TGFβ1 Mediates Collagen Ⅰ And Collagen Ⅳ Synthesis In Neonate Rat Cultured Cardiac Fibroblasts

Objective: To isolate rat cardiac fibroblasts followed by investigationof the effect of TGF-β₁ on synthesis of collagen typeâ… and collagen typeâ…£,and to investigate the possible mechanism of TGF-β₁ in cardiac fibrosis.Method: In this research we isolated cardiac fibroblasts from SD ratwhich then were treated using different concentrations of TGF-β₁ ie.lng/mland 10ng/ml then incubated and observed for a specific duration of time (T),Cells were divided in to three main groups A, B and C, as follows Group A=TGF-β₁ 0mg/ml(control group), Group B= TGF-β₁ lng/ml and Group C=TGF-β₁ 10ng/ml). As stated above that all groups were observed for a giventime (T) (5h) or (24h) Then RT-PCR and Immunocytochemistry methodswere used to detect biosynthesis of collagen typeâ… and collagen typeâ…£respectively.Results:1. After digestion with pancreatin and collagenaseâ…¡with thetechnique of differential adhesion twice in a short period, this can isolatecardiacmyocytes and myocardial fibroblasts from each other. Themyocardial fibroblasts stained with monoclonal antibodies against Vimentinwere positive, stained with monoclonal antibodies againstα-sarcomeric actinwere negative.2. The primary cultured rat myocardial fibroblasts were treated usingdifferent concentrations of TGF-β₁ (0ng/ml, for control group, 1ng/ml, and10ng/ml for test groups respectively) after 5h. The OD values of collagen typeâ… /β-actin checked with RT-PCR were 0.61±0.02, 0.73±0.03 and 0.86±0.04 respectively, and after 24h the OD values of collagen typeâ… /β-actin forcontrol group, 1ng/ml and 10ng/ml were 0.65±0.03, 0.91±0.02 and 0.98±0.02 respectively (all P＜0.01 compared with controls, and other groups).3. The primary cultured neonate rat myocardial fibroblasts were treatedusing different concentrations of TGF-β₁ (0ng/ml, 1ng/ml, 10ng/ml) 5h and24h and the OD values of RT-PCR for collagen typeâ…£/β-actin were 0.58±0.06, 0.71±0.02, 0.87±0.02 respectively, 24h the OD values of RT-PCRwere 0.62±0.01, 0.83±0.05, 0.96±0.02 respectively (all P＜0.01 comparedwith controls, and other groups).4. The primary cultured neonate rat myocardial fibroblasts which weretreated using different concentrations of TGF-β₁ were harvested andImmunocytochemistry stains were carried out for (0ng/ml for control group,1ng/ml and 10ng/ml for treated group respectively).Conclusion:1. The neonate rat cardiac fibroblast cells were isolated and culturedsuccessfully.2. TGF-β₁ can enhance biosynthesis of collagen typeâ… and collagen typeâ…£in a dose and time dependent manner.3. TGF-β₁ can enhance biosynthesis of the collagen typeâ… and collagentypeâ…£, which give an indication of that TGF-β₁, may play a role in cardiacfibrosis.