Analysis airway of remodeling induced by CTGF

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We investigate the direct effects of CTGF on airway tissue with use of recombinant CTGF. To perform this experiment, we made chimeric protein consisting of CTGF and transferrin (TE) and tried to enhance infiltration of CTGF into airway tissue via transferrin receptor. Since chance of the gene expression by gene transfer With adenovirus vector is very low, we analyzed the infiltration of CTGF protein into airway tissue and its biological effects via TF receptor in combination with enhancer. Results(1) We fused human CTGF cDNA constructed in pcDNA3.1 from Invitrogen with Biotin carrier protein. Secondly we expressed this gene as Histidine tag protein in E coli with use of pET29-b and purified CTGF protein with nickel column.(2) We evaluated the biological effects of recombinant CTGF protein on cultured human fetal fibroblasts and murine fibroblasts. Both fibroblast cell lines showed significant proliferative responses and clear dose-response curve was recorded with MTT assay.(3) We analy … More zed the infiltration of the chimeric protein into murine airway in vivo and its effects on the tissue. We injected the chimeric protein (10μ1 ) into tracheas directly by incision of neck and analyzed localizaion and number of the chimeric protein in airway in the time course as 12, 24, 48 and 72 hours. We detected the localization of chimeric protein in airway tissue with immunostaining, however, cytological changes in pulmonary tissue was not confirmed. In addition, ctlprocollagen mRNA level and collagen content in lung tissue did not changed significantly.DiscussionIn the present study, we detected the chimeric protein consisting of CTGF and transferrin in the murine airway by injection into trachea directly, however we did not confirm cytological or structural changes of airway tissues by infiltrated CTGP protein. In this experiment, the infiltrated chimeric proteins were fewer than expected and its number was not increased significantly with addition of enhancer. This is the reason that effective biological concentration of CTGF was not increased enough and its biological effect was not detected in the airway. Less