表达金属蛋白酶组织抑制因子-1反义核酸的质粒对大鼠肝纤维化的影响。

Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats.

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

Chin Med J (Engl). 2005 Feb 5;118(3):192-7.

PMID:15740646

Abstract

BACKGROUND

No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen.

METHODS

Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type I collagen. In addition to hepatic hydroxyproline content, hepatic collagen types I and III were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining.

RESULTS

Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P < 0.01), hepatic hydroxyproline content and the accumulation of collagen types I and III were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P < 0.01) as compared with the model group.

CONCLUSION

The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.

摘要

背景

目前尚无有效的肝纤维化治疗方法。本研究旨在提供证据，证明将表达金属蛋白酶组织抑制因子-1（TIMP-1）反义核酸的质粒导入免疫诱导的大鼠肝纤维化模型中，可导致间质胶原酶活性增加，从而增强胶原蛋白的降解。

方法

采用实时巢式聚合酶链反应（RT-Nested-PCR）和基因重组技术构建可在真核细胞中表达的大鼠TIMP-1反义重组质粒。将重组质粒和空载体（pcDNA3）用糖基化聚-L-赖氨酸包封后，注射到猪血清诱导的肝纤维化大鼠体内。通过Northern印迹、RT-PCR和Western印迹评估外源转染质粒的表达。使用异硫氰酸荧光素（FITC）标记的I型胶原检测肝间质胶原酶活性。除检测肝羟脯氨酸含量外，通过免疫组织化学染色检测肝I型和III型胶原，并通过Van Gieson染色评估肝纤维化分期。