反义转化生长因子βⅡ型受体表达质粒对实验性肝纤维化的影响

目的 观察反义转化生长因子βⅡ型受作(TGF βRⅡ)表达质粒对实验性肝纤维化的影响。方法 运用重组DNA技术中构建反义TGF βRⅡ真核细胞表达质粒,采用猪血清腹腔注射制备免疫性大鼠肝纤维化模型,实验动物分为肝纤维化模型组、反义TGF βRⅡ治疗组、pCDNA3对照组及正常对照组。反义TGF βRⅡ质粒和pCDNA3空质粒与糖化多聚赖氨酸偶联后经尾静脉分别导入大鼠体内,通过northern blot、RT-PCR、Western blot检测外源导入质粒在肝组织中的表达,检测血清转化生长因子β1(TGFβ1)、肝组织羟脯氨酸测定,Ⅰ、Ⅲ型胶原免疫组织化学与Van Gieson染色观察反义TGF βRⅡ质粒对大鼠肝纤维化的影响。 结果 反义TGF βRⅡ表达质粒可在肝组织中获得确切表达,其表达可使反义治疗组血清TGF β1含量下降,反义TGF βRⅡ治疗组为(23.16 ± 3.13)ng/ml,模型组为(32.96±3.79)ng/ml,F=36.73,P<0.01。肝组织羟脯氦酸含量下降,治疗组为(0.17±0.01)mg/g,模型组为(0.30±0.03)mg/g,F=15.48,P<0.01。减少了肝组织Ⅰ、Ⅲ型胶原的沉积,治疗组Ⅰ型胶原为650.26±51.51,Ⅲ型胶原为661.5 8±55.28,模型组Ⅰ型胶原为1209.44±16.60,Ⅲ型胶原为1175.14±121.44,F值分圳为69.87、70.46,P<0.01。并促进反义治疗组肝脏病理形态一定程度的改善。

Effects of antisense transforming growth factor β receptor- Ⅱ (TGF βR Ⅱ) expressing plasmid on experimental liver fibrosis

OBJECTIVE: To study the effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis. METHODS: RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TGFbetaRII recombinant plasmid which can be expressed in eukaryotic cells. Thirty-six male SD rats were randomly distributed into five groups: 10 in experimental liver fibrosis model induced by pig-serum as disease control group; 10 in antisense TGFbetaRII transfection as treatment group; 10 in pCDNA3 transfection as treatment control group and 6 in normal control group. The recombinant plasmid and empty vector (pCDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model respectively. Expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR and Western blot. We also tested ELISA of serum TGF-beta1, the contents of hepatic hydroxyproline, immunohistochemistry of type I and III collagen, and VG staining for pathological study. RESULTS: The antisense TGFbetaRII expressing plasmid could be well expressed in vivo, and could block the mRNA and protein expression of TGFbetaRII in the fibrotic liver induced by pig serum. Its expression also reduced the level of TGF-beta1 antisense treatment group (23.16+/-3.13) ng/ml, disease control group (32.96+/-3.79) ng/ml; F=36.73, 0.01]. Compared with the disease control group, the contents of hepatic hydroxyproline antisense treatment group (0.17+/-0.01) mg/g liver, disease control group (0.30+/-0.03) mg/g liver; F=15.48, 0.01] and the deposition of collagens type I and type III decreased in the antisense group (antisense treatment group collagen type I 650.26+/-51.51, collagen type III 661.58+/-55.28; disease control group type I 1209.44+/-116.60, collagen type III 1175.14+/-121.44; F values are 69.87, 70.46, 0.01). And its expression also improved the pathologic classification of liver fibrosis models (0.01). CONCLUSION: The results demonstrate that TGF-beta plays a key role in liver fibrogenesis and the prevention of liver fibrosis by antisense TGFbetaRII recombinant plasmid intervention may be therapeutically useful.