曲安奈德和酮咯酸氨丁三醇对缺氧视网膜Müller细胞上水通道蛋白-4及VEGF表达的影响  

Effects of triamcinolone acetonide and Ketorolac tromethamine on the expressions of aquaporin-4 andvascular endothelial growth factor in hypoxic Miiller cell

作　　者：蔡维[1] 程扬[3] 柯丽娜[2] 张有顺[2] 李国保[1] 

机构地区：[1]湖北医药学院附属东风医院眼科,湖北省十堰市442008 [2]湖北医药学院附属东风医院实验中心,湖北省十堰市442008 [3]华中科技大学同济医学院附属协和医院眼科,武汉430022

出　　处：《中华实验眼科杂志》2013年第8期744-748,共5页Chinese Journal Of Experimental Ophthalmology

摘　　要：背景 曲安奈德（TA）玻璃体腔内注射可引起高眼压、白内障及眼内炎等并发症.酮咯酸氨丁三醇（Ketorolac）是一种作用机制与TA相似、不良反应较少的新型非甾体类抗炎药,其在治疗视网膜水肿方面可能替代或部分替代TA. 目的 研究TA和Ketorolac对CoCl2诱导的缺氧条件下兔视网膜Müller细胞上水通道蛋白-4（AQP4）及血管内皮生长因子（VEGF）表达的影响,探讨其治疗视网膜水肿的作用机制. 方法 收集健康新西兰白兔20只,采用酶消化法培养兔视网膜Müller细胞并进行鉴定.取第2代细胞用于实验.分别用含500μmol/L CoCl2的DMEM 2 ml孵育细胞0、6、12、24 h建立视网膜Müller细胞缺氧模型,采用半定量逆转录PCR（RT-PCR）法观察缺氧对Müller细胞中AQP4 mRNA及VEGF mRNA表达的影响,确定造模的最佳时间点.将培养24 h的细胞分为正常对照组,缺氧对照组,缺氧＋50、100、200 mg/L TA组,缺氧＋50、100、200 mg/L Ketorolac组,分别在缺氧细胞培养基中加入相应的药物进行共培养,采用半定量RT-PCR法观察TA和Ketorolac对Müller细胞中AQP4 mRNA及VEGF mRNA表达的影响. 结果 培养的细胞呈不规则形、星形,细胞核呈卵圆形,培养14 ～25 d时细胞达到80％以上融合.95％的培养细胞中间丝波形蛋白（vimentin）、α-平滑肌肌动蛋白（α-SMA）和神经胶质纤维酸性蛋白（GFAP）阳性表达,呈绿色荧光.RT-PCR法检测结果显示,培养后各时间点间Müller细胞中AQP4 mRNA和VEGF mRNA表达的吸光度（A）值总体比较差异均有统计学意义（F=18.70、53.20,P＜0.01）,CoCl2作用6、12、24 h值均明显高于正常Müller,差异均有统计学意义（P＜0.05）,其中24 h时AQP4 mRNA和VEGF mRNA的表达量达峰值.缺氧24 h时8个组Müller细胞中AQP4 mRNA和VEGF mRNA表达（A）值的总体比较差异均有统计学意义（F=27.98、10.03,P＜0.01）；与缺氧对照组比较,除缺氧＋50 mg/L TA组、缺氧＋50 mg/L Ketorolac�Background Intravitreal injection with triamcinolone acetonide （TA） may cause complications,including increase of intraoculapressure （IOP）,cataracand endophthalmitis.Ketorolatromethamine （Ketorolac） inew,lesadverse reactionof non-steroidal anti-inflammatory drug.The action mechanism of Ketorolaisimilato TA.Therefore,Ketorolamay be completely opartly replace Tin the treatmenof retinal edema.Objective The purpose of thistudy wato investigate the effectof Tand Ketorolaon the expressionof aquaporin-4 （AQP4） and vasculaendothelial growth facto（VEGF） in hypoxiretinal Müllecellin vitro and to explore the mechanism of treating retinal edemwith Tand Ketorolac.MethodThe propose of research and use of the animalwere approved by Animal ExperimenResearch Review Committee of Hubei University of Medicine.Twenty eyeof New Zealand albino rabbitwere extracted and the retinal tissue waisolated.The Müllecellwere cultured and passaged using the enzymatidigestion method and Müllecellwere identified using glial fibrillary acidiprotein （GFAP）,vimentin and α-smooth muscle actin （α-SMA） by immunofluorescence staining.The hypoxicell modelwere established by culturing the cellin DMEM with 500 μmol/L CoCl2 fo0,6,12,24 hours.The cellof hypoxifo24 hourwere divided into normal control group,hypoxicontrol group,hypoxia＋50,100,200 mg/L To50,100,200 mg/L Ketorolagroups.Corresponding drugwere added into the medium in the differengroups.The expressionof AQP4 mRNand VEGF mRNin Müllecellwere detected by semi-quantitative reverse transcription PC（RT-PCR）.ResultThe cellgrew well and reached 80% confluence with the irregulashape and ovoid nuclei 14-15 dayaftecultured.More than 95% primary cellshowed positive reaction to GFAP,vimentin and α-SMA.The expressing levelof AQP4 mRNand VEGF mRNin Müllecell（values） were significantly differenin varioutime point（AQP4 mRNA：F=18.70,P〈0.01 ; VEGF mRNA：F =53.20,P〈0.01）,and those of 6,12 and 24 houraftecultured with CoCl2were increased than those withouCoCl2 �

关 键 词：曲安奈德 酮咯酸氨丁三醇 MÜLLER细胞 缺氧 水通道蛋白-4 血管内皮生长因子 

分 类 号：R774[医药卫生—眼科]