抑制核因子-κB对人眼角膜基质细胞分泌细胞因子的影响  

Inhibition effects of nuclear factor-κB on cytokines releasing from human corneal fibroblasts

作　　者：陈国玲[1,2] 陈双 刘艳丽[2] 魏璐婉[2] 杨丽霞[1] 刘执玉[2] 

机构地区：[1]山东大学眼科中心第二医院眼科,济南250033 [2]山东大学医学院人体解剖学与组织学胚胎学教研室,济南250012 [3]枣庄市山亭区人民医院药剂科,山东枣庄277200

出　　处：《解剖学报》2009年第6期914-918,共5页Acta Anatomica Sinica

基　　金：山东省教育厅科技计划项目(J08LH59)

摘　　要：目的观察体外抑制核因子-κB(NF-κB)对人眼角膜基质细胞分泌细胞因子的影响。方法四甲基偶氮唑盐(MTT)法检测不同浓度的大黄素对人眼角膜基质细胞活性的影响。用脂多糖(LPS)刺激体外培养的基质细胞,将细胞分为正常对照组(n=6)、LPS组(n=24)和大黄素组(n=24)。LPS组单纯给予LPS刺激,大黄素组于LPS刺激前先用大黄素预处理30min,其余处理同LPS组。分别用反转录聚合酶链反应(RT-PCR)和酶联免疫吸附实验(ELISA)检测LPS刺激对角膜基质细胞白细胞介素8(IL-8)基因表达和蛋白分泌的影响,Westernblotting检测角膜基质细胞中κB抑制因子α(IκB-α)蛋白的表达。结果所用浓度大黄素对体外培养的角膜基质细胞活性无影响。与刺激前相比,LPS刺激可引起人眼角膜基质细胞IL-8mRNA表达增加,促进IL-8蛋白分泌、下调细胞内IκB-α蛋白水平,P<0.01差异均有统计学意义。大黄素预处理可明显抑制LPS诱导的人眼角膜基质细胞表达IκB-α,下调细胞IL-8基因表达,减少IL-8蛋白分泌,明显低于同时间点LPS组的表达,P<0.01差异具有统计学意义。结论LPS可引起人眼角膜基质细胞IκB-α的降解,促进NF-κB核转位,引起IL-8表达。体外抑制NF-κB,能减少人眼角膜基质细胞分泌IL-8。Objective To investigate the inhibition effects of nuclear factor-roB （NF-kB） on cytokine expression of cultured human corneal fibroblasts in vitro. Methods The cultured human corneal fibroblasts were used in the present experiment. Cell viability insensitive to emodin on cultured human corneal fibroblasts in vitro was assessed using the MTF assay. Cells were randomly divided into three groups：control group（ group 0 hour, n = 6）, lipopolysaccharidel （LPS）group （ n = 24）and emodin pretreatment group（ n = 24） . Cells of the emodin pretreatment group were incubated with emodin for 30 minutes before LPS challenged. The cultured human corneal fibroblasts were then challenged with Lt~, the expression of interleukin-8（IL-8） mRNAs was detected by reverse transcription polymerase chain reaction analysis （RT-PCR） and the proteins of IL-8 secreted from these cells were assessed by enzyme-linked immunosorbent assays （ELISA）. Western blotting analysis was used for detecting the protein expression of inhibitor of nuclear factor-tcB-ct（InB-a）induced by LPS. At the same time, the effects of emodin on these expressions were also assessed in fibroblasts. Results The concentration of emodin used in this study was safe to these cultured human corneal fibroblasts in vitro. Both the protein release and mRNA expression of IL-8 in corneal fibroblasts increased significantly after challenged with LPS, however, the protein level of IkB-a significantly decreased after treated with LPS. These results indicated that LPS could mediate the activation of NF-kB and upregulate the expression of cytokines in corneal fibroblasts. Pretreated with emodin 30 min before challenged with LPS, the activation of NF-kB induced by LPS was markedly inhibited, and the mRNA expression as well as protein release of IL-8 were also inhibited（ P 〈 0.01 ）. Conclusion These results suggest that LPS takes part in the degeneration of IsB-α and the expression of IL-8 in cultured human corneal fibroblasts in vitro. Emodin, an

关 键 词：抑制剂 核因子-KB 白细胞介素 细胞培养 反转录聚合酶链反应 人 

分 类 号：R772.2[医药卫生—眼科]