoma B16 Cells by Total Alkaloid of Cynanchum Komarovii AI.Icjinski

目的研究老瓜头生物总碱对小鼠黑色素瘤B16细胞的增殖抑制与促进凋亡的作用。方法采用小鼠黑色素瘤B16细胞,MTT法检测老瓜头生物总碱对B16细胞增殖的抑制作用,通过琼脂糖凝胶电泳、Hoechst 33258染色、Caspase-3和Caspase-8活性检测,观察老瓜头生物总碱诱导细胞凋亡的作用和机制。结果 MTT检测显示老瓜头生物总碱抑制B16细胞的增殖,呈现时间依赖性和浓度依赖性,作用24、48、72h的IC50分别为0.97、0.98、0.16mg·L^(-1);选用浓度0.25、0.5、1mg·L^(-1)老瓜头生物总碱孵育B16细胞24h,经DNA Ladder检测出现阶梯状DNA Ladder,片段100~200bp,呈现细胞凋亡裂解;0.5、1mg·L^(-1)老瓜头生物总碱孵育B16细胞24h后Rhodamine 123染色与空白对照组相比显示荧光降低,且随浓度增加,荧光降低增加;凋亡蛋白Caspase-3、Caspase-8活性增强,0.5、1mg·L^(-1)老瓜头生物总碱药物组与对照组相比有显著性差异(P<0.05,P<0.01)。结论老瓜头生物总碱可抑制B16细胞增殖,诱导B16细胞凋亡,可通过增强凋亡蛋白Caspase-3和Caspase-8活性诱导凋亡。

Objective To study the inhibitory effect and promotion of apoptosis of melanoma B16 cells by total alkaloid of Cynanchum Komarovii AI.Icjinski.Methods B16 cells were exposed to total alkaloid of Cynanchum Komarovii AI.Icjinski treatment while growth inhibition of the cells was examined by MTT assay.The mechanism by which total alkaloid of Cynanchum Komarovii AI.Icjinski-induced apoptosis was detected by agarose gel electrophoresis,Hoechst 33258 staining,and by the activity of Caspase-3 and Caspase-8.Results Total alkaloid of cynanchum Komarovii AI.Icjinski inhibited the proliferation of B16 cells in a concentration-and time-dependent manner.The IC50 was 0.97,0.98,0.16 mg·L^-1 respectively when B16 cells were exposed to total alkaloid of Cynanchum Komarovii AI.Icjinski for 24,48 and 72 h.B16 cells were incubated for 24 hat the concentration of 0.25,0.5,1 mg·L^-1,and the DNA ladder was detected by DNA ladder detection kit.The fragments were 100-200 bp,suggesting apoptotic disapoptosis.The phosphorylation of Rhodamine 123 was lower than that of the blank control group.The activity of caspase-3 and caspase-8 increased with the concentration of total alkaloid of Cynanchum Komarovii AI.Icjinski,0.5,1 mg·L^-1 drug group was significantly different from the control group（P〈0.05,P〈0.01）.Conclusion Total alkaloid of Cynanchum Komarovii AI.Icjinski can inhibit proliferation of B16 cells and induce their apoptosis by enhancing caspase-3 and caspase-8 activity.