兔准分子激光角膜切削术后角膜血小板源性生长因子mRNA表达的变化
眼科研究 2000年第3期第18卷 实验研究
作者:钟一声 程枫 周颖明 廉井财 王康孙 邓伟
单位:钟一声 周颖明 廉井财 王康孙 邓伟(200025 上海第二医科大学瑞金医院眼科中心);程枫(上海第二医科大学人类基因治疗研究中心)
关键词:准分子激光屈光性角膜切削术;角膜上皮细胞;角膜基质;血小板源性生长因子;上皮下混浊
摘要 目的 研究角膜上皮下混浊(haze)形成的发生机制,检测准分子激光屈光性角膜切削术(PRK)后角膜上皮和基质血小板源性生长因子(PDGF)表达的变化。方法 新西兰白兔施行PRK后1,2,3月用裂隙灯显微镜观察haze形成情况,并用原位核酸分子杂交方法,检测角膜上皮和基质PDGFmRNA的表达。结果 正常角膜上皮细胞有PDGF mRNA表达,基质层无表达;PRK后角膜上皮细胞PDGF mRNA表达增加,术后2月表达最强,且基质中亦有轻微表达。上皮细胞PDGF mRNA表达强弱与haze形成的轻重密切相关。结论 PDGF参与PRK后伤口愈合过程,且调节着haze的形成和发展。
分类号 R772
Expression of platelet-derived growth factor mRNA in rabbit cornea after photorefractive keratectomy
Zhong Yisheng,Cheng Feng,Zhou Yingming
(Ophthalmic Center,Ruijin Hospital,Shanghai Second Medical University,Shanghai 200025)
Abstract Objective To study the mechanism of haze formation and to investigate the expression of platelet-derived growth factor (PDGF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK).Methods Twenty white rabbits were randomly divided into 4 groups,and PRK was performed on each eye of 15 rabbits.Haze formation was determined by a slit-lamp examination at the 1st,2nd and 3rd month after PRK,and the expression of PDGF mRNA was detected by in situ hybridization.ResultsThe corneal haze was first seen one month after PRK.The most obvious haze formation was observed at two months,and haze decreased or disappeared by the 3rd month after PRK.PDGF mRNA expression was present in the normal corneal epithelium,but was not present in the normal corneal stroma.Expression of PDGF mRNA in the corneal epithelium increased after PRK,with the peak appearing in the 2nd month.PDGF expression was detected in corneal stroma in the 2nd month.The extent of the expression of PDGF mRNA was related to the amount of haze formation.Conclusion PDGF takes part in promoting corneal wound healing after PRK,and may contribute to the formation and development of corneal haze. Key words photorefractive keratectomy corneal epithelium corneal stroma platelet-derived growth factor haze
准分子激光屈光性角膜切削术(photorefractive keratectomy,PRK)是治疗近视安全和有效的方法。然而,其术后发生的屈光回退和上皮下混浊(haze)影响着其预测性和稳定性。一般认为,它们与术后伤口愈合过程密切相关[1]。角膜伤口愈合是一复杂的生物学过程,细胞因子参与并调节其愈合。本文旨在研究PRK后角膜血小板源性生长因子(platelet-derived growth factor,PDGF)mRNA的表达变化,探讨其与术后常见并发症的关系。
1 材料与方法
1.1 动物模型建立
取新西兰白兔20只,体重2.5~3 kg,无眼疾病,雌雄兼用。随机分为正常对照组(5只)、PRK后1月组(5只)、2月组(5只)和3月组(5只)。用50 mg/kg戊巴比妥钠兔耳缘静脉麻醉后,以1%Alcaine滴眼表面麻醉,按常规方法将兔施行PRK,术后结膜囊涂金霉素眼膏。使用激光器为Chiron Vision 117-C型(USA),输入-8.0 D切削程序,切削深度156 μm,光学消融直径5.6 mm。激光参数为能量120 mJ/cm2,频率50 Hz。
1.2 术后观察及处理
PRK后1周内,每日用手持裂隙灯观察眼前段情况,并点0.25%氯霉素滴眼液1次。于PRK后1月、2月和3月分别将兔全身麻醉,裂隙灯检查角膜haze形成情况,并按Rai等[2]haze分级标准进行分级并记录。无菌操作下摘除眼球,剪下完整角膜,沿角膜中心将角膜一分为二,迅速投入液氮中保存备用。同样方法获得对照组角膜并保存。
1.3 PDGF mRNA检测
1.3.1 试剂:寡核苷酸探针PDGF[3]:5’-GACGTATTCCACCTTGGCCA-3’经DNA合成仪合成。DIG3’末端寡核苷酸标记试剂盒和显色系统(Boehringer Mannheim公司);顺丁烯二酸(Sigma公司);鲑鱼精DNA(Sigma公司),所有试剂均用DEPC水配制。
1.3.2 探针标记:按标记试剂盒说明进行3’末端标记,标上DIG-11-ddUTP。
1.3.3 实验器材处理:参照文献方法进行[3],载玻片使用前涂以多聚赖氨酸。
1.3.4 原位杂交:参照Boehringer公司试剂盒推荐的检测方法。角膜组织冰冻切片(厚约5 μm),并将对照组、术后1,2和3月组角膜切片按顺序粘于同一载玻片上,以保证杂交和显色条件一致。每份标本切片3张,干燥后4%多聚甲醛固定15 min,PBS洗涤;室温预杂交1 h;置湿盒中37℃,杂交16~18 h;杂交后依次用2×SSC 15 min,1×SSC 15 min,0.5×SSC 37℃ 15 min,0.5×SSC 15 min,以洗去多余未杂交探针;2%绵羊血清封闭30 min;加1∶1 000抗体室温2 h;加缓冲显色液1 ml(含NBT 4.5 μl,X-P 3.5 μl),置37℃暗处反应4~5 h;HE复染,显微镜观察结果;阴性对照:杂交液中使用未标记探针。
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