吉九里香碱体外诱导HCT-15细胞凋亡的研究

Study on induction of apoptosis by girinimbine in HCT-15 cell in vitro

目的:探讨中药单体吉九里香碱是否通过诱导 HCT-15细胞凋亡而发挥其抑制肿瘤细胞增殖的作用。方法:采用 MTT法检测细胞增殖抑制作用;采用透射电子显微镜观察细胞凋亡的形态学变化;库尔特全自动颗粒粒度分析药物引起 HCT-15细胞体积大小分布的变化;琼脂糖凝胶电泳测定 DNA ladder 的发生;通过流式细胞仪应用 Annexin V-FITC 和 Rhodamine123检测磷脂酰丝氨酸(PS)及线粒体跨膜电位(△ψm)。结果:MTT 结果显示吉九里香碱以浓度和时间依赖方式抑制 HCT-15细胞的增殖,抑制率依赖于吉九里香碱的浓度和作用时间;50 μmol·L^(-1)吉九里香碱处理 HCT-15细胞24 h 时,细胞出现凋亡典型的形态学特征;50 μmol·L^(-1)吉九里香碱分别处理 HCT-15细胞6,12,24 h 及不同浓度吉九里香碱处理 HCT-15细胞24 h 时,能够使细胞产生明显的 DNA ladder 和体积大小变化,呈一定的量效和时效关系;细胞 PS 外翻随作用时间的延长而增加,分别为15.34%(6 h),20.20%(12 h),39.37%(24 h);50 μmol·L^(-1)吉九里香碱处理 HCT-15细胞导致线粒体△ψm以时间依赖方式下降。结论:吉九里香碱通过诱导 HCT-15细胞凋亡而发挥其抗 HCT-15细胞增殖的作用,致凋亡机理可能与线粒体△ψm快速下降有关。

Objective：To investigate if girinimbine inhibits the proliferation of HCT - 15 by inducing its apoptosis. Methods：Cell proliferation inhibition was assessed by MTF assay;Morphological changes of apoptosis were observed with invert fluorescence microscope;The distribution of the cell volume sizes was assessed by Coulter Muhisizer 11 analytical instrument; DNA ladder was examined by DNA agarose gel electrophoresis; Phospholipid phosphatidyl- serine （PS） and mitochondrial transmembrane potential（ A ψm） were measured with Annexin V - FITC and fluo- rescent probe Rh123 ,respectively. Results ：MTF assay showed that girinimbine inhibited the proliferation of HCT- 15 cells in a time - and concentration - dependent manner. HCT - 15 cells treated with 50 μmol·L^-1 girinimbine for 24 hours showed typical morphological changes of apoptotic phase. After treated with 50 μmol · L^-1 girinimbine for 6,12 and 24 hours or with girinimbine in different concentrations for 24 hours, respectively, there were obvious changes of DNA ladder and the distribution of the cell volume size. The results obtained with Annexin V - FITC kit showed that the percentage of the apoptotic cells was 15.34% （6 h） ,20. 20% （ 12 h） ,and 39. 37% （24 h）. The treatment with 50μmol· L^-1girinimbine resulted in a rapid decrease of △ψm in a time -dependent manner. Conclusion ：These results suggest that girinimbine shows its anticancer activity by inducing apoptosis of HCT - 15 cells, and its apoptosis inducing mechanism may involve the rapid decrease of Aψm.