不同眼内灌注液对视网膜组织学及功能的影响  

Comparison of the effects of different intraocular infusion solutions on histology and function of retina

作　　者：朱莉 苗恒[1] 胡钦瑞 刘志明 白玉婧[1] 虞有智[1] 傅亚飞 夏会卡 黄旅珍[1] 齐赟[1] 邓洵[1] 李岩[1] 黎晓新[1] Zhu Li;Miao Heng;Hu Qinrui;Liu Zhiming;Bai Yujing;Yu Youzhi;Fu Yafei;Xia Huika;Huang Lvzhen;Qi Yun;Deng Xun;Li Yan;Li Xiaoxin(Department of Ophthalmology,Peking University People's Hospital,Eye Diseases and Optometry Institute,Beijing Key Laboratory of Diagnosis and Therapy of Retinal and Choroid Diseases,College of Optometry,Peking University Health Science Center,Beijing 100044,China)

机构地区：[1]北京大学人民医院眼科,眼视光中心,眼病与视光医学研究所,视网膜脉络膜疾病诊治研究北京市重点实验室,北京大学医学部眼视光学院,100044

出　　处：《中华实验眼科杂志》2021年第11期957-967,共11页Chinese Journal Of Experimental Ophthalmology

基　　金：国家重点基础研究发展计划项目(2011CB510200);国家自然科学基金项目(81670870)。

摘　　要：目的比较不同眼内灌注液对视网膜组织学及功能的影响。方法取人角膜内皮细胞(HCEC)、人视网膜色素上皮(HRPE)细胞和大鼠视网膜神经节细胞(RGC),分为正常对照组、平衡盐灌洗液(BSS)组和复方电解质眼内灌注液(CEIIS)组,分别在含体积分数10%DMEM/F12培养基、BSS和CEIIS中培养12、24、48 h,采用细胞计数试剂盒8(CCK8)法测定培养细胞的增生A值;采用细胞免疫荧光染色法检测培养细胞中凋亡相关蛋白表达;采用流式细胞术测定细胞凋亡率和细胞周期;采用乳酸脱氢酶(LDH)和琥珀酸脱氢酶(SDH)定量检测试剂盒检测细胞线粒体损伤。选用新西兰大耳白兔15只,采用抽签法随机分为对照组3只、BSS组6只和CEIIS组6只,均取左眼行玻璃体切割术,术中根据分组方法分别采用不同的眼内灌注液。分别于术前和术后24 h采用闪光视网膜电图(ERG)测定术眼视网膜功能,采用光相干断层扫描(OCT)检测实验眼视网膜各层结构变化。收集各时间点术眼眼球,采用TUNEL染色法检测视网膜各层细胞的早期凋亡情况;采用免疫组织化学染色法检测视网膜组织中细胞色素C和bax蛋白表达情况;透射电子显微镜下观察实验眼视网膜超微结构变化。结果BSS组和CEIIS组3种培养细胞均有不同程度的损伤,随培养时间延长,细胞增生减少,凋亡细胞数量增加;与BSS组相比,CEIIS组培养细胞排列致密整齐,细胞形态和大小均一。BSS组HRPE细胞和RGC的细胞凋亡率分别为(37.157±6.918)%和(29.993±12.330)%,明显高于CEIIS组的(4.163±1.310)%和(6.337±1.903)%,差异均有统计学意义(P=0.003、0.045)。正常对照组、BSS组和CEIIS组间HCEC和HRPE细胞的G0/G1+S期比例总体比较,差异均无统计学意义(HCEC:F=2.226,P=0.189;HRPE:F=2.634,P=0.151);BSS组RGC的G2/M分裂阻滞期比例高于正常对照组和CEIIS组,差异均有统计学意义(P=0.047、0.024)。各培养时间点CEIIS组HCEC、HRPE细胞和RGC的增生A值�Objective To compare the effects of different intraocular infusion solutions on histology and function of retina.Methods Human corneal endothelial cells(HCEC),human retinal pigment epithelium(HRPE)cells and rat retinal ganglion cells(RGC)were divided into normal control group,balanced saline solution(BSS)group and compound electrolyte intraocular irrigating solution(CEIIS)group,and the cells were cultured in 10%DMEM/F12 medium,BSS and CEIIS for 12,24 and 48 hours,respectively,according to grouping.The proliferation absorbance value of cultured cells was measured by cell counting kit-8(CCK8)method.The expression of apoptosis related proteins in cultured cells was detected by cellular immunofluorescence staining.The cell apoptosis rate and cell cycle were measured by flow cytometry.The mitochondrial damage was detected by lactate dehydrogenase(LDH)and succinate dehydrogenase(SDH)quantitative detection kit.Fifteen New Zealand white rabbits were randomly divided into control group(n=3),BSS group(n=6)and CEIIS group(n=6).The left eyes were taken for vitrectomy and different intraocular perfusion fluids were used during vitrectomy according to grouping.The retinal function of operative eyes was measured by flash electroretinogram(ERG)before operation and 24 hours after operation,and the structural changes of each layer of retina were detected by optical coherence tomography(OCT).The early apoptosis of retinal cells was detected by TUNEL staining.The expressions of cytochrome C and bax protein in retina were detected by immunohistochemical staining.The ultrastructural changes of retina were observed under a transmission electron microscope.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Peking University People's Hospital(No.2019PHE059).Results The three kinds of cultured cells in BSS and CEIIS groups were damaged in various degrees.With the extension of culture time,proliferated cells were decreased and the number of apoptotic cells was increased.C

关 键 词：玻璃体切割术 灌注液 细胞活性 视网膜功能 细胞凋亡 氧化应激 

分 类 号：R774.1[医药卫生—眼科]