中文摘要：

背景 研究发现一氧化氮（NO）及一氧化氮合成酶（NOS）参与新生血管的发生、发展,但其在角膜新生血管（CNV）发生过程中的具体作用机制是眼科界值得关注的问题. 目的 研究NOS及其抑制剂NW-硝基-L-精氨酸甲酯（L-NAME）在实验性CNV发生过程中的作用,并通过体外研究验证NOS及L-NAME对人血管内皮细胞（RECs）血管管腔形成的作用,为眼科新生血管性疾病的防治提供实验依据.方法7～8周龄雄性BALB/c小鼠36只用NaOH滤纸贴附角膜中央的方法构建小鼠左眼CNV模型,然后用随机数字表法将小鼠分为L-NAME干预组和PBS对照组,L-NAME干预组小鼠于造模前1周开始腹腔内注射10 g/LL-NAME 0.5 ml,每周3次,持续3周,而PBS对照组小鼠以同样的方法注射PBS.分别于造模后2、4、7、14 d在裂隙灯显微镜下观察CNV形成情况,用全角膜铺片法检测CD31在CNV中的表达以鉴定CNV面积；采用逆转录聚合酶链反应（RT-PCR）法检测2个组小鼠角膜组织中NOSmRNA的表达；采用Western blot法检测人RECs中血管内皮生长因子（VEGF）蛋白的表达；利用体外实验,观察人RECs的管腔形成能力.采用独立样本t检验对2个组小鼠上述各检测指标的结果进行差异比较,分类资料采用X2检验. 结果裂隙灯显微镜下可见小鼠造模后2周CNV生长达高峰,而L-NAME干预组小鼠CNV明显少于PBS对照组.造模后2、4、7 d,L-NAME干预组小鼠角膜中NOS mRNA的相对表达量均明显低于PBS对照组,差异均有统计学意义（t=19.481、22.059、10.961,P＜0.01）.全角膜铺片法检测显示,L-NAME干预组被CD31标记的RECs面积与总面积的比值为0.59 ±0.01,明显小于PBS对照组的0.78±0.10,差异有统计学意义（t=3.078,P＜0.05）.Western blot法检测表明,造模0、2、4、7d,L-NAME干预组人RECs中VEGF蛋白的相对表达量均明显降低,造模后4d、7d两组比较差异均有统计学意义（-7.696、17.953,P＜0.01）.管腔形成实验中,L-NAM

英文摘要：

Background Though nitric oxide （NO） and NO synthase （NOS） have a critical role in angiogenesis,their effects on corneal neovascularization （CNV） and mechanism need to be further explored.Objective The aim of this study was to explore the effects of NOS and its antagonist,Nw-nitro-L-arginine methyl ester （L-NAME） on experimental CNV in mice,and investigate the influence of NOS and L-NAME on the tube formation of human retinal endothelial cells （RECs） in vitro.Methods The CNV models were established in the left eyes of 36 male BALB/c mice aged 7-8 weeks by application of the filter paper with NaOH in the center of corneas.The mice were randomized into two groups.L-NAME of 10 g/L （0.5 ml） was intraperitoneally injected 1 week before induction of CNV three times a week for three weeks in the mice of the L-NAME injection,and PBS was used in the same way in the control group.CNV was examined under the slit lamp biomicroscope 2,4,7,14 days after NaOH burn.The expression of CD31 in the CNV was assayed to calculate the ratio of CNV area and total corneal area using whole mount technique.The expression of NOS mRNA in the corneal tissue was detected by reverse transcriptase polymerase chain reaction （PCR）,and VEGF expression in the human RECs was assayed by Western blot.The vessel formation number of cultured human RECs with or without L-NAME was performed by matrigel in vitro.Grouped t test was used to compare the differences of the parameters between the two groups.Results CNV developed and peaked 2 weeks after the application of NaOH on the mice corneas,and the CNV was obviously less in the L-NAME group compared with the control group.The expression of NOS mRNA in the corneas （NOS mRNA/ GAPDH mRNA）was significantly lower in the L-NAME group than that of the control group 2,4,7 days after CNV induction （t =19.481,t=22.059,t=10.961,all at P〈0.01）.The ratio of the CD31 positive area in whole corneal area was 0.59± 0.01 in the L-NAME group,and that of the control group was 0.78±0.10,sh