羟基喜树碱对大鼠肝星状细胞增殖抑制的最佳浓度

Optimum concentration of hydrocamptothecin for inhibiting proliferation of rat hepatic stellate cells

目的:探讨羟基喜树碱(HCPT)对体外培养的大鼠肝星状细胞(HSC)增殖的影响,并观察其对大鼠正常肝细胞的毒性作用,确定HCPT对大鼠肝星状细胞增殖抑制的最佳浓度.方法:大鼠肝星状细胞株(HSC-T6)和大鼠正常肝细胞株(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的培养液作用)及对照组(单纯培养)中体外培养24,48,72h.应用四甲基偶氮唑盐法(MTT法)检测细胞增殖情况,找出对HSC-T6细胞增殖抑制作用最大的HCPT最佳浓度;光镜下观察对照组和实验组HSC-T6细胞的形态学变化.结果:24,48,72h实验组HSC-T6细胞的增殖率均低于对照组,差异具有显著性(t=6.07-46.98,10.98-63.97,20.76-107.68,均P<0.01);HCPT对HSC-T6细胞和BRL-3A细胞的增殖抑制率均随着药物作用浓度的升高及药物作用时间的延长而逐渐升高;当HCPT浓度>0.5mg/L时,对BRL-3A细胞的毒性作用显著性增高(均P<0.05);0.25,0.5,1mg/L的HCPT作用HSC-T6细胞24h,光镜下均可见细胞数量减少、体积缩小、核浓缩等变化,且随着作用浓度的增加变化更加明显.结论:HCPT在体外可以明显的抑制HSC-T6细胞的增殖,作用强度呈剂量-时间依赖性;0.5mg/L为对HSC-T6细胞增殖抑制作用最大的HCPT最佳浓度.

AIM： To evaluate the effects of hydrocamptothecin （HCPT） on proliferation of rat hepatic stellate cells （HSC） in vitro, and meanwhile, to investigate the cytotoxicity of HCPT on rat hepatocytes, then to determine the optimum concentration of HCPT for inhibiting proliferation of rat HSC.METHODS： Rat HSC line （HSC-T6） and rat hepatocyte line （BRL-3A） were incubated with different concentrations of HCPT （0.008, 0.016, 0.031, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 mg/L respectively） or without HCPT for 24, 48, 72hours, respectively. The proliferation rates of the HSC-T6 and BRL-3A were determined by MTT assay. The optimum concentration of HCPT with highest inhibitory action on proliferation of HSC-T6 was examined. The morphological changes of the HSC-T6 were observed with light microscopy.RESULTS： MTT assay indicated that the pro- liferation rates of HSC-T6 induced with different concentrations of HCPT were significantly reduced compared with the control group incubated in vitro for 24, 48, 72 h （t = 6.07-46.98, 10.98-63.97, 20.76-107.68, all P 〈 0.01）. HCPT significantly inhibited the proliferation of HSC-T6 in a dose-dependent and time-dependent manner. On the other hand, HCPT also inhibited the proliferation of BRL-3A in a dose-dependent and time-dependent manner. When the concentration was higher than 0.5 mg/L, the cytotoxicity of HCPT on the BRL-3A had a significant raise （all P 〈 0.05）. After stimulation of HSC-T6 by HCPT with the concentrations of 0.25, 0.5, 1 mg/L for 24 hours, light microscopy showed the morphological changes of HSC-T6, including cells shrinkage, karyorrhexis and karyopycnosis, and the changes were more significant as the concentration increased.CONCLUSION： HCPT could significantly inhibit the proliferation of HSC-T6 in a dose-dependent and time-dependent manner; 0.5 mg/L is the optimum concentration of HCPT for inhibiting the proliferation of HSC-T6.