Effects of antisense tissue inhibitor metalloproteinase-1 on hepatic stellate cells
- VernacularTitle:反义金属蛋白酶抑制因子-1对肝星形细胞的影响
- Author:
Wenbin LIU
;
Jiyao WANG
;
Changqing YANG
- Publication Type:Journal Article
- Keywords:
Hepatic stellate cell;
Tissue inhibitor of metalloproteinase-1;
TypeⅠcollag en;
Type Ⅲ collagen
- From:
Chinese Journal of Digestion
2001;0(01):-
- CountryChina
- Language:Chinese
-
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
