【作者】 王秀青；

【导师】 贺翔鸽；

【作者基本信息】 第三军医大学 ， 眼科学， 2007， 博士

【摘要】 目的通过回顾性分析准本院准分子激光角膜屈光术(laser keratorefractive surgery,LKRS)后皮质类固醇性高眼压(glucocorticoid-induced ocular hypertension,GIOH)的发病率及临床相关因素,检测GIOH患者泪液总蛋白和各蛋白成分的变化,并分析其糖皮质激素受体(Glucocorticoid receptor,GR)基因多态性的变化,探讨GIOH的发病机制。方法实验分以下三部分第一部分LKRS后GIOH临床相关因素分析回顾性分析LKRS后2060例患者,计算GIOH的发病率;对其中88例GIOH患者及随机选择的125例正常眼压者,采用Logistic回归分析GIOH与年龄、性别、眼别、最大径线屈光度、最大径线曲率、切削深度、眼底杯/盘比(C/D)值及术前眼压的相关性。第二部分LKRS后GIOH泪液蛋白变化研究对象:LKRS术前组、术后1周地塞米松(dexamethasone,DEX)组、、术后1周氟米龙(fluorometholone,FML)组、术后1月组、术后3月组各10例,术后高眼压组23例。方法:1.毛细管法取基础泪液5ul,-20℃保存备用;2.BCA法测泪液总蛋白浓度;3.泪液SDS-PAGE,Bio-Rad图像分析系统分析泪液中各种蛋白的变化。第三部分GR基因多态性分析研究对象:重庆地区健康人266名,准分子激光角膜屈光术后GIOH患者39人,正常眼压者37人,POAG患者11人。方法:1.所有受试者均取外周静脉血1ml,提取全血DNA;2.对重庆地区健康人进行GR基因分型,并计算各位点SNP发生频率。基因分型方法:根据GR基因ER22/23EK、N363S和BclⅠSNP位点设计PCR引物,PCR扩增后,选择合适的限制性内切酶进行酶切及电泳鉴定;3.哈迪—温伯格(Hardy-Weinberg,H-W)平衡分析;4.根据健康人GR基因分型的结果,对LKRS后GIOH患者、正常眼压者及POAG患者进行GR基因BclⅠ位点、N363S位点SNP分型5.分别对各组进行哈迪—温伯格(Hardy-Weinberg,H-W)平衡分析6.计算各组SNP发生频率,并分析SNP与GIOH和POAG的关系,比较组间差异。结果1.LKRS后GIOH发病率为3.5%;GIOH的发生与患者眼底C/D值明显相关;2.泪液总蛋白:LKRS后1周DEX组明显高于其它各组;LKRS后高眼压组各时间点的变化趋势同正常眼压组;3.LKRS后GIOH患者泪液溶菌酶及乳铁蛋白浓度增加;4.重庆地区健康人中未发现GR基因编码区ER22/23EK、N363S多态性,而发现有内含子BclⅠ位点多态性存在,G等位基因发生频率为23.5%;H-W平衡分析发现所选人群符合H-W平衡。5.LKRS后正常眼压和高眼压患者GR基因BclⅠ位点SNP发生频率分别为21.62%、18.29%,两者间无显著差异;6.POAG患者GR基因N363S和BclⅠ位点SNP发生频率分别为0、33.3%,与其他各组间无显著差异。结论LKRS后GIOH的发病率为3.5%,GIOH的发生与患者视乳头杯/盘比值明显相关;LKRS后早期泪液总蛋白浓度增加,GC由DEX改为FML后泪液总蛋白浓度降低,推测与DEX的强效抗炎、抗免疫和对间叶组织的作用有关;观察到LKRS后GIOH患者泪液溶菌酶及乳铁蛋白浓度增加的现象,其机制有待进一步研究。重庆地区人群中GR基因无ER22/23EK及N363S多态性存在,与白种人不同,而与日本人相似;存在BclⅠ位点SNP,但发生频率为23.5%,较白种人低。LKRS后GIOH患者与正常眼压患者、健康人群、POAG患者在GR基因N363S位点和BclⅠ位点SNP发生频率上无显著差异;虽然GR基因N363S和BclⅠ位点多态性与GC敏感性增强有关,但与GIOH、POAG的发生之间没有统计学关联,推测GR基因内或GR基因外可能存在其它与GIOH、POAG发生有关的多态性。更多还原

【Abstract】 ObjectiveTo study the pathogenesy of glucocorticoid-induced ocular hypertension（GIOH） in patients after laser keratorefractive surgery（LKRS）,by analyzing retrospectively the incidence and clinical related factors of GIOH,investigating their variations of total tear protein and tear protein compositions,and detecting polymorphism changes within glucocorticoid receptor（GR） gene.MethodsThe whole experiment is consisted of 3 parts as follows.The 1^st part Analysis of the incidence and clinical related factors of GIOH in patients after LKRS2060 subjects after LKRS were recruited in our retrospective study to assess the incidence of GIOH;by logistic regression analysis,data of GIOH group and normal group were collected to analyse the association between GIOH and age,sex,right/left eye,max saggital diopter,max saggital curvature,depth of cut,papilla cup/disc ratio of ocular fundus and preoperative intraocular pressure.The 2^nd part Tear protein variations in patients with GIOH after LKRSSubjects10 subjects respectively in preoperative group,postoperative groups including of the 1st week with dexamethasone（DEX）,the 1st week with fluorometholone（FML）,the 1st month and the 3rd month;23 subjects with GIOH after LKRS.Methods1.5ul basal tear was collected by capillary tube method and stored at -20℃.2.Measurement of total tear protein with BCA kit.3.Variation of tear protein compositions by Bio-Rad image analysis system after SDS-PAGE. The 3^rd part Polymorphism analysis of GR geneSubjects:266 healthy individuals in Chongqing,39 patients with GIOH and 37 patients with normal intraocular pressure（IOP） after LKRS,12 patients with POAG.Methods:1.1ml peripheral venous blood was collected and genomic DNA was extracted from the whole blood.2.Genotyping:first,PCR primers were designed according to SNP within GR gene and suitable restriction enzymes were selected for PCR amplification,then genotyping of GR gene was finished in healthful subjects in Chongqing region,at last each SNP frequency was assessed.3.Hardy-Weinberg equilibrium assay4.According to SNP results from healthy population,genotyping of GR gene was done by BclⅠand N363S SNPs in subjects with GIOH and normal IOP after LKRS,and patients with POAG.5.Hardy-Weinberg equilibrium assay of all groups.6.Incidences of 2 kinds of SNPs were calculated,and then differences among groups and associations between these SNPs and GIOH,POAG were assessed.Results1.Incidence of GIOH after LKRS is 3.5%and is associated with C/D ratio of optic nerve head.2.Total tear protein level in postoperative group at 1^st week with DEX is higher than other groups;its variation tendency in GIOH group at different time is same to group with normal IOP.3.Tear lysozyme and lactoferrin level in patients with GIOH is higher.4.There is no ER22/23EK and N363S polymorphism of GR gene inChongqing’s healthy population,but BclⅠSNP exists and its G allele frequency is 23.5%.All groups in this study are in line with H-W equilibrium. 5.The incidence of BclⅠSNP of GR gene is 21.62%and 18.57%respectively in group of normal IOP and GIOH after LKRS. 6.The incidence of N363S and BclⅠSNP of GR gene is 0 and 18.57%respectively in group of POAG,and there is no significant difference with other groups. ConclusionIncidence of GIOH after LKRS is 3.5%and is associated significantly with C/D ratio of optic nerve head;total tear protein level in patients at early postoperative period increased but decreased when GC changed from DEX to FML.So we suppose this is induced by strong anti-inflammatory,anti-immunization and effects on mesenchymal tissue of DEX;We also found tear lysozyme and lactoferrin level in patients with GIOH after LKRS improved,which maybe caused by tear secretion changes of patients with GIOH.The Chongqing population hasn’t polymorphisms of ER22/23EK and N363S location but has BclⅠSNP,which is different from Caucasian but same to Japanese in the Asia. However,SNP frequency of BclⅠin Chongqing population is lower than Caucasian’s. There is no difference in SNP frequency of N363S and BclⅠamong patients with and without GIOH after LKRS and with POAG.Although SNPs of N363S and BclⅠwithin GR gene has been reported to be associated with increased sensitivity to GC,no statistical association was found between these two SNPs and GIOH.Maybe there are some other SNPs associated with GIOH,which needs further research.更多还原