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

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（ICAM-1,CD54）、人类白细胞抗原DR（HLA—DR）、白细胞介素1β（IL-1β）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（IFN-γ）0U／ml、IFN-γ300U／ml、IFN-γ300U／ml＋多西环素10μg／ml、IFN-γ300U／ml＋多西环素20μg／ml、IFN-γ300U／ml＋多西环素40μg／ml、IFN-γ300U／ml＋地塞米松100μg／ml,24h后收集细胞,流式细胞学检测CD54、HLA—DR及IL-1β的表达,Western blot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IL-1β,加入IFN-γ后表达明显增加,加入多西环素和地塞米松后CD54和IL-1β的表达下降（P〈0．01）。随着所加多西环素浓度的增加,CD54和IL-1β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HLA—DR的表达及凋亡的产生,加入IFN-γ及多西环素后HLA—DR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IL-1β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。（中华眼科杂志,2005,41：842-846）

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

OBJECTIVE: To evaluate the effects of doxycycline on the regulation of intercellular adhesion molecule-1 (ICAM-1, CD54), interleukin-1beta (IL-1beta), 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-gamma, 300 U/ml IFN-gamma, 300 U/ml IFN-gamma with 10 microg/ml doxycycline, 300 U/ml IFN-gamma with 20 microg/ml doxycycline, 300 U/ml IFN-gamma with 40 microg/ml doxycycline or 300 U/ml IFN-gamma with 100 microg/ml dexamethasone for 24 hours. The amount of CD54, HLA-DR and IL-1beta 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-1beta. IFN-gamma increased the amount of CD54 and IL-1beta (P < 0.01). Doxycycline and dexamethasone inhibited the IFN-gamma induced increase of express of CD54 and IL-1beta of cultured conjunctival epithelial cells, and the inhibiting effect was dependent on the concentration of doxycycline (P < 0.01). Very little HLA-DR and apoptosis were detected before and after treatment with IFN-gamma. CONCLUSION: Doxycycline can suppress the expression of inflammatory cytokine such as CD54 and IL-1beta, which suggests that doxycycline may be a potent drug for the treatment of ocular surface inflammatory disease.