【摘要】 目的 观察抗小鼠 Caspase-12小干扰 RNA(siRNA)对小鼠原代肝细胞凋亡的抑制作用。方 法 原位二步灌流法分离并培养 Balb/c 小鼠原代肝细胞，化学合成制备三种抗 Caspase-12不同位点(130， 214，521)的 siRNA，脂质体包裹转染小鼠原代肝细胞。毒胡萝卜素4μmol/L 诱导肝细胞凋亡。逆转录聚 合酶链反应、western blot 检测抑制效果；四甲基偶氮唑盐法检测对细胞凋亡的影响。结果 在浓度为 100 nmol/L 时，siRNA(130)对小鼠原代肝细胞 Caspase-12 mRNA 的抑制率为0，siRNA(214)为52．08%， siRNA(521)为30．73%(t=4．30，P＜0．05)；在浓度为200 nmol/L 时，三种 siRNA 的抑制率分别为88．07%、 86．22%，89．41%。200 nmol/L 的 siRNA(214)对凋亡细胞 procaspase-12的抑制率为51．43%(t=4．30， P＜0．01)；MTT 比色实验发现，凋亡细胞活力增高48．76%(t=2．23，P＜0．01)。结论 抗 Caspase-12 siRNA 对小鼠原代肝细胞凋亡有一定的抑制作用。更多还原

【Abstract】 Objective To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs)against caspase-12.Methods The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type Ⅳ,and apoptosis were induced with 4 μmol/L thapsigargin(TG).The three kingds of siRNAs targeting different gene sites(130,214,521)were synthetized chemically.The single-stranded RNAs were annealed to produce double-stranded siRNAs,then the mouse primary hepatocytes were transfected by oligofectamine package.The inhibition of caspase-12 was analyzed with RT-PCR and western blot.The viabie hepatocytes following the induction of apoptosis were evaluated with MTT.Results All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA.The siRNA(214)were more effective than the other two when the concentration was 100 nmol/L.The caspase-12 mRNA expression was inhibited by 52.08%,while that of siRNA(521)was 30.73% (t=4.30,P＜0.05).However when the concentration was 200 nmol/L,the inhibitions were similar(88.07%,86.22% and 89.41% respectively),siRNA(214)could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43%(t=4.30,P＜0.01).Contrasted with apoptotic hepatocytes,the cell activity,which was analyzed with MTT, increased by 48.76%(t=2.23,P＜0.01).Conelusion siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.更多还原