Inhibition on the production of collagen type I, III of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid.
OBJECTIVE
To investigate the inhibition effects on the production of collagen type I, III secreted by activated rat hepatic stellate cells (rHSCs) by antisense tissue inhibitors of metalloproteinase 1 (TIMP-1) recombinant plasmid through elevating interstitial collagenase activity.


METHODS
rHSCs were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days. RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryotic cells. The recombinant plasmid and the pcDNA3 empty plasmid were transfected in rHSCs by Effectene (QIAGEN) separately. Cells were selected after growing in DMEM containing 400 microg/ml G418 for 2-3 weeks. Expression of exogenous gene was assessed by Northern blot, and expression of TIMP-1 in rHSCs was determined by Northern blot and Western blot. We tested the interstitial collagenase activity with FITC-labled type I collagen as substrate. Ultimately, we quantified the type I, III collagen by Western blot.


RESULTS
The exogenous antisense TIMP-1 recombinant plasmid could be expressed in rHSCs well, which could block the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNA level (P<0.05); the ratio of TIMP-1/beta-actin was 0.31, 0.98 and 1.32 separately at protein level (P<0.05); It might elevate active and latent interstitial collagenase activity, the collagenase activity was 0.3049, 0.1411 and 0.1196 respectively. (P<0.05), which led to promotion the degradation of type I, III collagen, the ratio of collagen I/beta-actin was 0.63, 1.78 and 1.92 separately (P<0.05); and the ratio of collagen III/beta-actin was 0.59, 1.81 and 1.98 separately (P<0.05).


CONCLUSIONS
These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on the production of type I, III collagens secreted by activated rHSCs in vitro. It could be a novel method to reverse hepatic fibrosis in the future.
Liu
Wen-Bin
WB
Division of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Yang
Chang-Qing
CQ
Jiang
Wei
W
Wang
Yi-Qing
YQ
Guo
Jing-Sheng
JS
He
Bo-Ming
BM
Wang
Ji-Yao
JY
D000818:Animals; D016375:Antisense Elements (Genetics) / Q000494:pharmacology*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000037:antagonists & inhibitors*; D022781:Hepatocytes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011995:Recombination, Genetic; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics*
D016375:Antisense Elements (Genetics); D024042:Collagen Type I; D019715:Tissue Inhibitor of Metalloproteinase-1