中文摘要：

背景 基质金属蛋白酶-2（MMP-2）/MMP-13在脉络膜新生血管（CNV）生成过程中发挥重要作用,但CNV原位细胞中MMP-2/MMP-13的表达有限.骨髓来源细胞（BMCs）参与CNV生成,可能是CNV中MMP-2/MMP-13的重要来源,而微小RNA188-5p（miR188-5p）可能是调控BMCs表达MMP-2/MMP-13的关键节点. 目的 探讨参与CNV生成的BMCs表达MMP-2/MMP-13的情况及BMCs表达的MMP-2/MMP-13是否受miR188-5p调控.方法 将表达绿色荧光蛋白（GFP）的转基因雌性小鼠的骨髓细胞移植到野生型雌性C57BL/6J小鼠以建立C57BL/6J.GFP嵌合体小鼠模型,流式细胞术检测嵌合度大于85％的42只小鼠纳入实验作为实验组,未进行骨髓细胞移植的野生型雌性C57BL/6J小鼠作为对照组.两组小鼠均以视网膜激光光凝法诱导CNV,两个组分别于光凝前及光凝后第1、3、5、7、10、14、28天各取3只小鼠分离视网膜脉络膜组织,采用ELISA法检测小鼠视网膜脉络膜组织匀浆中MMP-2/MMP-13质量浓度的变化;采用实时荧光定量PCR（RT-qPCR）法检测上述时间点小鼠视网膜脉络膜组织中miR188-5p mRNA的相对表达量变化;采用免疫荧光染色和原位杂交技术观察上述时间点小鼠CNV区GFP阳性细胞中MMP-2/MMP-13和miR188-5p的表达情况,并进行定量分析.结果 实验组C57BL/6J.GFP嵌合体模型小鼠外周血单核细胞中表达GFP细胞所占比例平均为（90.67＆#177;3.02）％,符合实验要求.ELISA检测显示,对照组与实验组小鼠视网膜脉络膜匀浆中MMP-2/MMP-13的表达趋势一致,总体比较差异均无统计学意义（MMP-2：F=0.060,P=0.810;MMP-13：F=0.012,P=0.915）.ELISA和免疫荧光均显示在诱导CNV后1d,实验组和对照组MMP-2表达均快速增加,3d时表达量最高,实验组占总表达量的64.21％,随后两组MMP-2表达均下降.两组MMP-13表达量在诱导CNV后1d缓慢增加,7d时表达量最高,实验组占总表达量的79.61％,随后MMP-13总体表达下降.靶基因预测结果可显示MMP-2?

英文摘要：

Background Matrix metalloproteinases （MMPs） play important roles in the formation of choroidal neovascularization （CNV）,but its mail origin is not ocular cells in situ.Bone marrow-derived cells （BMCs） participate in the formation of CNV and is probably a primary source of expressing MMPs in CNV.MMP-2/MMP-13 is speculated to be the regulating target genes of miR188-5p.Objective This study was to verify whether BMCs are the main source of MMPs,and whether the MMP-2/MMP-13 expression is potentially regulated by miR188-5p.Methods BMCs expressed green fluorescent protein （GFP） from transgenic female C57BL/6J mice were transplanted to female wild-type C57BL/6J mice to establish C57BL/6J.GFP chimeras models,and 42 mice with chimerisms more than 85% by flow cytometry were included as the experimental group.Other 42 wild-type C57BL/6J mice without the BMCs transplantation were enrolled as the control group.CNV was induced by laser coagulation of retinas on the mice of both groups.MMP-2/MMP-13 levels in the retinochoroid tissue were quantified by ELISA at day 1,3,5,7,10,14,and 28 after photocoagulation.The expression of miR188-5p mR NA in the retinochoroid tissue was assayed by real-time quantitative PCR （RT-qPCR）.Immunofluorescence stain and fluorescent in situ hybridization were used to identify the MMP-2/MMP-13 and miR188-5p expressed by GFP-positive BMCs in CNV,and the expression level was quantified by images analysis.Results The proportion of GFP＋ mouse mononuclear cells was （90.67＆#177;3.02） % in the C57BL/6J.GFP chimeras.The concentration changes of MMP-2/MMP-13 in retinochoroid homogenate showed a same tendency with the lapse of time between the experimental group and the control group （MMP-2：F=0.060,P =0.810 ; MMP-13：F =0.012,P =0.915）.The expression level was zoomed in retinochoroid tissue after induce of CNV with the maximal value on the third day in both groups,and the proportion in the experimental group was 64.21% ;while the expression level of MMP-13 was slowly raised aft