恒河猴血管内皮细胞离心沉淀移植替代角膜内皮细胞的研究

Experimental study on the rhesus monkey corneal endothelial cells substituted by the allogeneic vascular endothelial cells cultivated in vitro

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.

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.