Study On The Biological Characteristics Of Rat Lung Fibroblast Seeded On ECM-Scaffold

Objective:To develope a reliable method for isolation,purification and primary culture of lung fibroblast from rats,and then investigate the biological characteristics of lung fibroblasts from rats seeded on ECM-scaffold. This study provides a new way for study on pathogenesis and treatment of interstitial lung disease in vitro.Methods:1.The lung tissue of 2 days old immature rats was digested with trypsin.LFs were isolated,purified,and then cultured.The nature of the cultures was identified by vimentin staining,electron micrography and Western blot.The growth cure was drawed for the obtained cells.2.Three materials including fibrinogen,laminin and fibronectin each at a certain concentration was used to constuct the monolayer ECM-scaffold,and the rat plasma was put into it for promoting coagulation.The lung fibroblasts purified from rats were planted on the monolayer ECM-scaffold and the monolayer ECM-scaffold with the hTGF-β₁ respectively.The biologic characteristics of lung fibroblasts were observed and analyzed by means of inverted microscope,immunofluorescence chemistry,electron micrography,MTT, Real-time PCR and Western blot.Results:1.Excellent yields of highly purified,culturable LFs could be obtained from 2 days immature rat lungs.The expression of vimentin in LFs was positive by immunofluorescence chemistry.Ramified stracture between purified LFs were observed by ultrastructural examination under electron micrography.Vimentin in purified LFs was revealed by Western blot.From the growth cure,the growth velocity of the cultured cells was stably.2.The biological characteristics of lung fibroblasts on both ECM-scaffold and ECM-scaffold with hTGF-β₁ demonstrated more active and higher expressing in cell proliferation, differentiation and collagen synthesis than that in the monolayer culture.Conclusions:1.This technique might be a reliable method for isolation,purification and primary culture of lung fibroblasts from rat lung tissues.2.The lung fibroblasts seeded on ECM-scaffold could grow and proliferate well.This three-dimensional culture model could be used for the study of interstitial lung disease in vitro.