Dighiero P, Behar-Cohen F, Courtois Y, Goureau O
Dévelopment, Vieillissement et Pathologie de la Rétine, U450 Institut National de la Santé et de la Recherche Médicale, Association Claude Bernard, Paris, France.
Invest Ophthalmol Vis Sci. 1997 Sep;38(10):2045-52.
To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors.
Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR).
Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells.
The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.
确定牛角膜内皮（BCE）细胞和角膜细胞在暴露于细胞因子和脂多糖（LPS）后是否表达诱导型一氧化氮合酶（NOS），并研究生长因子对NOS的调节作用。
将牛角膜内皮细胞和角膜细胞培养物暴露于浓度递增的LPS、干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）。在暴露后的选定时间间隔，通过格里斯反应评估上清液中的亚硝酸盐水平。从细胞培养物中提取总RNA，并通过逆转录-聚合酶链反应（RT-PCR）测量诱导型NOS（NOS-2）的信使RNA水平。
BCE细胞和角膜细胞暴露于LPS和IFN-γ导致亚硝酸盐水平升高，添加TNF-α可增强这种升高。RT-PCR分析表明，亚硝酸盐释放与BCE细胞和角膜细胞中NOS-2信使RNA的表达相关。NOS的立体选择性抑制剂和环己酰亚胺抑制两种细胞中LPS-IFN-γ诱导的亚硝酸盐释放，而转化生长因子-β（TGF-β）则略有增强作用。成纤维细胞生长因子-2（FGF-2）抑制角膜细胞中LPS-IFN-γ诱导的亚硝酸盐释放和NOS-2信使RNA积累，但对BCE细胞无此作用。
结果表明，LPS和细胞因子在体外激活角膜细胞和BCE细胞可诱导NOS-2表达并释放大量NO。体内大量的NO可能与角膜炎症性疾病有关。
