Analysis airway of remodeling induced by CTGF

CTGF诱导的气道重塑分析

• 批准号: 14570560
• 负责人: YAMAUCHI Kohei
• 金额: $ 2.24万
• 依托单位: Iwate Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570560/
• 关键词: CTGF; Transterrin; airway; chimeric protein; transferrin receptor; remodeling; remodeling; トランスフェリン

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

我们利用重组CTGF研究了CTGF对呼吸道组织的直接作用。为了进行本实验，我们制备了CTGF和转铁蛋白(TE)的嵌合蛋白，并试图通过转铁蛋白受体增强CTGF对呼吸道组织的渗透。鉴于腺病毒载体基因转移表达的几率很低，我们分析了CTGF蛋白在呼吸道组织中的渗透及其通过TF受体和增强子的生物学效应。结果(1)成功地将Invitgen构建的人CTGF基因与生物素载体蛋白融合。用pET29-b表达该基因，镍柱纯化CTGF蛋白。(2)检测重组CTGF蛋白对人胎儿成纤维细胞和小鼠成纤维细胞的生物学效应。两种成纤维细胞系均表现出明显的增殖反应，并用四甲基偶氮唑盐比色法记录了清晰的剂量-反应曲线。(3)…分析进一步研究了嵌合蛋白在体内对小鼠呼吸道的渗透及其对组织的影响。将嵌合蛋白(10μ-1)经颈部切开直接注入气管内，分别于12、24、48、72h分析嵌合蛋白在气管内的定位和数量。免疫组织化学染色检测到嵌合蛋白在呼吸道组织中的定位，但肺组织中的细胞学改变尚未得到证实。此外，肺组织中ctlproollagen mRNA水平和胶原含量无明显变化。本研究通过气管内直接注射CTGF和转铁蛋白的方法，在小鼠气道内检测到由CTGF和转铁蛋白组成的嵌合蛋白，但未证实CTGP蛋白对呼吸道组织的细胞学或结构的影响。在本实验中，渗透的嵌合蛋白比预期的要少，并且其数量并没有随着增强剂的加入而显著增加。这就是CTGF的有效生物浓度没有提高到足够的程度，在呼吸道内检测不到其生物学效应的原因。较少

项目成果

Pan LH, et al.: "Pulmonary lymphangioleiomyomatosis : a case report with immunohistochemical details and DNA analysis"Tohoku J.Exp.Med.. 199. 119-126 (2003)

Pan LH 等人：“肺淋巴管平滑肌瘤病：免疫组织化学细节和 DNA 分析的病例报告”Tohoku J.Exp.Med.. 199. 119-126 (2003)

Pan LH et al.: "Neoplastic cells and proliferating endothelial cells express CTGF in glioblastoma"Neurol Res. 24. 677-683 (2002)

Pan LH 等人：“胶质母细胞瘤中的肿瘤细胞和增殖内皮细胞表达 CTGF”Neurol Res。

Pan LH et al.: "Pulmonary Lymphangioleiomyomatosis"Tohoku J Exp. 199. 119-126 (2003)

Pan LH 等：“肺淋巴管平滑肌瘤病”Tohoku J Exp.

Yamauchi K: "Up-regulation of connective tissue Growth Factor gene expression in murine Model with allergcal way inflamation"proceedings of the 7th Congress of Asian Pacific Society of Recpirology. 65-66 (2002)

Yamauchi K：“过敏性炎症小鼠模型中结缔组织生长因子基因表达的上调”亚太呼吸学会第七届大会论文集。

K Yamauchi, et al.: "Up-regulation of connective tissue growth factor gene expression in murine model with allergic airway inflammation."Proceeding of the 7^<th> Congress of Asian Pacific Society of Respirology.. 65-66 (2002)

K Yamauchi 等人：“过敏性气道炎症小鼠模型中结缔组织生长因子基因表达的上调。”亚太呼吸学会第 7 届大会论文集.. 65-66 (2002)