多西环素对培养的人结膜上皮细胞炎性反应及凋亡的影响  

Effect of doxycycline on inflammation-related cytokines and apoptosis in human conjunctival epithelial cells

作　　者：肖启国[1] 刘祖国[1] 张梅[1] 郑健樑[1] 张志红[1] 罗丽辉[1] 陈家祺[1] 

机构地区：[1]中山大学中山眼科中心中山大学眼表疾病研究中心

出　　处：《中华眼科杂志》2005年第9期842-846,共5页Chinese Journal of Ophthalmology

基　　金：广东省高教厅千百十工程基金资助项目(粤教科[2000]21号);广东省科委重点科技基金资助项目(2KM052045);国家自然科学基金资助项目(30070804);国家杰出青年基金资助项目(30225044)

摘　　要：目的探讨多西环素对体外培养的人结膜上皮细胞细胞黏附分子1(ICAM1,CD54)、人类白细胞抗原DR(HLADR)、白细胞介素1β(IL1β)表达及凋亡的影响。方法混合消化液培养法培养人结膜上皮细胞,免疫组织化学方法进行细胞鉴定。培养至第3代或第4代,在细胞培养液中分别加入γ干扰素(IFNγ)0U/ml、IFNγ300U/ml、IFNγ300U/ml+多西环素10μg/ml、IFNγ300U/ml+多西环素20μg/ml、IFNγ300U/ml+多西环素40μg/ml、IFNγ300U/ml+地塞米松100μg/ml,24h后收集细胞,流式细胞学检测CD54、HLADR及IL1β的表达,Westernblot检测CD54的表达。72h后收集细胞,碘化丙啶(PI)染色,流式细胞学检测凋亡。结果培养的正常人结膜上皮细胞可以表达少许CD54和IL1β,加入IFNγ后表达明显增加,加入多西环素和地塞米松后CD54和IL1β的表达下降(P<0.01)。随着所加多西环素浓度的增加,CD54和IL1β的表达逐渐降低(P<0.01)。培养的正常人结膜上皮细胞未发现明显的HLADR的表达及凋亡的产生,加入IFNγ及多西环素后HLADR的表达及凋亡水平无明显变化(P>0.05)。结论多西环素可抑制培养的正常人结膜上皮细胞CD54和IL1β等炎性相关因子的产生,提示多西环素对干眼等非感染性眼表炎性疾病的治疗可能具有一定的应用前景。Objective To evaluate the effects of doxycycline on the regulation of intercellular adhesion molecule-1 （ ICAM-1 ,CD54） , interleukin-1β （IL-1β） , human leukocyte antigen DR （HLA-DR） and apoptosis in human conjunctival epithelial cells. Methods Human primary conjunctival epithelial cells were isolated and cultured from donors and identified by immunohistochemistry. Cultured epithelial cells were treated with either 0 U/ml IFN-γ, 300 U/ml IFN-γ, 300 U/ml IFN-γ with 10 μg,/ml doxycycline, 300 U/ml IFN-γ with 20 μg,/ml doxycycline, 300 U/ml IFN-γ with 40 μg/ml doxycycline or 300 U/ml IFN-γ with 100 μg,/ml dexamethasone for 24 hours. The amount of CD54, HLA-DR and IL-1β was measured by flow cytometry and western blot analysis. Apoptosis was evaluated by flow cytometry after the cultured epithelial cells were treated for 72 hours. Results Cultured conjunctival epithelial cells can express CD54 and IL-1β. IFN-γ, increased the amount of CD54 and IL-1β （P 〈 0. 01 ）. Doxcycline and dexamethasone inhibited the IFN-γ induced increase of express of CD54 and IL-1β of cultured conjunctival epithelial cells, and the inhibiting effect was dependent on the concentration of doxycycline （P 〈0. 01 ）. Very litde HLA-DR and apoptosis were detected before and after treatment with IFN-γ. Conclusion Doxycycline can suppress the expression of inflammatory cytokine such as CD54 and IL-1β, which suggests that doxycycline may be a potent drug for the treatment of ocular surface inflammatory disease. （Chin J Ophthalmol , 2005,41：842-846 ）

关 键 词：多西环素 细胞培养 结膜上皮细胞 炎性反应 细胞凋亡 分子量标志 

分 类 号：R77[医药卫生—眼科]