改良前房注射法移植血管内皮细胞替代角膜内皮细胞的实验观察

Transplanted vascular endothelial cells to replace corneal endothelial cells by improved anterior chamber injection

目的 探讨血管内皮细胞（VEC）移植治疗角膜内皮损伤的可行性.方法 实验研究.体外培养增殖VEC.将恒河猴12只,按照随机数字表随机分为两组：实验组（6只）、对照组（6只）.实验组通过超声乳化破坏角膜内皮细胞层之后,将体外培养的VEC利用前房注射法移植到恒河猴角膜内表面;对照组超声乳化破坏角膜内皮细胞层后不注射VEC.术后7、14、30、60、90 d分别行术眼前节照相;术前及术后7、14、30、60、90 d分别用A超测量角膜厚度、Goldman眼压计测量眼压,对数据进行独立样本t检验及方差分析;术后30、60、90 d分别摘取术眼行病理切片HE染色、扫描电镜观察VEC在角膜植片内表面的分布.结果 实验组角膜比对照组有较好的透明度,未出现大泡性角膜病变,术后3个月时实验组角膜缘可见少量新生血管.术眼前房深度均正常;术后3个月时,实验组角膜厚度为（500±14） μm,明显低于对照组角膜厚度[（618±11）μm],同时期实验组和对照之间角膜植片厚度值变化差异有统计学意义（术后1、2、3个月t值分别为-3.256、-4.419、-12.896,均P＜0.05）;同时期实验组和对照之间眼压变化差异无统计学意义（术后1、2、3个月t值分别为-1.179、-2.166、-2.536,均P＞0.05）;实验组角膜内表面可见细胞层;对照组角膜内表面未见细胞样结构.扫描电镜：实验组角膜植片内表面可见VEC分布均匀,生长良好,呈不规则多边形,表面可见微绒毛,VEC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.结论 超声乳化法能很好的建立恒河猴角膜内皮损伤模型;前房注射法能够将VEC移植到角膜内表面,且细胞能在撕出后弹力层的角膜内表面生长,并在一定程度上发挥屏障作用维持角膜的脱水状态和透明性.移植手术对前房角的组织结构形态在短期内不产生病理性影响.

Objective To observe form and function changes of vascular endothelial cells （VEC） which were transplanted to the posterior surface of rhesus monkey cornea without Descemet＇s Membrane by anterior chamber injection,explore the feasibility of transplanting VEC to treat corneal endothelial injury,and find new method of corneal endothelial cell transplantation.Methods Cultured VEC to proliferate in vitro.Rhesus monkeys were randomly divided into two groups：Experimental group（6） and control group（6）according to a random number table.The experimental group：transplant the culured VEC to the posterior surface of rhesus monkey cornea without Descemet＇s membrane by anterior chamber injection.The control group：Tear out the Descemet＇s membrane by capsulotomy needle without VEC transplantation.A ultrasound apparatus was adopted to measure the postoperative thicknes of the cornea and Goldman intraocular pressure meter measuring intraocular pressure in the postoperative 7 days,14 days,30 days,60 days,90 days,organize the data and entered into the computer,applicated the software of SPSS 11.5 for data independent samples t-test and analysis of variance.The eyes were removed respectively in postoperative 30,60,90 days to do pathological HE dyeing and scanning electron microscopy （SEM） observation of VEC in the posterior surface of cornea graft.Results Corneal transpareney：In the experimental observation period （3 months）,the experimental group had better transparency than the control group with normal anterior chamber depth and without bullous kerat-opathy.And the corneal neovascularization was exist in the cornea graft in experimental group in the third month.After 3 months,the corneal thickness of experimental group （500 ± 14） μm was significantly lower than the control group （618 ± 11） μm,Corneal thickness values between experimental and control groups were statistically significant differences in changes （all P ＜ 0.05,t-values were-3.256,-4.419,-12.896 postoperative 1,2,3 months）.Changes of intraocular pressure difference between the experimental group and the control was not statistically significant （all P ＞ 0.05,t-values were-1.179,-2.166,-2.536 postoperative 1,2,3 months）.The pathological：the cell layer was visible in the posterior surface of cornea graft.The control group：can＇t find the cell sample structure.SEM：Experimental group showed that VEC 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 VEC,and part of cellular debris exist in the trabecular meshwork.Control group showed a fiber material without VEC.Conclusions Ultrasonic emulsification can established a repeatability and simple model of corneal endothelial injury in Rhesus monkeys.VEC can be transplanted to the corneal surface by Anterior chamber injection and the cells can grow on the surface and play a barrier role in maintaining the state of dehydration and transparency of the cornea to a certain extent.After transplantation,organizational structure and morphology of the anterior chamber angle does not produce pathological effects in the short term.Prompt that transplant the culured VEC to the posterior surface of rhesus monkey cornea without Descemet＇s Membrane by anterior chamber injection to substitute the function of the corneal endothelial cells may be a new idea for treatment of cornealendothelial damage.