高血糖对小鼠骨髓来源细胞参与脉络膜新生血管形成的体内动态观察  

In vivo dynamical monitoring of rat bone marrow-derived cells participating in choroidal neovascularization under hyperglycemia

作　　者：王瑜[1] 王雨生[1] 曹丰[2] 张健[3] 吕洋[1] 王海燕[1] 药立波[3] 

机构地区：[1]第四军医大学西京医院眼科全军眼科研究所,西安710032 [2]第四军医大学西京医院心内科,西安710032 [3]第四军医大学西京医院基础部生物化学和分子生物学教研室,西安710032

出　　处：《中华实验眼科杂志》2014年第4期298-302,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：国家重点基础研究发展计划（973计划）项目（2011CB510200）;国家自然科学基金项目（81070748、81200708、81300770）

摘　　要：背景 我们先前的研究提示高血糖可加重脉络膜新生血管（CNV）的严重程度,并有可能通过促进骨髓来源细胞（BMCs）的趋化参与血管发生,且已证实在体生物发光成像（BLI）技术能实时、活体观察CNV的动态变化,但糖尿病与CNV发生的关联性尚无明确的研究证据.目的 应用BLI技术并结合组织学研究方法,动态观察高血糖状态下BMCs参与CNV的形成过程. 方法 将荧光素酶-绿色荧光蛋白（FlucGFP）双转基因小鼠的BMCs移植至野生型C57BL/6J小鼠构建嵌合体,嵌合度＞85％的小鼠纳入实验并按照随机数字表法分为对照组和糖尿病组,每组9只.糖尿病组嵌合体小鼠采用腹腔内注射链脲佐菌素法[（STZ60 mg/（kg＆#183;d）,连续5d]建立糖尿病模型,血糖浓度＞15 mmol/L者视为建模成功,对照组不进行注射.2个组嵌合体小鼠均应用532 nm倍频激光视网膜光凝法诱导CNV形成.采用IVIS Kinetics小动物成像系统,分别于CNV模型建立后第1、3、5、7、14、21和28天对2个组小鼠进行BLI检测,动态观察CNV小鼠眼部发光信号.于CNV模型建立后第7天处死动物,制备视网膜脉络膜组织切片行苏木精-伊红染色,比较2个组小鼠CNV的长度和厚度.结果 流式缅胞仪分析显示,C57BL/6J小鼠行Fluc-GFP双转基因小鼠BMCs移植后28 d,纳入实验的嵌合体小鼠平均嵌合度为（88.85±2.46）％;糖尿病组嵌合体小鼠平均血糖浓度为（17.88±0.86） mmol/L.小鼠视网膜脉络膜组织病理学检查显示,CNV造模后第7天,新生血管穿过Bruch膜到达视网膜下腔,糖尿病组小鼠CNV长度为（338.67±33.17） μm,明显长于对照组小鼠的（180.33±24.68） μm,差异有统计学意义（t=8.943,P＜0.05）;2个组间小鼠CNV的厚度差异无统计学意义（t=1.790,P＞0.05）.CNV建模后第1天,两组小鼠眼部出现光学信号,第7天光学信号值均达最高,且糖尿病组明显强于对照组;第14天后两组小鼠眼部信号减弱.CNV建Background Our previous study demonstrated that hyperglycemia aggravate the choroidal neovascularization （CNV） by promoting the chemotaxis process of bone marrow-derived cells （BMCs）.Bioluminescence imaging （BLI） can dynamically monitor CNV in vivo.However,how diabetes mellitus （DM）participate in CNV is still in research.Objective This study was to dynamically observe the influence of BMCs to CNV under hyperglycaemia by using BLI combined with histopathology.Methods BMCs from luciferase-green fluorescent protein （Fluc-GFP） double transgenic mice were injected to adult wild type C57BL/6J mice （nine mice per group） via caudal vein to create the chimera models with a chimerism degree higher than 85%,and the chimeric mice were randomized into the control group and DM group based on randomized number table.Streptozotocin [60 mg/（kg ＆#183; d）] was intraperitoneally injected daily for 5 days to establish the DM models in the chimeric mice of the DM group.CNV was induced in the chimeric mice of both control group and DM group with 532 nm laser photocoagulation.BLI signal of BMCsFluc＋GFP＋ was in vivo examined by IVIS Kinetics system 1,3,5,7,14,21 and 28 days after CNV modeling.At the seventh day after laser,part of mice were sacrificed,and choroidal and retinal sections were prepared for histopathological examination.The length and thickness of CNV were compared between the control group and DM group.The use and care of experimental followed Statement of ARVO.Results The chimerism degree of the chimeric mice was （88.85 ± 2.46） % 28 days after BMCs transplantation,and the blood glucose concentration in the DM group was （17.88±0.86）mmol/L.Histopathological examination revealed that CNV broke through the Bruch membrane toward subretinas.The length of the CNV was （338.67±33.17） μm in the DM group and （180.33±24.68）μm in the control group,showing a significant difference between the two groups （t =8.943,P〈0.05）.However,no significant difference was seen in the CNV t

关 键 词：高血糖 药物诱导 脉络膜新生血管 糖尿病 实验性 骨髓来源细胞 激光 光凝 不良反应 发光测量 方法 在体生物发光成像 

分 类 号：R773.4[医药卫生—眼科]