The Effects Of Thrombospondin 1 In Transforming Growth Factorβ1 Induce Rat Cardiac Fibroblasts

Objective : To investigate the expression of thrombospondin 1 and its influence on the expression of vascular endothelial growth factor during the process of the proliferation,collagen synthesis of rat cardiac fibroblasts induced by transforming growth factorβ1,so as to introduce theoretic proof for prevention of myocardial fibrosis.Methods: Cardiac fibroblasts were isolated from the ventricles of one to three day Sprague-Dawley neonatal rats and primarily cultured(digestion,different speed adherence method),then passaged to three to four generation for experiment. These cells were divided into five groups according to the different intervention factors: 1.Normal control guoup: CFs were cultured with free serum medium(FSM) for 24h. 2.The dose-dependent effects of TGF-β1 groups; CFs were treated with FSM contained different dose (10ng/ml,20ng/ml,30ng/ml) TGF-β1 and cultured for 24h; 3.The time–dependent effect of TGF-β1 groups;CFs were treated with FSM containing TGF-β1 cultured with different time:12h,24h,48h; 4.Antisense oligonucleotide groups:CFs were treated with FSM contained TGF-β1 and TSP-1 Antisense oligonucleotide cultured for 24h. 5.Sense oligonucleotide groups: CFs are treated with FSM contained TGF-β1 and TSP-1 sense oligonucleotide cultured for 24h.The proliferation,collagen synthesis of rat cardiac fibroblasts were observed with MTT (A value) and Hydroxyproline (mg/L).The expression of VEGFmRNA and TSP-1mRNA were analyzed by RT-PCR. Linear correlation analysis will be used to evaluate the relationship between the relative expression of VEGF mRNA and TSP-1 mRNA.Results:1 The proliferation,collagen synthesis:1.1 Compared with control, TGF-β1 group stimulated the proliferation,collagen synthesis of CFs(P <0.01).1.2 Compared with TGF-β1 group, TSP-1ASODN inhibited the proliferation,collagen synthesis of CF(sP <0.01).1.3Compared with TGF-β1 group, TSP-1SODN didn't significantly change on the proliferation , collagen synthesis of (P>0.05) 2. Expression of mRNA.2.1,The dose-dependent effects of TGF-β1, Expression of VEGFmRNA were declined significantly(P<0.01)and TSP-1 mRNA were increased significantly(P <0.01) compared with that in control groups ;2.2The time-dependent effect of TGF-β1groups: Expression of VEGF mRNA were declined significantly(P <0.01)and TSP-1 mRNA were increased significantly(P <0.01) compared with that in control groups.2.3 Antisense oligonucleotide groups: VEGF mRNA expression were increased significantly compared with those in TGF-β1 groups ( P <0.01).TSP-1mRNA expression were decreased significantly compared with that on TGF-β1 groups(P<0.01).2.4 Sense oligonucleotide groups : expression of VEGF mRNA and TSP-1 mRNA were similar to in TGF-β1 groups(P<0.05).3 The expression of VEGF mRNA were negatively related to the TSP-1mRNA (r=-0.537, r=-0.714, P<0.01) Conclusions: 1.TSP-1 plays a role in positive feedback regulation of TGF-β1 bioactivity in the proliferation ,collagen synthesis of CFs induced by TGF-β1.2.VEGF expression could be inhibited by TSP-1.3.The proliferation ,collagen synthesis of CFs induced by TGF-β1 could be inhibited by TSP-1.4.TSP-1 Antisense oligonucleotide may exert helpful effect on anti-fibrosis.