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。
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 viable 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）. Conclusion siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2005年第12期923-926,共4页
Chinese Journal of Hepatology
基金
国家自然科学基金(30371268)上海市教委曙光计划基金(03SG37)
