PIAS1基因沉默对胰腺腺泡细胞炎症反应的影响

Effect of PIAS1 gene silencing on inflammatory response of pancreatic acinar cell

目的 观察活化信号转导和转录激活因子的蛋白抑制因子-1（PIAS1）基因特异性siRNA干扰大鼠胰腺腺泡细胞株AR42J后对雨蛙素诱导炎症反应的影响,探讨其在胰腺炎发病中的作用.方法 采用脂质体法将靶向PIAS1的siRNA和阴性siRNA转染AR42J细胞,24 h后分别加入雨蛙素继续培养24 h.同时设脂质体＋雨蛙素组、雨蛙素组及仅加PBS的对照组.Western blotting检测p38丝裂原激活蛋白激酶（p38MAPK）及磷酸化p38MAPK（P-p38MAPK）表达;RT-PCR及Western blotting检测各组细胞肿瘤坏死因子α（TNF-α）、白介素1β（IL-1β）、IL-6、基质金属蛋白酶9（MMP-9）的mRNA和蛋白表达.结果 siRNA＋雨蛙素组、阴性siRNA＋雨蛙素组、脂质体＋雨蛙素组、雨蛙素组及对照组细胞p38MAPK表达量分别为1.93±0.11、1.22±0.10、1.30±0.17、1.32±0.21、0.12±0.02;P-p38MAPK表达量分别为2.10±0.25、1.36±0.20、1.26±0.15、1.23±0.25、0.58±0.48,siRNA＋雨蛙素组较其余各组明显增加（P值均＜0.05）.siRNA＋雨蛙素组细胞TNF-α、IL-1β、IL-6、MMP-9 mRNA表达量分别为1.66±0.15、1.66±0.15、1.90±0.01、1.56±0.20;蛋白的表达量分别为2.06±0.37、2.20±0.34、1.80±0.10、1.17±0.05,均较其他雨蛙素处理组表达上调（P值均＜0.05）.结论 PIAS1参与雨蛙素诱导的胰腺腺泡细胞p38MAPK活性与下游炎症介质的表达调控.

Objective To investigate the effect of protein inhibitor of activated signal transducer and activator of transcription 1 （ PIAS1 ） gene silencing on the inflammatory response of rat pancreatic acinar cell lines AR42J with cerulein stimulation, to study its role in the pathogenesis of acute pancreatitis.Methods The siRNA targeting PIASI was designed, synthesized, transfected into AR42J cells by lipofectmine 2000.24 h later, cerulean was added and cultured for another 24 h.Subsequent AR42J cells with cerulein stimulation were divided into 4 groups： cerulein, liposome, negative-siRNA and PIAS1-siRNA groups.In addition, a group with PBS was as control group.The activity of p38 mitogen- activated protein kinase （p38MAPK） was detected by western blotting.TNF-α, IL-1β, IL-6, matrix metalloproteinase （MMP） 9 expression were analyzed by RT-PCR and western blotting, respectively.Results The expression of p38MAPK in PIAS1-siRNA, negative-siRNA, liposome, cerulein,and control group was 1.93 ±0.11, 1.22 ±0.10, 1.30 ±0.17,1.32 ± 0.21, 0.12 ± 0.02;while the expression of phosphorylated p38MAPK was 2.10 ± 0.25, 1.36 ± 0.20,1.26 ±0.15, 1.23 ±0.25, 0.58 ±0.48, the expression in PIAS1-siRNA group was significantly increased when compared with other groups （P〈0.05）.The levels of TNF-α, IL-1β, IL-6, MMP-9 mRNA were 1.66 ±0.15,1.66 ± 0.15,1.90 ±0.01, 1.56 ±0.20 in PIAS1-siRNA group, while the expression of protein was 2.06 ±0.37,2.20 ±0.34, 1.80 ±0.10, 1.17 ±0.05, which was markedly higher than those in other group （P 〈0.05）.Conclusions PIAS1 gene silencing could enhance p38MAPK activity, and improve inflammatory mediator expression in pancreatic acinar cells with cerulein stimulation.