摘要
目的观察超低温环境下长期保存后人角膜基质透镜的透明度及结构变化,探讨简单可行且有效的角膜基质透镜长期保存方案。方法收集2013-2020年于中山大学中山眼科中心海南眼科医院行飞秒激光小切口角膜透镜取出术(SMILE)200眼,手术中获取完整人角膜基质透镜标本200份。将人角膜基质透镜标本置于-80℃超低温冰箱中保存,按照保存时间的不同将标本分为1个月组、24个月组、60个月组和84个月组,每组50份。采用超微量分光光度计测量各组在300~800 nm波长范围内角膜基质透镜的透光率,每个透镜共连续检测10次,每次间隔50 nm;分别采用苏木精-伊红染色和Masson染色观察各组角膜基质透镜的组织学形态变化和胶原纤维结构改变;采用透射电子显微镜观察各组角膜基质透镜中胶原纤维排列方式及角膜基质细胞的超微结构变化;采用TUNEL染色法检测各组人角膜基质透镜中角膜基质细胞凋亡情况。结果各组450~800 nm波长范围内角膜基质透镜透光率比较差异均无统计学意义(均P>0.05)。Masson染色结果显示,1个月组角膜基质透镜胶原纤维排列整齐、紧密,24个月组部分胶原纤维间出现空泡,60个月组胶原纤维排列疏松,空泡增多,84个月组部分胶原纤维脱落,角膜基质透镜变薄;苏木精-伊红染色显示角膜基质透镜形态改变与胶原纤维变化相对应。透射电子显微镜下可见1个月组角膜基质透镜胶原纤维排列规则,角膜基质细胞呈长梭形,核膜完整,胞质丰富;24个月组胶原纤维排列稍疏松,角膜基质细胞变形,核膜不完整;60个月组胶原纤维排列疏松、不整齐,细胞核萎缩变形;84个月组胶原纤维排列紊乱,角膜基质细胞皱缩,核膜不连续,核质裂解。TUNEL染色结果显示,1个月组、24个月组、60个月组和84个月组透镜中基质细胞凋亡率分别为(87.80±1.17)%、(89.50±1.05)%、(89.30±1.51)%和(90.20±1.47)%,总体比较
Objective To observe the transparency and tissue structure changes of human corneal stromal lenticules after long-term cryopreservation and explore a simple and feasible method for long-term effective preservation of corneal stromal lenticules.Methods Two hundred samples of intact human corneal stromal lenticules from 200 eyes were obtained during femtosecond laser small-incision lenticule extraction(SMILE)in Hainan Eye Hospital,Zhongshan Ophthalmic Center from 2013 to 2020.The samples were divided into 1-month,24-month,60-month and 80-month group and were stored in an ultra-low temperature freezer for 1,24,60 and 84 months respectively at-80℃according to grouping,with 50 samples in each group.Transmittance of the corneal lenticules at wavelength of 300-800 nm was measured with an ultra-micro spectrophotometer and every lenticule was measured for 10 times with a 50 nm interval.The histomorphology and collagen fiber structure of the corneal lenticules were examined by hematoxylin-eosin staining and Masson staining,respectively.The arrangement of collagen fibers and ultrastructure changes of keratocytes in the samples were inspected with a transmission electron microscope.The apoptosis rate of keratocytes was determined by TUNEL staining.The study protocol was approved by an Ethics Committee of Hainan Eye Hospital at Zhongshan Ophthalmic Center(No.2013-003).This study complied with the Declaration of Helsinki.Written informed consent was obtained from each subject before surgery.Results The corneal lenticules were clear and intact in all groups and no significant difference in the transmittance within 450-800 nm wavelength was seen among the 4 groups(all at P>0.05).Masson staining revealed that the collagen fibers in the lenticules were neatly arranged and tightly packed in the 1-month group.In the 24-month group,interfibrous vacuoles were found in some collagen fibers.The arrangement of the collagen fibers was loose and more vacuoles were displayed in the 60-month group,and the loss of some collagen fibers appea
作者
韦琦
丁辉
钟探
余瀚洋
杨镇朵
范红明
何林艺
钟兴武
Wei Qi;Ding Hui;Zhong Tan;Yu Hanyang;Yang Zhenduo;Fan Hongming;He Linyi;Zhong Xingwu(State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center,Sun Yat-sen University,Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science,Guangzhou 510060,China;Hainan Eye Hospital and Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center,Sun Yat-sen University,Haikou 570300,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2021年第12期1031-1037,共7页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目(81870681)
海南省临床医学中心和海南省重点研发计划项目(ZDYF2020151)。
关键词
角膜疾病
角膜移植
角膜基质
生物保存
低温贮藏
超微结构
凋亡
透明度
Corneal diseases
Corneal transplantation
Corneal stroma
Preservation,biological
Cryopreservation
Ultrastructure
Apoptosis
Transparency
