体内RNA干扰抑制盘状受体1对急性肝损伤枯否细胞功能的影响

Biological changes of Kupffer cells in response to suppression of discoidin domain receptor 1 by in vivo delivery of small interfering RNA following acute hepatic injury

目的观察体内注射小干扰RNA（siRNA）技术抑制盘状结构域受体1（DDRl）在急性肝损伤时对枯否细胞（KC）功能的影响。方法雄性BALB／c小鼠随机分为对照组、肝损伤模型组、Non-silencing siRNA和DDRlsiRNA处理组。采用静脉注射刀豆蛋白A（ConA）15mg／kg诱发小鼠特异性肝损伤模型，按分组情况给予或不给予尾静脉高压注射裸siRNA（50μg，2．0～2．5mg／kg）或等渗盐水。Western印迹法分析DDR1蛋白表达变化、ELISA法检测肝内促炎性细胞因子的表达，同时检测血浆丙氨酸氨基转移酶（ALT）活性和KC的吞噬功能。结果肝脏DDR1蛋白在肝损伤早期6h才开始明显增高，24h达到高峰，48h后又逐渐减少。提前给予DDR1siRNA后明显抑制DDR1蛋白表达，同时抑制了KC吞噬指数、ALT活性的升高和促炎性细胞因子TNF—α、IL-1β的表达。结论采用体内siRNA技术抑制DDR1在肝脏中的表达，可以明显减轻ConA诱发的小鼠特异性肝损伤，其机制可能是通过抑制KC活化减少了炎性细胞因子的表达，从而减轻了后期肝免疫性损伤。

Objective To investigate the effect of small interfering RNA （siRNA） suppressing discoidin domain receptor 1 （ DDR1 ） gene on biological behaviour of Kupffer cells （KC） in acute hepatic injury. Methods Male BALB/c mice were randomly divided into control, hepatic injury model, nonsilencing siRNA and DDR1 siRNA groups. Hepatic injury model induced by intravenous injection of Con- canavalinA （ConA） 15 mg/kg, with or without hydrodynamic tail intravenous injection of naked siRNA （50μg,2.0-2.5 mg/kg）/mouse or 1.5 ml normal saline. The expression of DDR1 was assayed by Western blot and pro-inflammatory cytokine expression analyzed by ELISA. In the meantime, alanine amin- otransferase （ALT） and Kupffer cells＇elearance to carbon granules was detected. Results The expression of DDR1 obviously increased at 6 h after hepatic injury, reached peak at 24 h and began to decrease at 48 h. Pretreatment with DDR1 siRNA could obviously inhibit the expression of DDR1 and abrogate the high levels of ALT, expressions of TNF-α and IL-1β as well as phagocytosis of Kupffer cells. Conclusions Inhibition of discoidin domain receptor 1 by in vivo delivery of siRNA attenuates ConA-induced hepatic injury. Possible mechanism is that the inhibition of activity of KC inhibits the expression of pro-inflammatory cytokines and thus alleviates hepatic injury.