中文摘要：

背景 角膜新生血管（CNV）是导致角膜盲的主要原因之一,研究表明紧密连接蛋白中的闭锁小带蛋白1（ZO-1）对病理性新生血管的发生有调节作用,能通过紧密连接结构构成有效的生理屏障,抑制病理性新生血管的生长,但ZO-1对CNV是否发挥作用尚不清楚. 目的 探讨ZO-1在实验性CNV发生及发展中的作用及其机制.方法 将清洁级7～8周龄雄性BALB/c小鼠24只按随机数字表法随机分为碱烧伤15s组和碱烧伤40 s组,用NaOH滤纸贴附小鼠左眼中央角膜的方法构建CNV模型,于碱烧伤后2周在裂隙灯显微镜下观察并比较两组小鼠CNV生长情况,采用逆转录PCR（RT-PCR）法检测并比较两组小鼠角膜组织中ZO-1 mRNA的表达.另取鼠龄和性别相匹配同种小鼠54只随机分为3个组,均用NaOH滤纸贴附小鼠左眼中央角膜40 s的方法构建CNV模型,造模后分别用质量分数0.2％透明质酸钠（HA）配制的10 mg/L的ZO-1抗体和0.2％ HA配制的5 mg/L缺氧诱导因子-1α（HIF-1α）重组蛋白点眼1周,每日3次.于实验后2周摘除大鼠左眼角膜,用免疫组织化学法检测CD31在CNV中的表达,鉴定CNV的形成数目和面积;采用RT-PCR法检测3个组小鼠角膜组织中血管内皮生长因子（VEGF） mRNA的表达;采用流式细胞术检测碱烧伤角膜中巨噬细胞特异性标志物F4/80、中性粒细胞特异性标志物Ly-6G的阳性细胞比例,评价炎性细胞的浸润情况.结果 裂隙灯显微镜下可见造模后2周小鼠CNV达高峰;碱烧伤15 s组小鼠轻度、中度、重度CNV的眼数分布明显少于碱烧伤40 s组,差异有统计学意义（x2=6.032,P=O.049）;碱烧伤15s组和碱烧伤40 s组小鼠角膜中ZO-1 mRNA的相对表达量分别为1.53±0.04和1.15±0.08,差异有统计学意义（t=4.157,P=0.014）.免疫组织化学法检测显示,ZO-1抗体干预组和HIF-1α阳性对照组小鼠角膜组织中CD31阳性细胞数多于0.2％ HA组,差异均有统计学意义（t=-129.590、-226.820,均

英文摘要：

Background Corneal neovascularization （CNV） is one of the causes of corneal blindness.Studies showed that zonula occludens-1 （ZO-1） can inhibit pathological angiogenesis through physical barrier formed by tight junction structure.However,whether ZO-1 plays a role in CNV is unclear.Objective The aim of this study was to explore the effect of ZO-1,a tight junction protein on experimental CNV.Methods The CNV models were established in the left eyes of 24 clear male BALB/c mice aged 7-8 weeks by putting NaOH filter paper in the center of corneas for 15 seconds （15 s group） or 40 seconds （40 s group）.CNV was examined and evaluated under the slit lamp microscope,and the expression of ZO-1 mRNA in the corneas were detected and compared by reverse transcription PCR （RT-PCR） between the two groups 2 weeks after modeling.In addition,54 models created by the same method were assigned to 3 groups according to randomized number table,0.2% hyaluronic acid （HA）,antiZO-1 neutralizing antibody （10 mg/L） ＋0.2% HA and mouse hypoxia inducible factor-1α （HIF-1α） recombinant protein （5 mg/L）＋0.2% HA were topically administrated in the mice three times a day for 1 week after modeling respectively.The corneas were extracted 2 weeks after application of the drugs.Expression of CD31 in the CNV was assayed to calculate the number and the area of CNV by immunohistochemistry.The expression of VEGF mRNA in the corneas was detected by RT-PCR.The percentages of macrophage-specific F4/80 positive cells and neutrophilsspecific Ly-6G positive cells were calculated to evaluate the infiltrations of inflammatory cells in the corneas by flow cytometry.Results In 2 weeks after alkali burn of corneas,the number of severe CNV was more in the 40 s group than that in the 15 s group （x2 =6.032,P=0.049）,and the expression level of ZO-1 mRNA was lower in the 40 s group than that in the 15 s group （1.15±0.08 versus 1.53±0.04） （t=4.157,P=0.014）.CD31 positive cell number was more and the staining area was l