中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
4期
298-302
,共5页
王瑜%王雨生%曹丰%张健%吕洋%王海燕%药立波
王瑜%王雨生%曹豐%張健%呂洋%王海燕%藥立波
왕유%왕우생%조봉%장건%려양%왕해연%약립파
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
高血糖/藥物誘導%脈絡膜新生血管%糖尿病/實驗性%骨髓來源細胞%激光%光凝/不良反應%髮光測量/方法%在體生物髮光成像
고혈당/약물유도%맥락막신생혈관%당뇨병/실험성%골수래원세포%격광%광응/불량반응%발광측량/방법%재체생물발광성상
Hyperglycemia/chemically induced%Choroidal neovascularization%Diabetes mellitus/experimental%Bone marrow cell%Laser%Light coagulation/adverse effect%Luminescent measurement /method%Bioluminescence imaging
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·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建模后第5、7、14和21天,糖尿病组小鼠眼部光学信号均明显强于对照组小鼠,差异均有统计学意义(t=3.411、5.594、5.067、2.663,P＜0.05).结论 高血糖可促使更多的BMCs参与CNV的形成过程,加重CNV的严重程度.BLI技术可用于评估和比较高血糖条件下BMCs在CNV形成过程中的动态变化.
揹景 我們先前的研究提示高血糖可加重脈絡膜新生血管(CNV)的嚴重程度,併有可能通過促進骨髓來源細胞(BMCs)的趨化參與血管髮生,且已證實在體生物髮光成像(BLI)技術能實時、活體觀察CNV的動態變化,但糖尿病與CNV髮生的關聯性尚無明確的研究證據.目的 應用BLI技術併結閤組織學研究方法,動態觀察高血糖狀態下BMCs參與CNV的形成過程. 方法 將熒光素酶-綠色熒光蛋白(FlucGFP)雙轉基因小鼠的BMCs移植至野生型C57BL/6J小鼠構建嵌閤體,嵌閤度＞85％的小鼠納入實驗併按照隨機數字錶法分為對照組和糖尿病組,每組9隻.糖尿病組嵌閤體小鼠採用腹腔內註射鏈脲佐菌素法[(STZ60 mg/(kg·d),連續5d]建立糖尿病模型,血糖濃度＞15 mmol/L者視為建模成功,對照組不進行註射.2箇組嵌閤體小鼠均應用532 nm倍頻激光視網膜光凝法誘導CNV形成.採用IVIS Kinetics小動物成像繫統,分彆于CNV模型建立後第1、3、5、7、14、21和28天對2箇組小鼠進行BLI檢測,動態觀察CNV小鼠眼部髮光信號.于CNV模型建立後第7天處死動物,製備視網膜脈絡膜組織切片行囌木精-伊紅染色,比較2箇組小鼠CNV的長度和厚度.結果 流式緬胞儀分析顯示,C57BL/6J小鼠行Fluc-GFP雙轉基因小鼠BMCs移植後28 d,納入實驗的嵌閤體小鼠平均嵌閤度為(88.85±2.46)％;糖尿病組嵌閤體小鼠平均血糖濃度為(17.88±0.86) mmol/L.小鼠視網膜脈絡膜組織病理學檢查顯示,CNV造模後第7天,新生血管穿過Bruch膜到達視網膜下腔,糖尿病組小鼠CNV長度為(338.67±33.17) μm,明顯長于對照組小鼠的(180.33±24.68) μm,差異有統計學意義(t=8.943,P＜0.05);2箇組間小鼠CNV的厚度差異無統計學意義(t=1.790,P＞0.05).CNV建模後第1天,兩組小鼠眼部齣現光學信號,第7天光學信號值均達最高,且糖尿病組明顯彊于對照組;第14天後兩組小鼠眼部信號減弱.CNV建模後第5、7、14和21天,糖尿病組小鼠眼部光學信號均明顯彊于對照組小鼠,差異均有統計學意義(t=3.411、5.594、5.067、2.663,P＜0.05).結論 高血糖可促使更多的BMCs參與CNV的形成過程,加重CNV的嚴重程度.BLI技術可用于評估和比較高血糖條件下BMCs在CNV形成過程中的動態變化.
배경 아문선전적연구제시고혈당가가중맥락막신생혈관(CNV)적엄중정도,병유가능통과촉진골수래원세포(BMCs)적추화삼여혈관발생,차이증실재체생물발광성상(BLI)기술능실시、활체관찰CNV적동태변화,단당뇨병여CNV발생적관련성상무명학적연구증거.목적 응용BLI기술병결합조직학연구방법,동태관찰고혈당상태하BMCs삼여CNV적형성과정. 방법 장형광소매-록색형광단백(FlucGFP)쌍전기인소서적BMCs이식지야생형C57BL/6J소서구건감합체,감합도＞85％적소서납입실험병안조수궤수자표법분위대조조화당뇨병조,매조9지.당뇨병조감합체소서채용복강내주사련뇨좌균소법[(STZ60 mg/(kg·d),련속5d]건립당뇨병모형,혈당농도＞15 mmol/L자시위건모성공,대조조불진행주사.2개조감합체소서균응용532 nm배빈격광시망막광응법유도CNV형성.채용IVIS Kinetics소동물성상계통,분별우CNV모형건립후제1、3、5、7、14、21화28천대2개조소서진행BLI검측,동태관찰CNV소서안부발광신호.우CNV모형건립후제7천처사동물,제비시망막맥락막조직절편행소목정-이홍염색,비교2개조소서CNV적장도화후도.결과 류식면포의분석현시,C57BL/6J소서행Fluc-GFP쌍전기인소서BMCs이식후28 d,납입실험적감합체소서평균감합도위(88.85±2.46)％;당뇨병조감합체소서평균혈당농도위(17.88±0.86) mmol/L.소서시망막맥락막조직병이학검사현시,CNV조모후제7천,신생혈관천과Bruch막도체시망막하강,당뇨병조소서CNV장도위(338.67±33.17) μm,명현장우대조조소서적(180.33±24.68) μm,차이유통계학의의(t=8.943,P＜0.05);2개조간소서CNV적후도차이무통계학의의(t=1.790,P＞0.05).CNV건모후제1천,량조소서안부출현광학신호,제7천광학신호치균체최고,차당뇨병조명현강우대조조;제14천후량조소서안부신호감약.CNV건모후제5、7、14화21천,당뇨병조소서안부광학신호균명현강우대조조소서,차이균유통계학의의(t=3.411、5.594、5.067、2.663,P＜0.05).결론 고혈당가촉사경다적BMCs삼여CNV적형성과정,가중CNV적엄중정도.BLI기술가용우평고화비교고혈당조건하BMCs재CNV형성과정중적동태변화.
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 · 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 thickness between the two groups (t =1.790,P＞0.05).Light signals appeared 1 day and reach strongest 7 days after CNV modeling in both groups.The Light signals were stronger in the DM group than those in the control group on 5,7,14 and 21 days after CNV modeling (t =3.411,5.594,5.067,2.663,all at P＜0.05).Conclusions Hyperglycemia can promote more BMCs to participate in the pathogenesis and aggravation of CNV.The behavior of BMCs in CNV can be evaluated using BLI in vivo.
