摘要
目的:探讨针对fas的siRNA对大鼠原位肝移植供肝冷保存和缺血再灌注损伤的拮抗作用及机制。方法:(1)化学合成针对大鼠fas基因的3对siRNA,转染大鼠正常肝细胞(BRL细胞),筛选抑制效果最高的siRNA序列。(2)对SD大鼠用水压注射法转染fassiRNA,48 h后取肝,供肝冷保存2、4、6 h后行细胞凋亡指数及fas表达检测。(3)对照组、fassiRNA组各25对SD雄性大鼠,供肝冷保存3 h后行原位肝移植,再灌注后1、3、6、12和24 h每组各处死5只大鼠,查血谷丙转氨酶(ALT);取供肝查fasmRNA表达、Fas蛋白水平和细胞凋亡计数。结果:315位点的siRNA抑制BRL细胞fas基因效率最高。供肝冷保存实验中,fassiRNA组冷保存2、4、6 h的肝脏fas表达与细胞凋亡指数低于对照组(P<0.01)。大鼠肝移植复流后,fassiRNA组各检测点的fas基因表达、蛋白水平均明显低于、凋亡细胞少于、血清ALT低于对照组。结论:针对fas的siRNA对大鼠肝移植中的供肝冷保存和缺血再灌注损伤有一定的拮抗作用。
AIM： To investigate the silencing effect offas siRNA to alleviate ischemic -reperfusion （I/R） injury in liver transplantation. METHODS： Three pairs of 21 - nt synthesized fas siRNAs were transfected into BRL cells respectively for evaluation of silence efficacy, and the most effectivefas siRNA was chosen in vivo for experiment. In cold preservation experiment, siRNA was transfected in vivo by hydrodynamics method. After 48 h, livers offas siRNA group and control group were harvested and cold preserved, and cell apoptosis andfas expression was evaluated at 2, 4 and 6 h. Orthotopic liver transplantation was performed infas siRNA group and blank control group. At 1,3, 6, 12 and 24 h after transplantation, blood and liver samples were collected for evaluation of serum ALT levels, Fas protein and mRNA expression, and apoptosis by TUNEL staining. RESULTS： fas siRNA2, which began at nt 315, inhibitedfas gene expression much more than other siRNAs. As to cold preservation, apoptosis index （AI） and fas expression in fas siRNA group was lower than that in control group at each checked point （P 〈 0.01 ）. At 1,3, 6, 12, and 24 h after blood reperfusion of liver transplantation, the serum ALT level infas siRNA group was much less than that in control group. The cell apoptosis in fas siRNA group was substantially decreased, and the expressions offas mRNA and protein were dramatically reduced. CONCLUSION ： fas - mediated apoptosis plays an important role in I/R injury of rat liver transplantation. Silencingfas by siRNA holds therapeutic promise to limit I/R injury.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第2期315-319,共5页
Chinese Journal of Pathophysiology
基金
国家教育部博士点基金新教师资助项目(No.20070558267)
广东省科技计划资助项目(No.2007B031511009)
