Analysis airway of remodeling induced by CTGF

我们研究了CTGF对气道组织的直接作用。为了进行该实验，我们制备了由CTGF和转铁蛋白（TE）组成的嵌合蛋白，并试图通过转铁蛋白受体增强CTGF向气道组织的浸润。由于腺病毒载体介导的基因转移表达的机会很低，我们分析了CTGF蛋白通过TF受体和增强子在气道组织中的浸润及其生物学效应。结果（1）将构建在pcDNA3.1中的人CTGF cDNA与Biotin载体蛋白融合。其次，利用pET 29-B在大肠杆菌中表达了CTGF的组氨酸标签蛋白，并用镍柱纯化了CTGF蛋白。(2)本实验观察了重组CTGF蛋白对体外培养的人胎儿成纤维细胞和小鼠成纤维细胞的生物学效应。MTT法显示两种成纤维细胞株均表现出明显的增殖反应，并记录到清晰的量效曲线。(3)我们分析了 ...更多信息 目的：观察嵌合蛋白在小鼠气道中的浸润情况及其对气道组织的影响。将嵌合蛋白（10μ l）经颈部切口直接注入气管内，观察12、24、48和72小时气道内嵌合蛋白的定位和数量变化。免疫组化检测嵌合蛋白在气道组织中的定位，但肺组织的细胞学变化未得到证实。此外，肺组织中CTGF前胶原mRNA水平和胶原含量均无明显变化。DiscussionIn本研究中，我们通过气管内直接注射的方法检测到CTGF和转铁蛋白组成的嵌合蛋白在小鼠气道中的表达，但我们没有证实CTGP蛋白浸润后气道组织的细胞学或结构变化。在本实验中，浸润的嵌合蛋白比预期的要少，并且其数量没有随着增强子的添加而显著增加。这是CTGF有效生物浓度未充分增加，在气道中未检测到其生物学效应的原因。少

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