【作者】 陈亚娟；

【导师】 文富强；

【作者基本信息】 四川大学 ， 内科学， 2007， 硕士

【摘要】 目的气道重构及肺纤维化形成是引起呼吸系统疾病致残率和死亡率上升的主要原因，亦是疾病防治的重点及难点。许多研究证实，炎症损伤及组织修复失衡是导致支气管肺部纤维化的主要成因，其中可能涉及血管新生及相应生长因子。本实验通过应用转化生长因子（transforming growth factor-β1，TGF-β1）刺激人胎肺成纤维细胞（human fetal lung fibroblast-1，HFL-1），检测血管内皮生长因子（vascular endothelial growth factor，VEGF）在其中的表达，以及观察布地奈德（Budesonide，Bud）对HFL-1分泌VEGF的干预作用。方法本实验采用细胞培养法，分三个部分：第一部分探讨TGF-β诱导HFL-1分泌VEGF的时间依赖效应，实验组加入0.25ng/ml TGF-β1刺激，分别在12、24、48、72小时收集细胞培养上清液，经酶联免疫分析法（enzyme immunoassay，EIa）检测VEGF含量，同时进行细胞计数；第二部分探讨TGF-β1诱导HFL-1分泌VEGF的浓度依赖效应，实验组加入不同浓度（0、0.025、0.05、0.25、0.5、2.5、5ng/mL）TGF-β1刺激，孵育72小时后收集细胞培养上清液，经EIA检测VEGF含量；第三部分探讨布地奈德对HFL-1分泌内源性和外源性VEGF的影响，实验组随机分为对照组、不同浓度(10^-11M、10^-9M、10^-7M)布地奈德干预组、0.5ng/mlTGF-β1+不同浓度(10^-11M、10^-9M、10^-7M)布地奈德干预组，孵育72小时后检测上清液VEGF含量。结果在第一部分实验中，随着细胞孵育时间延长，TGF-β1刺激组成纤维细胞生长加快，细胞生长密度增加；VEGF分泌水平也增加，在72小时点达到分泌高峰，并且各时间点VEGF相对含量均高于对照组，两组比较有统计学差异（P＜0.05）。在第二部分实验中，低浓度TGF-β1（≤0.5ng/ml）上调VEGF的表达量与对照组比较差异无显著性（P＞0.05）；随着TGF-β1刺激浓度加大，VGEF含量也逐渐增加，当TGF-β1浓度为5ng/ml时，VEGF相对含量与空白对照组相比增加了近七倍，两组比较差异有统计学意义（P＜0.01）。在第三部分实验中，低浓度(10^-11M)Bud刺激内源性VEGF分泌轻微增加，但与空白对照组比较无明显差异（P＞0.05）；而高浓度(10^-7M)Bud不仅能明显阻断内源性VEGF的表达，与空白对照组相比差异有统计学意义（P＜0.01），还可明显下调TGF-β1诱导的VEGF分泌增加的效应，与同时相相同浓度单独TGF-β1刺激组比较，VEGF含量下降明显，二者比较具有统计学意义（P＜0.01）。结论人胎肺成纤维细胞自身可分泌内源性VEGF，TGF-β1能够诱导HFL-1增殖及VEGF分泌增加，并具有时间和浓度依赖效应。提示成纤维细胞是支气管肺部纤维化病中主要的功能细胞，TGF-β1诱导VEGF分泌增加可能是纤维化形成新的重要原因之一。高浓度(10^-9M，10^-7M)布地奈德不仅能够抑制HFL-1自身分泌VEGF，还能阻断TGF-β1诱导VEGF水平上调的效应，而低浓度(10^-11M)布地奈德没有抑制VEGF分泌的作用。提示抑制VEGF生成及血管新生可能是布地奈德发挥抗纤维化作用中新的重要分子机制。更多还原

【Abstract】 ObjectiveAirway remodeling and lung fibrosis are the leading causes result in increasingly high mutilation and mortality in respiratory disease. Studies show that inflammatory tissue damage and inappropriate repair lead to bronch-lung fibrosis, in which angiogenesis relating growth factors maybe involved. In the current study, we examined the effect of budesonide on transforming growth factor-β1 （TGF-β1） induced vascular endothelial growth factor （VEGF） release by human fetal lung fibroblast （HFL-1）.MethodsBy using cultured human fetal lung fibroblasts, we firstly examed the time-dependency of TGF-Bl induced VEGF production by human fetal lung fibroblasts. HFL-1 fibroblasts treated with 0.25ng/ml TGF-β1 were incubated for 12, 24, 48 and 72 hrs.The post-culture media were collected for enzyme immunoassay （ EIA） of VEGF. Cell counts were also done.Secondly, we examed the dose-dependency of TGF-Bl induced VEGF production by human fetal lung fibroblasts. HFL-1 fibroblasts were cultured with various concentrations of TGF-β1 for 72 hrs.The post-culture media were collected for EIA.Finally, we examed the effect of Budesonide on basal and TGF-β1 induced VEGF release by HFL-1 fibroblasts. HFL-1 fibroblasts were treated with various concentrations of budesonide in the absence and presence of 0.5ng/ml TGF-β1 for 72 hrs. Post-culture media were collected for the assay of VEGF at 72 hrs.ResultsHFL-1 fibroblasts increasingly release VEGF as incubation goes on. With the stimulation of TGF-β1, increased release of VEGF was noted at different time points.A significant increase in VEGF was observed in HFL-1 fibroblasts treated with 0. 5-5ng/ml of TGF-β1 （ P<0.05）. A sevenfold increase in VEGF release was observed in cells treated with 5ng/ml TGF-β1 compared with control. However, cells treated with TGF-β1 below 0.5ng/ml had no stimulatory effect on VEGF release by HFL-1 fibroblasts.10^-7M of Budesonide significantly decreased VEGF release by HFL-1 cells. Furthermore, TGF-81 argumented release of VEGF was attenuated by Bud at 10^-9M and 10^-7M, which shows a dose-dependent manner.ConclusionsHFL-1 fibroblasts release VEGF spontaneously. TGF-β1 increased the release of VEGF by HFL-1 fibroblasts in a time and dose-dependent manners. Budesonide inhibited basal VEGF release, and TGF-β1 enhanced VEGF production by HFL-1 fibroblast. Suppession of VEGF production may be a mechanism that budesonide modulates fibroblasts’ function in pulmonary fibrosis.更多还原