结膜成纤维细胞由来的警报素对角膜成纤维细胞的促炎作用  

Effects of alarmins from necrotic human conjunctival fibroblasts(NHCS)on the production of inflammatory mediactors by human corneal fibroblasts(HCFs)

作　　者：陈琳 刘苹苹[1] 杨秀霞[1] 李勤[1] 刘洋[1] CHEN Lin;LIU Ping-ping;YANG Xiu-xia(Department of Ophthalmology.Fifth Affiliated Hospital,Sun Yat-sen University,Zhuhai 519000,China)

机构地区：[1]中山大学附属第五医院眼科,广东珠海519000

出　　处：《中国实验诊断学》2020年第6期1021-1026,共6页Chinese Journal of Laboratory Diagnosis

基　　金：广东省科技厅自然科学基金面上项目,2017A030313774。

摘　　要：目的严重烧伤可致眼表结膜组织大量坏死,并导致角膜基质持续性炎症反应及溃疡形成。本研究探讨结膜成纤维细胞坏死上清产生的警报素(alarmins)对人角膜基质成纤维细胞(HCFs)释放促炎因子及粘附分子的影响。方法通过冻融方法制备结膜成纤维细胞坏死上清(NHCS),用NHCS、IKK2抑制剂、IL-1及TNF-α受体拮抗剂对HCFs进行处理。ELISA检测IL-6、IL-8及MCP-1的表达,Western blot分析ICAM-1表达及IκB-α磷酸化,免疫荧光染色检测NF-κB p65亚基定位。结果 NHCS可刺激HCFs表达IL-6、IL-8、MCP-1和ICAM-1,激活IκB-α磷酸化,并介导NF-κB p65亚基的核内转移。此外,IKK2抑制剂、IL-1及TNF-α受体拮抗剂可抑制NHCS诱导的上述炎性介质的表达。结论由结膜成纤维细胞坏死上清产生的警报素包括IL-1和TNF-α,可通过激活NF-κB信号通路促进角膜基质成纤维细胞表达促炎因子和粘附分子。IL-1和TNF-α可能成为改善严重眼烧伤角膜基质炎症反应的潜在治疗靶点。Objective Severe burns can cause necrosis of ocular surface tissue and lead to persistent inflammation and ulceration in the cornea.Effects of alarmins produced by necrotic human conjunctival fibroblasts on the release of inflammatory mediactors by human corneal fibroblasts(HCFs)were investigated.Methods A necrotic cell supernatant(NHCS)was prepared by subjecting human conjunctival fibroblasts to three cycles of freezing and thawing.HCFs were cultured with or without NHCS in the presence or absence of inhibitors of NF-κB signaling pathway and IL-1 or TNF-αreceptor antagonists.The expressions of IL-6,IL-8 and MCP-1 were detected by ELISA.The phosphorylation and degradation of the NF-κB–inhibitory protein IκB-αand ICAM-1 was assessed by western blot.Subcellular localization of the p65 subunit of the transcription factor NF-κB was examined by immunofluorescence analysis.Results NHCS induced the expression of IL-6,IL-8,MCP-1 and ICAM-1 by HCFs and stimulated the phosphorylation and degradation of IκB-αas well as the nuclear translocation of p65 in these cells.The inhibitors of NF-κB signaling pathway as well as IL-1 or TNF-αreceptor antagonist significantly inhibited the expressions of these inflammatory mediators in HCFs.Conclusion The alarmins generated by NHCS,including IL-1 and TNF-α,triggered the secretion of cytokine,chemokine and adhesion molecules through NF-κB signaling pathway.IL-1 and TNF-αare potential therapeutic targets for improving corneal stromal ulceration and inflammatory reactions in severe ocular burns.

关 键 词：警报素 炎症 结膜成纤维细胞 人角膜基质成纤维细胞 

分 类 号：R722.21[医药卫生—儿科]