Effect Of TGF-?1 On Collagen Synthesis Of Bronchial Fibroblasts In Rats

Objective:In this experiment,the role of TGF-?1 and rat bronchial fibroblasts in airway fibrosis of chronic obstructive pulmonary disease?COPD?was investigated by observing the regulation of transforming growth factor-?1?TGF-?1?on rat bronchial fibroblasts Collagen type ?/? synthesis in vitro.Methods:Bronchi of male SD rats of 5-6 weeks of age,150-180g or so,were rapidly separated.Rat bronchial fibroblasts were cultured in vitro by enzyme digestion and tissue block adhesion.The third generation of rat bronchial fibroblasts with good growth status was selected for the experiment.The control group was treated with 0ng/ml TGF-?1 for different time,and the experimental group was treated with TGF-?1 concentration gradients of 5ng/ml,10ng/ml and 20ng/ml for 2h,12h,24h and 48 h.Then we collect supernatants,and use rat type ? collagen?COL I?ELISA detection kit and rat type ? collagen?Col ??ELISA detection kit to respectively detect the levels of type ? and type ? collagen in supernatants of each group of samples.Results:1.Cell culture was carried out by enzyme digestion and tissue block adhesion.The cells were long fusiform,triangular and protruding.It was identified that the anti-vimentin staining of the cell was positive.The rat bronchial fibroblasts were successfully isolated and cultured.2.CCK-8 was used to detect the proliferation activity of the 3rd generation bronchial fibroblasts,then OD_450nm50nm was detected and the growth curve was drawn.The average value of OD_450nm50nm was taken as the ordinate and the days of cultivation as the abscissa.The cell growth was relatively slow at 1d?2d,which was the adaptation period of the cell.The cells grew relatively fast at the 3d?6d,which was the logarithmic growth phase of the cells.The cell growth reached its peak on the 6th day.On the 7th day,cell proliferation slowed down and entered a plateau of growth.The growth curve approximates to an"S"shape.3.Under the effect of 0ng/ml TGF-?1,the expression levels of type ? collagen in rat bronchial fibroblasts at 2h,12h,24h and 48h were 1.454±0.005 ng/ml,1.664±0.016 ng/ml,1.862±0.013ng/ml and 1.982±0.017 ng/ml respectively.The expression levels of type ? collagen were 0.206±0.00 ng/ml,0.288±0.007 ng/ml,0.354±0.008 ng/ml and 0.430±0.009ng/ml respectively.4.Compared with the corresponding group of 0ng/ml TGF-?1,the expression of type ? collagen in bronchial fibroblasts of rats with any concentration of TGF-?1 increased in all time periods?P<0.05?,and was correlated with the concentration of TGF-?1 in general.However,the expression of type ? collagen in5ng/ml group was higher than that in 10ng/ml group at 2h?P<0.05?.There was no significant difference between 10ng/ml and 20ng/ml group at 24h?P>0.05?.At 48h,the expression of type ? collagen in 10ng/ml group was higher than that in 20ng/ml group?P<0.05?.5.Compared with the corresponding group of 0ng/ml TGF-?1,the expression of type ? collagen in bronchial fibroblasts of rats with any concentration of TGF-?1 increased in all time periods?P<0.05?,and was correlated with the concentration of TGF-?1 in general.However,the expression of type ? collagen in5ng/ml group was higher than that in 10ng/ml group at 2h?P<0.05?.Conclusion:1.Rat bronchial fibroblasts can be cultured by enzyme digestion and tissue adhesion,which can provide experimental basis for in vitro study of airway remodeling mechanism of COPD.2.TGF-?1 can promote the synthesis of type ? and type ? collagen in rat bronchial fibroblasts,which is related to time and dose.