中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
8期
715-720
,共6页
陈春丽%周仲楼%闫东升%郑景伟%宋宗明
陳春麗%週仲樓%閆東升%鄭景偉%宋宗明
진춘려%주중루%염동승%정경위%송종명
表皮生长因子%Müller细胞%氧化损伤%年龄相关性黄斑变性
錶皮生長因子%Müller細胞%氧化損傷%年齡相關性黃斑變性
표피생장인자%Müller세포%양화손상%년령상관성황반변성
Epidermal growth factor%Müller cell%Oxidative damage%Age-related macular degeneration
背景 目前认为氧化损伤机制在年龄相关性黄斑变性(AMD)的发病中起着重要作用,主要与血-视网膜屏障的破坏有关,而Müller细胞是稳定血-视网膜内屏障功能的主要细胞成分.研究表明,表皮生长因子(EGF)可促进实验动物视网膜Müller细胞的增生和迁移,但其对人Müller细胞的作用研究较少. 目的 探讨EGF对体外氧化损伤的人眼Müller细胞增生和迁移的影响及其作用机制. 方法 将人眼Müller细胞系MIO-M1细胞进行培养并用胶质纤维酸性蛋白(GFAP)、第Ⅷ因子、α-平滑肌肌动蛋白(α-SMA)、人角蛋白及S -1 00免疫组织化学法进行鉴定.在无血清DMEM中加入不同质量浓度(0、1、10、30、100 mg/L) EGF,用5-溴脱氧尿嘧啶核苷( BrdU)标记法测定MIO-MI细胞的阳性率.根据干物方式的不同将培养细胞分为EGF组、H2O2损伤组、EGF+H2O2组、葡萄糖氧化酶(GO)组、GO+EGF组、EGF+ LY294002+H2 O2组,采用MTT比色法测定各组培养细胞的增生情况(A590).对培养的人眼Müller细胞采用划痕实验观察H2O2损伤条件下0、1、10、30、100 mg/L EGF作用24、48、72 h后对Müller细胞增生、迁移的影响;采用Western blot技术检测EGF对体外不同培养条件下M üller细胞Akt信号传导通路的作用. 结果 正常培养条件下10、30、100 mg/L EGF作用后Müller细胞的增生率分别为28.0％、32.9％、39.0％,明显高于0 mg/L、1 mg/L EGF组(24.5％、26.2％).在H2O2和GO分别培养细胞的条件下,高质量浓度的EGF组Müller细胞的吸光度(A570)值明显大于低质量浓度组,各组总体差异有统计学意义(F=23.582,P=0.000).与EGF+H2O2组比较,EGF+ LY294002+H2O2组Müller细胞的吸光度(A570)值明显下降.10 mg/L EGF促进Müller细胞迁移的作用最强.0.08 mmol/LH2O2作用Müller细胞后Akt信号通路激活,提前2h加入外源性EGF后,100 mg/L EGF对抗氧化所致Müller细胞的损伤作用最明显,同时提前2h加入外源性EGF和Akt信号通路阻断剂LY294002后,Akt信号传导通路的保护作用减弱. 结论 EGF对体外培养的Müller细胞具有促进增生及迁移的作用,10 mg/L EGF促进Müller细胞迁移作用最强.100 mg/L外源性EGF抗Müller细胞氧化损伤作用最强,其作用机制是激活Akt信号传导通路.
揹景 目前認為氧化損傷機製在年齡相關性黃斑變性(AMD)的髮病中起著重要作用,主要與血-視網膜屏障的破壞有關,而Müller細胞是穩定血-視網膜內屏障功能的主要細胞成分.研究錶明,錶皮生長因子(EGF)可促進實驗動物視網膜Müller細胞的增生和遷移,但其對人Müller細胞的作用研究較少. 目的 探討EGF對體外氧化損傷的人眼Müller細胞增生和遷移的影響及其作用機製. 方法 將人眼Müller細胞繫MIO-M1細胞進行培養併用膠質纖維痠性蛋白(GFAP)、第Ⅷ因子、α-平滑肌肌動蛋白(α-SMA)、人角蛋白及S -1 00免疫組織化學法進行鑒定.在無血清DMEM中加入不同質量濃度(0、1、10、30、100 mg/L) EGF,用5-溴脫氧尿嘧啶覈苷( BrdU)標記法測定MIO-MI細胞的暘性率.根據榦物方式的不同將培養細胞分為EGF組、H2O2損傷組、EGF+H2O2組、葡萄糖氧化酶(GO)組、GO+EGF組、EGF+ LY294002+H2 O2組,採用MTT比色法測定各組培養細胞的增生情況(A590).對培養的人眼Müller細胞採用劃痕實驗觀察H2O2損傷條件下0、1、10、30、100 mg/L EGF作用24、48、72 h後對Müller細胞增生、遷移的影響;採用Western blot技術檢測EGF對體外不同培養條件下M üller細胞Akt信號傳導通路的作用. 結果 正常培養條件下10、30、100 mg/L EGF作用後Müller細胞的增生率分彆為28.0％、32.9％、39.0％,明顯高于0 mg/L、1 mg/L EGF組(24.5％、26.2％).在H2O2和GO分彆培養細胞的條件下,高質量濃度的EGF組Müller細胞的吸光度(A570)值明顯大于低質量濃度組,各組總體差異有統計學意義(F=23.582,P=0.000).與EGF+H2O2組比較,EGF+ LY294002+H2O2組Müller細胞的吸光度(A570)值明顯下降.10 mg/L EGF促進Müller細胞遷移的作用最彊.0.08 mmol/LH2O2作用Müller細胞後Akt信號通路激活,提前2h加入外源性EGF後,100 mg/L EGF對抗氧化所緻Müller細胞的損傷作用最明顯,同時提前2h加入外源性EGF和Akt信號通路阻斷劑LY294002後,Akt信號傳導通路的保護作用減弱. 結論 EGF對體外培養的Müller細胞具有促進增生及遷移的作用,10 mg/L EGF促進Müller細胞遷移作用最彊.100 mg/L外源性EGF抗Müller細胞氧化損傷作用最彊,其作用機製是激活Akt信號傳導通路.
배경 목전인위양화손상궤제재년령상관성황반변성(AMD)적발병중기착중요작용,주요여혈-시망막병장적파배유관,이Müller세포시은정혈-시망막내병장공능적주요세포성분.연구표명,표피생장인자(EGF)가촉진실험동물시망막Müller세포적증생화천이,단기대인Müller세포적작용연구교소. 목적 탐토EGF대체외양화손상적인안Müller세포증생화천이적영향급기작용궤제. 방법 장인안Müller세포계MIO-M1세포진행배양병용효질섬유산성단백(GFAP)、제Ⅷ인자、α-평활기기동단백(α-SMA)、인각단백급S -1 00면역조직화학법진행감정.재무혈청DMEM중가입불동질량농도(0、1、10、30、100 mg/L) EGF,용5-추탈양뇨밀정핵감( BrdU)표기법측정MIO-MI세포적양성솔.근거간물방식적불동장배양세포분위EGF조、H2O2손상조、EGF+H2O2조、포도당양화매(GO)조、GO+EGF조、EGF+ LY294002+H2 O2조,채용MTT비색법측정각조배양세포적증생정황(A590).대배양적인안Müller세포채용화흔실험관찰H2O2손상조건하0、1、10、30、100 mg/L EGF작용24、48、72 h후대Müller세포증생、천이적영향;채용Western blot기술검측EGF대체외불동배양조건하M üller세포Akt신호전도통로적작용. 결과 정상배양조건하10、30、100 mg/L EGF작용후Müller세포적증생솔분별위28.0％、32.9％、39.0％,명현고우0 mg/L、1 mg/L EGF조(24.5％、26.2％).재H2O2화GO분별배양세포적조건하,고질량농도적EGF조Müller세포적흡광도(A570)치명현대우저질량농도조,각조총체차이유통계학의의(F=23.582,P=0.000).여EGF+H2O2조비교,EGF+ LY294002+H2O2조Müller세포적흡광도(A570)치명현하강.10 mg/L EGF촉진Müller세포천이적작용최강.0.08 mmol/LH2O2작용Müller세포후Akt신호통로격활,제전2h가입외원성EGF후,100 mg/L EGF대항양화소치Müller세포적손상작용최명현,동시제전2h가입외원성EGF화Akt신호통로조단제LY294002후,Akt신호전도통로적보호작용감약. 결론 EGF대체외배양적Müller세포구유촉진증생급천이적작용,10 mg/L EGF촉진Müller세포천이작용최강.100 mg/L외원성EGF항Müller세포양화손상작용최강,기작용궤제시격활Akt신호전도통로.
Background Oxidative damage plays an important role in pathogenesis of age-related macular degeneration( AMD ),and its mechanism is the destroy of blood-retinal barrier.Müller cells is a primary component to stabilize the inner barrier of the blood-retina.Researches showed that epidermal growth factor(EGF) can promote the proliferation and migration of animal Müller cells,but less study was found in the effect of EGF on human Müller cells. Objective The present study was to investigate the effects of EGF on the proliferation and migration of human Müller cells and its molecular mechanism. Methods Human Müller cell line MIO-M1 cells were cultured and incubated,and cultured cells were identified using glial fibrillory acidic protein (GFAP),factor Ⅷ,α-smooth muscle actin( α-SMA ),keratin and S-100.Different concentrations of EGF( 0,1,10,30,100 mg/L)was added in freeserum DMEM,and the positive rate of the cells was calculated using 5-bromo-2-deoxyuridine(BrdU) method.The cells were divided into EGF group,H2 O2 group,EGF + H2 O2 group,glucose oxidase ( GO ) group,GO + EGF group,EGF + LY294002+H2O2 group according to the different intervention,and the effects of LY294002 on the proliferation of Müller cells (A590 )were detected by colorimetric assay for cellular growth and survival( MTT assay).The scratch test of Müller cells was used to assess the influence of EGF(0,1,10,30,100 mg/L)on H2 O2-induced damage of human Müller cell.Western blot was used to detect the cell proliferation under the protection of EGF on co-cultured cells using LY294002 and H2O2 and the activation of Akt signal pathways. Results The proliferative rates of the cells were 28.0％,32.9％,39.0％ in 10,30,100 mg/L EGF groups respectively and obviously higher than those in 0,1 mg/L EGF groups (24.5 ％,26.2 ％ ).Under the H2O2 culture,GO culture,respectively,the A570 value of the Müller cell in high concentrations of EGF groups was significantly increased in comparison with lower concentrations EGF groups with the statistical significance among the groups( F=23.582,P=0.000).Compared with EGF+H2O2 group,the A570value of the Müller cells was lowed in EGF+LY294002+H2O2 group.The maximum migration rate of Müller cells was found in 10 mg/L EGF group.Western blot revealed that the presence of H2O2 reinforced the expression of Akt in Müller cells,however,pretreatment with 100 mg/L EGF antagonized the harmful effect of H2O2 on Müller cells.Meanwhile,pretreatment with EGF and LY294002 reduced the expression of Akt in Müller cells. Conclusions EGF can induce the proliferation and migration of human Müller cells with the strongest effect in 10 mg/L.100 mg/L exogenous EGF has a stronger protection to the Müiller cells against H2O2-induced cell damage by activating the PI3KAkt cell survival pathway.
