LV-EGFP标记兔角膜基质细胞体外基质层移植的实验观察  

Experimental observation of transplantation of rabbit corneal stromal cells with LV-EGFP in vitro

作　　者：张璐 李妍 李梦怡 王爔烔 李楠钰 孙子雯 胡竹林 Lu Zhang;Yan Li;Meng-Yi Li;Xi-Tong Wang;Nan-Yu Li;Zi-Wen Sun;Zhu-Lin Hu(Fourth Affiliated Hospital of Kunming Medical University,Yunnan Second People's Hospital,Yunnan Ophthalmology Research Institute,Key Laboratory of Ophthalmic Disease Research of Yunnan Province,Kunming 650021,Yunnan Province,China)

机构地区：[1]昆明医科大学第四附属医院云南省第二人民医院云南省眼科研究所云南省眼科疾病研究重点实验室

出　　处：《国际眼科杂志》2020年第1期32-36,共5页International Eye Science

基　　金：云南省眼科研究所、云南省眼科疾病研究重点实验室(No.2017DG008);云南省科技计划项目(No.2017IC064);云南省创新团队计划任务书(No.2017HC010)~~

摘　　要：目的:探讨兔角膜基质细胞(CSCs)体外移植兔角膜后的存活时间。方法:体外培养原代兔CSCs,并行细胞免疫组化鉴定,利用慢病毒载体(LV)携带标记基因增强型绿色荧光蛋白(EGFP)转染兔CSCs,倒置荧光显微镜下观察转染后细胞生长状态及荧光强度,体外动物实验,随机分为2组,实验组行LV-EGFP标记的兔CSCs细胞悬液角膜基质注射,对照组等量生理盐水角膜基质注射,转染后1wk,1mo取材冰冻切片观察移植的CSCs荧光,石蜡切片行苏木素-伊红(HE)染色观察组织形态。结果:LV-EGFP转染兔CSCs在倒置荧光显微镜下24h后可见少量荧光,96h和110h荧光较强,转染后的CSCs与正常CSCs细胞形态无明显差异;转染后1wk,1mo实验组中角膜基质层中可见绿色荧光,对照组中无绿色荧光;石蜡切片1wk实验组见明显上皮细胞增生及角膜轻微水肿,少量炎症细胞浸润,转染后1mo实验组上皮细胞增生减弱,未见角膜层水肿;对照组1wk,1mo均未见明显异常。结论:LV-EGFP标记的兔CSCs行体外角膜基质移植,可在角膜中至少存活1mo,且与邻近组织相容性较好。AIM:To investigate the survival time and distribution of rabbit corneal stromal cells(CSCs)after transplantation of rabbit corneal in vitro.METHODS:Primary rabbit CSCs was cultured in vitro and identified by immunohistochemical staining.using lentivirus(LV)with marker gene enhanced green fluorescent protein(EGFP)transfection rabbit CSCs,the growth status and fluorescence intensity of the transfected cells were observed under an inverted fluorescence microscope.The in vitro animal experiments were randomly divided into 2 groups.experimental group lines of LV-EGFP tag of rabbit CSCs suspension stromal injection,control group amount of normal saline injection corneal stroma,Frozen sections were taken 1wk and 1mo after surgery to observe the fluorescence of transplanted CSCs,and hematoxylin-eosin(HE)was used to observe the tissue morphology of paraffin sections.RESULTS:LV-EGFP transfected rabbit CSCs showed a small amount of fluorescence after 24h under an inverted fluorescence microscope,with the strongest at 96h and 110h.There was no significant difference in the morphology of the transfected CSCs and normal CSCs.Green fluorescence can be seen in the stromal layer of the cornea in the experimental group at 1wk and 1mo,while there is no green fluorescence in the control group.Paraffin section for 1wk showed obvious epithelial cell hyperplasia and slight corneal edema in the experimental group,and a small amount of inflammatory cell infiltration.1mo after surgery,the epithelial cell hyperplasia was weakened in the experimental group,and no corneal layer edema was observed.No obvious abnormality was found in the control group for 1wk and 1mo.CONCLUSION:Extracorporeal corneal stroma transplantation of LV-EGFP labeled rabbit CSCs can survive at least 1mo in the corneal and is compatible with adjacent tissues.

关 键 词：角膜基质细胞 慢病毒 增强型绿色荧光蛋白 细胞移植 

分 类 号：R73[医药卫生—肿瘤]