摘要
目的分析利用离心沉淀法移植体外培养的恒河猴血管内皮细胞(vascular endothelial cells,VEC)替代角膜内皮细胞(corneal endothelial cells,CEC)的可行性。方法将恒河猴9只随机分为2组:实验组(6只)、对照组(3只)。实验组:角膜环钻取下中央角膜组织,显微镜下撕除后弹力层,将角膜植片形成约45°倾斜、内表面向上方置于0.6mLEP管中,再向管中加入配置好的密度为40×106L-1的恒河猴VEC悬液0.5mL,管中形成棉球在下、角膜植片在中间、细胞悬液在上面的三明治结构,盖紧EP管,放置于离心机中,以400r·min-1、800r·min-1、1200r·min-1的转速各离心4min后将植片原位缝回植床;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1个月、2个月、3个月分别摘取术眼,行扫描电镜观察移植的VEC在角膜植片内表面的分布情况。结果离心沉淀后倒置显微镜观察,可见移植的类圆形细胞均匀贴附于角膜植片内表面;术后1个月扫描电镜显示种植的VEC贴附于角膜内表面,细胞表面积较大,呈长梭形,细胞间连接不紧密,可见细胞间隙及少量裸露的胶原纤维区;随着时间推移,术后2个月时,可见植片内表面的VEC密度逐渐增大,细胞间出现紧密连接;术后3个月时,可见细胞密度进一步增大,大片状均匀贴附于角膜植片内表面,细胞呈不规则多边形,高倍镜下,细胞表面可见典型的长短不一微绒毛。对照组未见纤维裸露区,胶原前表面偶见白细胞及变形红细胞。结论离心沉淀法可以将VEC成功移植于角膜内表面并均匀分布,移植后的VEC可异位生长并大量分裂增殖。
Objective To analyze the feasibility of transplanting the rhesus monkeys vascular endothelial cells（VEC） cultured in vitro to replace corneal endothelial cells （CEC） by centrifugal sedimentation. Methods Nine rhesus monkeys were randomly divided into 2 groups： the experimental group （ 6 rhesus monkeys） and the control group （ 3 rhesus monkeys）. In the experimental group, corneal trephinations were performed to obtain a piece of central corneal issue, the descemet＇s membrane were removed un- der the microscope. The corneal grafts were set inclined at 45°with the inner surface upwards and put into the EP robes（0.5 mL）. 0.5 mL of VEC cell suspension（40 × 10^6 L^-1 ） was put into each tubes to created a sandwich structure with the corneal graft in the middle of the cell suspension above it and the cotton ball beneath it. Then the EP tubes were put in the centrifugal machine and centrifuged at 400 r · min^-1,800 r · min^-1 and 1200 r · min^-1 ,respectively, each centrifugal lasted 4 minutes. Then implant the corneal grafts into the plant beds as they were. In the control group ,after the descemet＇ s membrane were removed, the corneal grafts were implanted into the plant beds as they were. The target eyes were removed at the lst,2nd,3rd month postoperatively to observe the distribution of the VEC on the surface of the corneal grafts by electronic microscopy. Resultu After centrifugation, invert the tubes, transplanted round cells were observed evenly attached to the inner surface of corneal grafts. The electronic microscopy results showed that at the 1st month postoperatively, VEC were attached to the inner surface of corneal grafts with larger cellular superficial area and loose intercellular connection,intercellular space and bare collagenous fiber areas could be seen;At the 2nd month postoperatively,the cellular density of VEC was higher and intercellular connection was closer; At the 3rd month postoperatively, the cellular density of VEC was even higher, great tracts of VEC were attached to the inner surface of corneal grafts in irregular shape. Microvillus on the cellular surface could be observed under high power lens. No bare coUagenous fiber areas could be seen on inner surface of corneal grafts in the control group. A small number of the leukocytes and distorted red cells were observed on front surface of the collagen. Conclusion VEC can be successfully transplanted to the inner side of cornea and show an even distribution, the transplanted VEC could grow and proliferate enormously.
出处
《眼科新进展》
CAS
北大核心
2013年第5期405-409,共5页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:30960413)
云南省科技厅社会发展计划基金(编号:2009CA008)~~
关键词
离心沉淀法
角膜内皮移植
恒河猴
centrifugal sedimentation
corneal endothelial transplantation
rhesus monkey
