[表达反义转化生长因子β受体II（TGFβRII）的质粒对实验性肝纤维化的影响]

[Effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis].

作者信息

Jiang Wei, Wang Ji-yao, Yang Chang-qing, Liu Wen-bin, Wang Yi-qing, He Bo-ming

机构信息

Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.

出版信息

Zhonghua Gan Zang Bing Za Zhi. 2004 Mar;12(3):137-40.

PMID:15059294

Abstract

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.

摘要

目的

研究反义转化生长因子β受体II（TGFβRII）表达质粒对实验性肝纤维化的影响。

方法

采用RT-Nest-PCR和基因重组技术构建可在真核细胞中表达的大鼠反义TGFβRII重组质粒。36只雄性SD大鼠随机分为五组：10只作为疾病对照组，采用猪血清诱导建立实验性肝纤维化模型；10只作为治疗组，进行反义TGFβRII转染；10只作为治疗对照组，进行pCDNA3转染；6只作为正常对照组。将重组质粒和空载体（pCDNA3）用糖基化聚-L-赖氨酸包裹，然后分别转导入猪血清诱导的肝纤维化模型大鼠体内。通过Northern印迹、RT-PCR和Western印迹评估外源转染质粒的表达。我们还检测了血清TGF-β1的ELISA、肝羟脯氨酸含量、I型和III型胶原的免疫组化以及用于病理研究的VG染色。

结果

反义TGFβRII表达质粒在体内能良好表达，并能阻断猪血清诱导的纤维化肝脏中TGFβRII的mRNA和蛋白表达。其表达还降低了TGF-β1水平[反义治疗组（23.16±3.13）ng/ml，疾病对照组（32.96±3.79）ng/ml；F=36.73，P<0.01]。与疾病对照组相比，反义组肝羟脯氨酸含量[反义治疗组（0.17±0.01）mg/g肝脏，疾病对照组（0.30±0.03）mg/g肝脏；F=15.48，P<0.01]以及I型和III型胶原的沉积减少（反义治疗组I型胶原650.26±51.51，III型胶原661.58±55.28；疾病对照组I型胶原1209.44±116.60，III型胶原1175.14±121.44；F值分别为69.87、70.46，P<0.01）。并且其表达还改善了肝纤维化模型的病理分级（P<0.01）。