羟基喜树碱对HSC-T6细胞增殖与凋亡的影响

目的 探讨羟基喜树碱(HCPT)对体外培养大鼠肝星状细胞(HSC)的增殖与凋亡的影响.方法 大鼠肝星状细胞(HSC-T6)和大鼠正常肝细胞(BRL-3A)分别在实验组(分别以含HCPT浓度为0.008、0.016、0.031、0.063、0.125,0.25、0.5、1、2、4、8、16、32mg/L的培养液培养)和对照组(单纯培养)体外培养24 h.用四甲基偶氮唑盐法检测细胞增殖情况,找出HCPT对HSC-T6细胞增殖抑制的最佳作用浓度;流式细胞仪检测细胞凋亡率;透射电子显微镜下观察细胞凋亡的形态学变化情况;琼脂糖凝胶电泳法检测DNA片段化.多个样本均数的比较采用单因素方差分析,两样本均数的比较采用t检验.结果 HCPT对HSC-T6细胞和BRL-3A细胞的增殖抑制率随着药物浓度的升高而逐渐升高;当HCPT浓度＞0.5mg/L时,对BRL-3A细胞的毒性作用显著增高(P值均<0.05);0.5 mg/L对HSC-T6细胞增殖抑制作用最大,为HCPT对HSC-T6细胞增殖的最佳抑制浓度.0.125、0.25、0.5 mg/L的HCPT作用HSC-T6细胞24h,流式细胞仪检测显示细胞凋亡率分别为13.46%±2.42%、26.25%±5.65%、47.05%±8.76%,与对照组(4.89%±1.80%)相比,差异有统计学意义(F=34.24,P<0.01).0.5mg/L的HCPT作用HSC-T6细胞24 h,透射电子显微镜下可见细胞体积缩小,核仁消失,染色质浓缩聚集成团块状,沿核膜排列等凋亡形态学改变;琼脂糖凝胶电泳可见明显的DNA梯度带形成.结论 HCPT在体外可以明显地抑制HSC-T6细胞的增殖、诱导HSC-T6细胞凋亡,作用强度呈剂量依赖性.

Effect of hydrocamptothecin(HCPT)on proliferation and apoptosis of rat hepatic stellate cells

Objective To investigate the effects of hydrocamptothecin(HCPT)on proliferation and apoptosis of rat hepatic stellate cells(HSC).Methods Rat HSC line(HSC-T6)and rat hepatocyte line (BRL-3A)were treated with different concentrations of HCPT(0,0.008,0.016,0.031,0.125,0.25,0.5,1,2,4,8,16,32 mg/L respectively)for 24 h.Cell proliferation was assessed by MTT colorimetric assay,apoptosis was detected with PI staing followed by flow cytometry,and by DNA ladder assay.The morphological change of apoptosis was observed under transmission electron microscopy(TEM).Results MTT assay indicated that HCPT significantly inhibited the proliferation of HSC-T6 and BRL-3A in a dose-dependent manner.24 h after the treatment with different concentrations of HCPT(0.25,0.5,1 mg/L),the apoptosis rate(13.46%±2.42%,26.25%±5.65%,47.05%±8.76%,respectively)in HSC-T6 was significantly higher than that in control cells(4.89%±1.80%,F=34.24,P < 0.01).24 h after 0.5 mg/L HCPT treatment,cell shrinkage,nucleoli disappearance,chromatin condensation were found under TEM,and DNA ladder was demonstrated by agarose gel electrophoresis.Conclusion HCPT could significantly inhibit proliferation and induce apoptosis of HSC-T6 in a dose-dependent manner.