摘要
目的 观察反义金属蛋白酶组织抑制因子 1(TIMP 1)表达质粒对实验性肝纤维化的影响。方法 运用重组DNA技术构建反义TIMP 1真核细胞表达质粒 ,经与糖化多聚赖氨酸偶联后 ,尾静脉注射将其导入猪血清诱导的免疫性大鼠肝纤维化模型体内 ;通过Northern印迹法、RT PCR、Western印迹法检测外源导入基因的表达 ,并通过肝组织间质胶原酶活性和羟脯氨酸测定 ,以及肝组织Ⅰ、Ⅲ型胶原免疫组织化学与VanGieson染色观察反义TIMP 1质粒对大鼠肝纤维化的影响。结果 外源导入基因可在肝组织中获得确切表达 ,其表达可增加间质胶原酶的活性 (P <0 .0 1) ,减少羟脯氨酸含量 (P <0 .0 1) ,促进Ⅰ、Ⅲ型胶原的降解 (P <0 .0 1) ,并促进肝脏病理形态一定程度的改善 (P <0 .0 1)。结论 反义TIMP 1表达质粒对肝纤维化有较好的改善作用。
Objective To observe the effects of antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) expressing plasmid on rat liver fibrosis.Methods RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TIMP-1 recombinant plasmid which could be expressed in eucaryotic cells. The recombinant plasmid and empty vector (pcDNA3) were encapsulated by glycosyl-poly-L-lysine and then transduced into the porcire serum-induced liver fibrosis rat model. Expression of exogenous transfected gene was assessed by Northern blot, and the hepatic expression of TIMP-1 was evaluated by RT-PCR and Western blot. We also tested the hepatic interstitial collagenase activity with FITC-labled type Ⅰ collagen, and used the immunohistochemistry of hepatic type Ⅰ and Ⅲ collagen and VG staining for pathological study.Results The exogenous antisense TIMP-1 could be expressed in vivo, and could block the gene and protein expression of TIMP-1 in the liver of porcine serum-induced hepatic fibrosis rats at mRNA level, which might elevate the active and latent hepatic interstitial collagenase activity (P<0.01); decrease the hydroxyproline content of liver tissue (P<0.01), and promote the degradation of collagens tyep Ⅰ and Ⅲ (P<0.01). Its expression also improved the hepatic pathology to a certain extent (P<0.01). Conclusion The results demonstrate that the antisense TIMP-1 recombinant plasmid has some reversal effect on liver fibrosis which enables it to be a possible candidate of gene therapy in the future.
出处
《肝脏》
2003年第3期4-8,共5页
Chinese Hepatology
基金
国家自然科学基金资助项目 (39970 339)
