眼科研究
眼科研究
안과연구
CHINESE OPHTHALMIC RESEARCH
2010年
3期
243-247
,共5页
水通道蛋白-4%Müller细胞%视网膜水肿%缺氧
水通道蛋白-4%Müller細胞%視網膜水腫%缺氧
수통도단백-4%Müller세포%시망막수종%결양
aquaporin-4%Müller cell%retinal edema%hypoxia
目的 利用体外培养的视网膜Müller细胞,研究在缺氧条件下视网膜Müller细胞上水通道蛋白-4(AQP-4)表达的变化.方法 采用组织块培养法从新西兰大白兔视网膜中获取Müller细胞,在含20%胎牛血清的DMEM培养液中原代培养,培养的细胞通过胶质纤维酸性蛋白(GFAP)免疫细胞化学染色及透射电子显微镜进行鉴定.取第2代细胞进行实验,将化学缺氧诱导剂CoCl_2联合DMEM培养液培养24h的细胞作为缺氧组;将DMEM培养液单独培养24h的细胞作为对照组.采用免疫细胞化学法及半定量RT-PCR法分别检测2组Müller细胞上AQP-4蛋白及AQP-4 mRNA的表达.结果 Müller细胞(第2代)上GFAP的阳性率为90%以上,细胞质染色呈棕色.细胞内含特征性的中间丝,细胞表面有微绒毛,细胞质内含丰富的细胞器.CoCl_2联合DMEM培养液培养24h后Müller细胞上AQP-4蛋白的表达较对照组明显增加(t=6.74,P＜0.05);AQP-4 mRNA的表达亦明显增加(t=21.79,P＜0.05).结论 缺氧能增强Müller细胞上AQP-4的表达,进而使视网膜内液体的积聚增加.提示Müller细胞在糖尿病视网膜病变(DR)或增生性视网膜病变的视网膜水肿形成过程中起重要作用.
目的 利用體外培養的視網膜Müller細胞,研究在缺氧條件下視網膜Müller細胞上水通道蛋白-4(AQP-4)錶達的變化.方法 採用組織塊培養法從新西蘭大白兔視網膜中穫取Müller細胞,在含20%胎牛血清的DMEM培養液中原代培養,培養的細胞通過膠質纖維痠性蛋白(GFAP)免疫細胞化學染色及透射電子顯微鏡進行鑒定.取第2代細胞進行實驗,將化學缺氧誘導劑CoCl_2聯閤DMEM培養液培養24h的細胞作為缺氧組;將DMEM培養液單獨培養24h的細胞作為對照組.採用免疫細胞化學法及半定量RT-PCR法分彆檢測2組Müller細胞上AQP-4蛋白及AQP-4 mRNA的錶達.結果 Müller細胞(第2代)上GFAP的暘性率為90%以上,細胞質染色呈棕色.細胞內含特徵性的中間絲,細胞錶麵有微絨毛,細胞質內含豐富的細胞器.CoCl_2聯閤DMEM培養液培養24h後Müller細胞上AQP-4蛋白的錶達較對照組明顯增加(t=6.74,P＜0.05);AQP-4 mRNA的錶達亦明顯增加(t=21.79,P＜0.05).結論 缺氧能增彊Müller細胞上AQP-4的錶達,進而使視網膜內液體的積聚增加.提示Müller細胞在糖尿病視網膜病變(DR)或增生性視網膜病變的視網膜水腫形成過程中起重要作用.
목적 이용체외배양적시망막Müller세포,연구재결양조건하시망막Müller세포상수통도단백-4(AQP-4)표체적변화.방법 채용조직괴배양법종신서란대백토시망막중획취Müller세포,재함20%태우혈청적DMEM배양액중원대배양,배양적세포통과효질섬유산성단백(GFAP)면역세포화학염색급투사전자현미경진행감정.취제2대세포진행실험,장화학결양유도제CoCl_2연합DMEM배양액배양24h적세포작위결양조;장DMEM배양액단독배양24h적세포작위대조조.채용면역세포화학법급반정량RT-PCR법분별검측2조Müller세포상AQP-4단백급AQP-4 mRNA적표체.결과 Müller세포(제2대)상GFAP적양성솔위90%이상,세포질염색정종색.세포내함특정성적중간사,세포표면유미융모,세포질내함봉부적세포기.CoCl_2연합DMEM배양액배양24h후Müller세포상AQP-4단백적표체교대조조명현증가(t=6.74,P＜0.05);AQP-4 mRNA적표체역명현증가(t=21.79,P＜0.05).결론 결양능증강Müller세포상AQP-4적표체,진이사시망막내액체적적취증가.제시Müller세포재당뇨병시망막병변(DR)혹증생성시망막병변적시망막수종형성과정중기중요작용.
Background Hypoxia is an important cause resulting in many retinal diseases,such as retinal edema,diabetic retinopathy,proliferative retinopathy and so on.ObjectiveThis study is to investigate the effects of hypoxia on the expression of AQP-4 in Müller cells in vitro.MethodsMüller cells were isolated from New Zealand white rabbits and primarily cultured in DMEM containing 20% fetal bovine serum by the explant culture method.The cells were identified by immunostaining for the glial fibrillary acidic protein(GFAP).Generation 2 of cells was cultivated with the chemical hypoxia inducer,CoCl_2,for 24 hours in the hypoxic group and only with DMEM in the control group.The expression of the AQP-4 protein in Müller cells was detected by immunocytochemistry.The expression of AQP-4 mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).ResultsAbout 90% of Müller cells(generation 2) presented a positive immunoreactivity for GFAP,showing a brown staining in the cytoplasm.Cultured cells displayed the presence of intermediate filaments,microvillus and various cellular organs.The Integralabsorbance of the AQP-4 protein in Müller cells was markedly increased 24 hours after incubation with CoCl_2 in comparison with the control group (t=6.74,P＜0.05).The expression level of AQP-4 mRNA in Müller cells was significantly enhanced 24 hours after incubation with CoCl_2 in comparison with the control group (t=21.79,P＜0.05). ConclusionHypoxia enchances the expression of AQP-4 in Müller cells and further increases fluid accumulation in the retina.These results suggest that Müller cells play an important role in the formation of retinal edema in diabetic retinopathy or proliferative retinopathy.
