不同固定剂对高渗状态下角膜上皮细胞中YAP核质定位的影响  

Effects of different fixatives on the nuclear-cytoplasmic localization of YAP in human corneal epithelial cells under hyperosmotic stress

作　　者：戴义芹 张婧 徐建江 Dai Yiqin;Zhang Jing;Xu Jianjiang(Eye Institute and Department of Ophthalmology,Eye&ENT Hospital,Fudan University,NHC Key Laboratory of Myopia and Related Eye Diseases,Key Laboratory of Myopia and Related Eye Diseases,Chinese Academy of Medical Sciences,Shanghai 200031,China)

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,国家卫生健康委员会近视眼及相关眼病重点实验室,中国医学科学院近视眼及相关眼病重点实验室,上海200031

出　　处：《中华实验眼科杂志》2025年第4期308-314,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金(82201150、82171020)。

摘　　要：目的探讨不同固定剂对高渗状态下角膜上皮细胞中Yes相关蛋白(YAP)核质定位的影响。方法将永生化人角膜上皮细胞分为对照组和高渗组,常规培养1 d后高渗组细胞采用450 mOsM渗透压的氯化钠溶液处理1 h,对照组不做特殊处理。采用4种不同的固定剂(4%多聚甲醛、-20℃预冷无水乙醇、-20℃预冷甲醇丙酮1∶1混合液、Zamboni固定液)固定细胞20 min,行免疫荧光染色,观察对照组与高渗组细胞YAP在细胞内的定位变化情况。结果4%多聚甲醛固定后,角膜上皮细胞形态较好,对照组与高渗组细胞中YAP主要集中在细胞核内,对照组与高渗组细胞核内YAP平均荧光强度比较,差异无统计学意义(t=1.803,P=0.121);无水乙醇固定后角膜上皮细胞呈现一定程度的皱缩变形,YAP荧光染色弥散,2个组细胞中YAP阳性信号主要定位在细胞质中。与对照组相比,高渗组角膜上皮细胞核中YAP平均荧光强度有所降低,差异无统计学意义(t=0.803,P=0.453)。甲醇丙酮1∶1混合液固定后角膜上皮细胞形态不佳,YAP主要定位在细胞质中,高渗组细胞核内YAP荧光强度较对照组有所降低,差异无统计学意义(t=1.067,P=0.327)。Zamboni固定液固定后细胞结构完整,轮廓清晰,能明显观察到不同状态下角膜上皮细胞中YAP核质转位现象。高渗组YAP平均荧光强度为197.5±34.5,明显高于对照组的62.2±10.0,差异有统计学意义(t=7.530,P<0.001)。结论在免疫荧光染色实验中,角膜上皮细胞中YAP的核质定位会受到不同固定剂处理的影响,应用Zamboni固定液比4%多聚甲醛、无水乙醇、甲醇丙酮1∶1混合液能更好地观察到高渗刺激后YAP的核移位现象。Objective To explore the impact of various fixatives on the nuclear-cytoplasmic localization of Yes-associated protein(YAP)in human corneal epithelial cells under hyperosmotic stress condition.Methods Immortalized human corneal epithelial cells were divided into control group and hypertonic group.After 1 day of normal culture,cells of the hypertonic group were exposed to hyperosmotic medium at 450 mOsM by adding sodium chloride for 1 hour.No special treatment was given to the control group.Both groups of cells were fixed with four different fixatives,including 4%paraformaldehyde(PFA),-20℃precooled absolute ethanol,-20℃precooled methanol-acetone 1∶1 mixture,and Zamboni fixative solution for 20 minutes.Subsequent to fixation,immunofluorescent staining procedures were performed to identify the intracellular localization of YAP in the two groups.Results After fixation with 4%PFA,human corneal epithelial cells showed normal morphology with YAP mainly in the nucleus in both groups,and there was no significant difference in the mean nuclear YAP fluorescence intensity between the two groups(t=1.803,P=0.121).After fixation with absolute ethanol,cells showed some degree of shrinkage and deformation,diffuse YAP fluorescence staining with YAP-positive signals mainly localized in the cytoplasm in both groups,and the mean nuclear YAP fluorescence intensity was slightly decreased in the hypertonic group compared with the control group,but the difference was not statistically significant(t=0.803,P=0.453).After fixation with methanol-acetone 1∶1 mixture,cells were crenulated with YAP mainly in the cytoplasm,and the mean nuclear YAP fluorescence intensity in the hypertonic group was slightly decreased compared with the control group,but the difference was not statistically significant(t=1.067,P=0.327).After fixation with Zamboni solution,the cell structure was complete and clearly outlined,and the YAP nucleoplasmic translocation phenomenon could be clearly observed in cells in different states.The mean nuclear YAP fluoresc

关 键 词：干眼 泪液渗透压 角膜上皮细胞 免疫荧光技术 固定剂 Yes相关蛋白核质定位 

分 类 号：R772.2[医药卫生—眼科]