Effects of antisense tissue inhibitor metalloproteinase-1 on hepatic stellate cells / 中华消化杂志

ABSTRACT

Objective To study the expressing status of antisense tissue inhibitor of metalloproteina se-1(TIMP-1) in hepatic stellate cells (HSC) constructed in vitro, and to eval u ate the effects on the production of type Ⅰ and Ⅲ collagens secreted by activated rat HSC. Methods HSC were extracted from normal rat liver by pronase and co llagenase digestion and purified by centrifugal elutriation, and were cultured pla stic 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 ant isense TIMP-1 expression plasmid which can express in eukaryotic cells, and seg uenced after being counstructed. The expressing plasmid and the pcDNA3 empty pla smid were transfected into HSC by Effectene reagent separately. The cells were sel ected after growing in DMEM containing 400 ?g/ml G418 for 3 to 4 weeks. Exp ression of TIMP-1 in HSC was d etermined by Northern blot and Western blot. We tested the interstitial collagen ase activity in culture media with FITC-labled type Ⅰ collagen as substrate. U ltimately, we quantified the typeⅠ and type Ⅲ collagen in HSC by Wester n blot. Results The exogenous antisense TIMP-1 recombinant plasmid could block the expression of TIMP-1 greatly, while there were not the same outcome i n pcDNA3 empty plasmid g roup and non-transfecting control group. The ratio of TIMP-1/GAPDH was 0.67, 2 .41 and 2.97 respectively at mRNA level( P