小鼠胚胎干细胞条件培养液培养的人角膜内皮细胞在脱细胞猪角膜基质上单层细胞片的构建

Formation of cell sheet on acellular porcine corneal stroma with human corneal endothelial cells cocultured by mouse embryonic stem cell conditioned medium

背景 目前角膜供体来源短缺,且体外培养的人角膜内皮细胞（HCECs）难以再生和扩增,为临床上角膜移植的开展带来了很大困难,组织工程角膜的构建仍是研究的主要课题.前期研究已证实,小鼠胚胎干细胞条件培养基（ESC-CM）能够促进体外HCECs的增生,脱细胞猪角膜基质（APCS）是较好的支架材料,但ESC-CM培养的HCECs是否能在APCS上融合成单层细胞鲜见研究. 目的 研究ESC-CM培养的HCECs在APCS上是否能够形成单层细胞. 方法 用小鼠ESC-CM培养液培养小鼠ES-E14细胞,收集培养上清液,离心后按体积比1∶3与人角膜内皮细胞培养液（CEM）混合,制备体积分数25％ ESC-CM液.取穿透角膜移植术后剩余的供体人角巩膜缘组织,采用组织块培养法用ESC-CM对HCECs进行培养和传代,采用逆转录PCR法检测HCECs中Ⅷ型胶原蛋白（ColⅧ）与神经元特异性烯醇化酶（NSE）的表达以鉴定细胞.取猪角膜,利用磷脂酶A2和碳酸氢盐溶液脱细胞法制备APCS,将第2代HCECs混合液按照800/mm2的密度种植于灭菌的APCS上进行培养,于倒置相差显微镜下观察细胞形态,采用苏木精-伊红染色法观察培养细胞的组织结构;采用免疫荧光法检测构建的HCECs单细胞片中闭锁小带蛋白-1（ZO-1）和Na＋-K＋-ATP酶的表达. 结果 体外培养的HCECs呈典型的六角形,内皮特异性标志物ColⅧ和NSE mRNA表达阳性.脱细胞APCS呈白色透明状,苏木精-伊红染色显示APCS中无角膜细胞,但角膜胶原纤维排列规则.第2代HCECs复水后可在APCS上生长并贴附,培养后7d融合成单层细胞片,细胞片上HCECs的细胞密度为（2 694± 143）/mm2.构建的HCECs片中内皮细胞泵功能相关蛋白ZO-1和Na＋-K＋-ATP酶均呈阳性表达,呈红色荧光. 结论 25％ESC-CM可促进HCECs的生长并维持细胞的正常形态,APCS可为HCECs片的构建提供支架和较好的生存微环境.在APCS形成的HCECs单细胞片可表达HCECs泵功能,是角膜移植的良好供体.

Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells （HCECs） regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium （ESC-CM） improved the proliferative capacity of HCECs in vitro,and acellular porcine corneal stroma （APCS） was a good saffold material.However,whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied.Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet.Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured,and the cultured medium was centrifuged and mixed with 75% human corneal endothelium medium （CEM）at a proportion of 1 ∶ 3 to prepare the 25% ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet＆#39;s membrane,and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ （Col Ⅷ） mRNA and neuron-specific enolase （NSE） mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution,and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 （ZO-1）and Na＋-K＋-ATPase in the cell sheet were detected by immunofluorescence staining.Results The second generation of HCECs cultured with 25% ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent,and no corneal cells were seen,the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the cell density of （2 694±143）/mm2.ZO-1 and Na＋-K＋-ATPase were positively expressed on the HCECs sheet.Conclusions Twenty-five percent ESC-CM promotes the proliferation and maintains the normal morphology of HCECs.APCS provide a good scoffold and microenvironment for the formation of HCEC sheet.The HCEC sheet Possesses the pump function of HCECs and is a good corneal donor for transplantation.