CAJ | 학술논문

目的探讨恒河猴血管内皮细胞(RMVEC)移植替代角膜内皮细胞(CEC)治疗角膜内皮损伤的可行性.方法实验研究.体外培养增殖RMVEC并用5-溴脱氧尿嘧啶核苷(BrdU)标记.将恒河猴9只采用随机数字表法分为两组:实验组(6只)、对照组(3只).实验组将体外培养的BrdU标记的RMVEC利用离心沉淀法移植到撕除后弹力层的恒河猴角膜内表面后缝回植床;对照组将撕除后弹力层的术眼角膜植片原位缝回植床.术后观察各组角膜植片透明情况.术后30、60、90 d分别摘取术眼行病理切片、抗BrdU单克隆抗体免疫组织化学染色、扫描电镜及透射电镜观察RMVEC在角膜植片内表面的分布及细胞超微形态结构.结果在实验观察期内,实验组角膜植片比对照组角膜植片有较好的透明性,未出现新生血管及大泡性角膜病变.病理学检查发现实验组角膜内表面可见细胞层,抗BrdU单克隆抗体免疫组织化学染色阳性,证实该细胞为培养的BrdU标记的血管内皮细胞;对照组角膜内表面未见细胞样结构.扫描电镜下可见实验组角膜植片内表面可见RMVEC分布均匀,生长良好,呈不规则多边形,表面可见微绒毛,RMVEC间可见少量白细胞,小梁网处部分细胞碎片聚集;对照组角膜内表面呈纤维物质样,未见细胞生长.透射电镜:培养的RMVEC及实验组角膜内表面的RMVEC呈不规则扁圆状,细胞间大量桥粒链接,胞质内细胞器丰富,可见特征性WP小体,提示其具备活体血管内皮细胞的特征与活性,对照组角膜内表面未见细胞结构.结论 RMVEC能够在撕除后弹力层的角膜内表面生长并替代CEC的功能,可能成为治疗角膜内皮损伤的一种新思路.
목적 탐토항하후혈관내피세포(RMVEC)이식체대각막내피세포(CEC)치료각막내피손상적가행성.방법 실험연구.체외배양증식RMVEC병용5-추탈양뇨밀정핵감(BrdU)표기.장항하후9지채용수궤수자표법분위량조:실험조(6지)、대조조(3지).실험조장체외배양적BrdU표기적RMVEC이용리심침정법이식도시제후탄력층적항하후각막내표면후봉회식상;대조조장시제후탄력층적술안각막식편원위봉회식상.술후관찰각조각막식편투명정황.술후30、60、90 d분별적취술안행병리절편、항BrdU단극륭항체면역조직화학염색、소묘전경급투사전경관찰RMVEC재각막식편내표면적분포급세포초미형태결구.결과 재실험관찰기내,실험조각막식편비대조조각막식편유교호적투명성,미출현신생혈관급대포성각막병변.병이학검사발현실험조각막내표면가견세포층,항BrdU단극륭항체면역조직화학염색양성,증실해세포위배양적BrdU표기적혈관내피세포;대조조각막내표면미견세포양결구.소묘전경하가견실험조각막식편내표면가견RMVEC분포균균,생장량호,정불규칙다변형,표면가견미융모,RMVEC간가견소량백세포,소량망처부분세포쇄편취집;대조조각막내표면정섬유물질양,미견세포생장.투사전경:배양적RMVEC급실험조각막내표면적RMVEC정불규칙편원상,세포간대량교립련접,포질내세포기봉부,가견특정성WP소체,제시기구비활체혈관내피세포적특정여활성,대조조각막내표면미견세포결구.결론 RMVEC능구재시제후탄력층적각막내표면생장병체대CEC적공능,가능성위치료각막내피손상적일충신사로.
Objective To Explore the feasibility of rhesus monkey vascular endothelial cell (RMVEC) transplantation to substitute the allogeneic corneal endothelial cells(CEC),through observe the morphologic and functional change of the vascular endothelial cell which was transplanted to the inner surface of cornea without Descemet's membrane.Methods It was an experimental study.The rhesus monkey vascular endothelial cell was cultivated to proliferation and marked by BrdU in vitro.The experimented monkeys are divided into 2 groups:experimental group (6 monkeys),control group (3 monkey).In experimental group:the cultured RMVEC,marked by BrdU,were transplanted onto the posterior surface of rhesus monkey cornea without Descemet's membrane though centrifugal sedimentation,then sew back the corneal graft.In control group:detach the corneal Descemet's Membrane of rhesus monkey but without cultured RMVEC transplantation.Corneal transparence of every monkey was frequently observed postoperation.On 30,60,90 postoperative days,corneal graft were respectively detached to observe the distribution,appearance and ultrastructures morphological structure of RMVEC on the inner surface of cornea,by pathological section,anti BrdU monoclonal antibody immunohistochemistry,scanning electronic microscope(SEM) and transmission election microscopy (TEM).Results Corneal transparency:In the experimental observation period (three months),the corneal graft of experimental group had better transparency than control group and without corneal neovascularization and bullous keratopathy.Pathological section:A layer of cells with BrdU staining positive was found on the posterior surface of cornea in experimental group,indicated the cells are RMVEC.And no cell-like structure was found in control group.SEM:Experimental group showed that RMVEC with irregular shape uniformly distributed on the inner surface of cornea and growing well,a small amount of white blood cells can be seen between RMVEC,and part of cellular debris exist in the trabecular meshwork.Control group showed a fiber material without RMVEC.TEM:The cultured RMVEC and which in posterior surface of cornea was irregular oblateness,a large number of desmosomes link were been seen between RMVEC.Abundant organelles and characteristic WPBs appear in cytoplasm,which suggest the characteristics and vitality of vascular endothelial cells in vivo,and no cell structure in the control group.Conclusions Rhesus monkey endothelial cells can growth on the posterior surface of cornea without Descemet's membrane.The cultured cells,with similar ultrastructure to RMVEC in vivo,can play a role of barrier to keep the corneal dehydration and transparency to some extent.