中文摘要：

背景 巨噬细胞在脉络膜新生血管（CNV）中的作用尚存在争议,与其在不同微环境中的功能异质性有关,Notch信号通路参与眼内新生血管生长的调控,但其对CNV中巨噬细胞功能的调控作用尚未证实. 目的 探讨巨噬细胞在CNV生成中极化表型的变化与Notch信号通路对巨噬细胞极化表型的调控.方法 在体实验选择58只成年雄性C57BL小鼠,以随机数字表法按照造模后取材时间的不同随机分为光凝后3d组和7d组.每组选23只小鼠用视网膜光凝法诱导小鼠CNV模型,分别于光凝后3d和7d制备脉络膜铺片,采用免疫荧光染色法观察CNV面积和巨噬细胞阳性染色面积,血管内皮生长因子（VEGF）及肿瘤坏死因子α（TNF-α）的分泌情况;光凝后3d和7d对眼杯冰冻切片行免疫荧光染色,观察CNV中巨噬细胞的极化表型及其分泌VEGF、TNF-α的情况,评估巨噬细胞上Notch信号胞内段（NICD）相应分子标志物的表达情况.每组设定6只小鼠的左眼为正常对照眼,其右眼以激光光凝法建立CNV模型,采用实时荧光定量PCR（qRT-PCR）法检测眼杯组织中VEGF、TNF-α和巨噬细胞极化相关因子的表达情况.体外分离和培养C57BL小鼠的骨髓单核前体细胞,用粒细胞-巨噬细胞集落刺激因子（GM-CSF）诱导其分化为巨噬细胞,加入Notch信号通路抑制剂γ-分泌酶抑制剂（GSI）和极化诱导因子,24 h后收集细胞及培养液上清,通过qRT-PCR及ELISA法检测M1型、M2型巨噬细胞分子表面标志物的表达水平. 结果 与光凝后第3天比较,光凝后第7天CNV面积明显增大,但巨噬细胞浸润面积缩小,差异均有统计学意义（t=8.138、5.272,均P=0.000）.光凝后第3天,巨噬细胞在CNV周边和中央均有分布,第7天时局限于CNV中央部.光凝后第3天,色素上皮-脉络膜-巩膜复合体中精氨酸合酶1（Arg1）、甘露糖受体（MR）及白细胞介素-6（IL-6）mRNA的相对表达量明显高于第7天,而诱导型一氧?

英文摘要：

Background The role of macrophages （Mφ） in choroidal neovascularization （CNV） is still controversial due to the heterogeneity of Mφ in different microenvironments.Notch signaling is involves in a variety of ocular neovascularization including CNV.However,whether or how Notch signaling regulates the polarity convention and function of Mφ during CNV is below understood.Objective This study aimed to investigate the alteration of Mφ polarization and the regulation mediated by Notch signaling in CNV.Methods In an in vivo experiment,58 adult male C57B6 mice were grouped to post-photocoagulative 3 day group and 7 day group randomly based on the sampling time.CNV was induced by photocoagulation of retinas by 532 nm frequency doubling laser in 23 mice in each group.The CNV area,Mφ infiltrated area,secretion of vascular endothelial growth factor （VEGF） and tumor necrosis factor-α（TNF-α） were detected by choroidal flat mounts and immunofluorescence staining.The polarization of Mφ,the secretion of VEGF and TNF-α and the expression of activated Notch intracellular domain （NICD） were detected by immunofluorescence in frozen sections.The CNV was induced in the remained 6 mice in each group,with 14 laser spots in the right eyes and the left eyes as controls.Quantity real-time PCR （qRT-PCR） was performed to detect the polarization of Mφ and the expression of angiogenesis related factors.In an in vitro experiment,bone marrow derived macrophage （BMDM） was isolated and induced with granulocyte-macrophage colony stimulating factor （GMCSF） to differentiate into Mφ.The γ-secretion inhibitor （GSI） was added to BMDM culture environment with the polarization stimulus.The cells and the culture supernatant were collected to detect the molecule markers of M1 and M2 polarization by qRT-PCR and ELISA 24 hours later.Results The CNV area was significantly increased and the Mφ infiltrated area was decreased in the post-photocoagulative 7 day group compared with the 3 day group （t =8.138,5.272,bot