DDR2RNA干扰对CCl4诱导肝纤维化动物模型的实验研究

Effects of DDR2 siRNA on carbon tetrachloride-induced liver fibrosis in rats

目的探讨沉默大鼠盘状结构域受体2（discoidin domain receptor2，DDR2）基因表达对CCl4诱导大鼠肝纤维化的影响及其机制。方法SD大鼠随机分为正常组（8）、纤维化组（18）、阴性对照组（18）和治疗组（18），每组又据干预时间不同平均分为4周、6周两组。化学合成经胆固醇修饰的siRNA-DDR。尾静脉体内注射，干扰CCl4诱导大鼠纤维化模型DDR2基因表达。用荧光实时定量PCR及Westernblot分别检测干扰DDR2基因后DDR2、MMP-2和Ⅰ型胶原纤维（COL Ⅰ）的表达；同时行肝脏组织学观察及肝功能检测。结果经siRNA-DDR2干扰后，纤维化组大鼠DDRz、MMP-2和COLI的mRNA水平（t4=6．78，t6=9．02；t4=4．71，t6=6．37；t4=．81，t6=6．50，均P〈0．01）及蛋白表达水平（t=6．11，t=4．39，t=5．23，均P〈0．01，4周；t=7．82，t=4．80，t=7．64，均P〈0．01，6周）均显著降低；同时，肝脏的组织学病变及肝功能指标也显著改变。结论尾静脉注射化学合成经胆固醇修饰的抗DDR2 siRNA能显著降低DDR：基因表达，促进细胞外基质降解，具有潜在的抗肝纤维化作用。

Objective To explore the effects of silencing DDRz expression by siRNA on CCl4- induced liver fibrosis and its mechanism in rats. Methods Liver fibrosis model was induced by intraperitoneal injection of CCl4 twice a week for 6 consecutive weeks. Some rats were administered siRNA targeting DDR2 （0.3 mg/kg）, saline or control siRNA every three days from the beginning of CCl4 injection via tail vein injection, while other rats were treated in the same pattern after 2-week CC14 injection. Quantitative real-time polymerase chain reaction （QRT-PCR） and Western blot were used to detect the mRNA and protein expressions of DDR2, MMP-2 and COL Ⅰ. Meanwhile, the pathological changes of liver tissues and the levels of liver function were also observed. Results QRT-PCR showed that the DDR2, MMP-2 and COL I mRNA in the chemically synthetic cholesterol-modified siRNA- DDR2 group were significantly decreased as compared with those in the control group （P〈0. 01） ,and the protein expressions of DDR2, MMP-2 and COL Ⅰ were also significantly decreased （P〈0.01,4 w and 6w）. In addition, in comparison with those in the control group, the pathological changes of liver tissues in the siRNA-DDR2 treated group were markedly attenuated,and the levels of ALT（1356.17±83.80 nkat/L vs 2532.70±145.11 nkat/L,4w,1367.60±321.76 nkat/L vs 2604.37±255.02 nkat/L, 6w,P〈0.01 ） and AST（2460. 80±207. 58 nkat/L vs 3983. 70 ± 253. 08 nkat/L, 4w, P〈0. 01, 2383.27±290.16 nkat/L vs 3227.70±353.34 nkat/L,6w,P〈0. 05）were also significantly lowered, while the level of TBIL （7.97±1.60 μmol/L vs 3.80±0.60 μmol/L,4w, 10.40±1.61 μmol/L vs 6.10-4-0.79 μmol/L,6w,P〈0. 01）was markedly increased. Conclusion Systemic administration of cholesterol-modified siRNA targeting DDR2 could significantly suppress the expression of DDR2, decrease the contents of the extracellular matrix,and thus has a potential antifibrotic effect.