高危角膜移植排斥兔模型的建立及对比研究

背景 理想的角膜移植排斥动物模型是研究高危角膜移植免疫排斥机制的基础,具有重要的意义. 目的 比较各种建立兔高危角膜移植排斥模型方法的临床特点,探索合适的角膜移植排斥动物模型的建立方法.方法 45只新西兰白兔作为角膜移植受体,并按照造模方法的不同按随机数字表法随机分为缝线组、碱烧伤组和异种移植组,每组15只.分别用在角膜4个象限各间断缝1根5-0丝线法和1 mol/LNaOH碱烧伤法诱导角膜新生血管(CNV),再建立兔同种异体角膜移植;另一组以猫角膜为供体,建立猫-兔异种角膜移植模型.于第2周和第4周观察植片的组织学情况,对3个组角膜植片裂隙灯下观察植片排斥反应、炎症和新生血管,对植片水肿程度及炎症指数(IF)进行评分,根据角膜混浊、水肿及新生血管合计值计算排斥指数(RI).用免疫组织化学法检测CD4+T细胞和CD8+T细胞在植片组织中的表达. 结果 缝线组、碱烧伤组和异种移植组分别有14、15、15只兔完成穿透角膜移植术.术后2周,3个组IF中位数分别为0.556、0.778、0.222,差异有统计学意义(H=25.736,P=0.000),异种移植组IF值低于缝线组和碱烧伤组,差异均有统计学意义(Z=3.841、3.993,P=0.000),缝线组IF值低于碱烧伤组,差异有统计学意义(Z=3.568,P=0.000).术后2周,3个组RI中位数分别为2、6、3,差异有统计学意义(H=22.432,P=0.000),异种移植组RI高于缝线组而低于碱烧伤组,差异均有统计学意义(Z=2.373,P=0.018;Z=3.936,P=0.000),缝线组RI低于碱烧伤组,差异有统计学意义(Z=3.729,P=0.000).3个组植片存活时间分别为(17.9±2.0)、(13.4±2.4)、(15.5±2.0)d,差异有统计学意义(F=9.474,P=0.001).异种移植组的新生血管面积均低于缝线组和碱烧伤组(P＜0.05).术后2周和4周,组织病理学检查可见异种移植组植片中的炎性细胞少于缝线组和碱烧伤组,3个组植片中均出现以CD4+T细胞为主的细胞浸润. 结论 猫-兔异种角膜移植模型较缝线和碱烧伤法制作的角膜移植模型炎症反应轻、新生血管少,角膜免疫排斥反应稳定、适度,是理想的高危角膜移植动物模型.

Comparison among three modeling methods of high-risk rejection corneal transplantation

Background An ideal animal model is very important for the investigation of the immune mechanism of high-risk rejection corneal transplantation. Objective This study was to compare three methods of creating a high-risk corneal transplantation model in rabbits to study high-risk rejection corneal transplantation.Methods Forty-five New Zealand white rabbits were utilized and assigned randomly to three groups of different modeling methods,with 15 rabbits for each group.The high-risk corneal transplantation models were created by suturing with 5-0 silk thread in 4 quadrants,inducing alkali burn with 1 mol/L NaOH or corneal xenotransplantation.In the suturing group and alkali burning group,the rabbits received a unilateral 7.25 mm diameter corneal allograft after corneal neovascularization was induced,and in the xenotransplantation group,corneas from cats were used as donors.Rabbits were followed-up for 4 weeks in all groups.Corneal neovascular area was calculated and compared among the three groups.The amount of rejection,inflammatory index ( IF),neovascularization and histology of grafts were clinically scored to calculate the reject index (RI). Results There were 14,15 and 15 rabbits that survived the high-risk penetrating corneal transplantation,respectively,in the suturing group,alkali burning group and xenotransplantation group.Two weeks after operation,the IF scores were 0.543 ± 0.103,0.811 ± 0.054 and 0.191 ±0.087,and the RI were 2.111±0.928,7.0±0.816 and 3.182±0.751 in the suturing group,alkali burning group and xenotransplantation group,respectively,showing statistically significant differences among the three groups (x2 =25.736,22.432,P =0.000).The IF value was lower in the xenotransplantation group compared with the suturing group and alkali burning group (Z =3.841,3.993,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.568,P =0.000).The RI value of the xenotransplantation group was significantly raised in comparison with the suturing group and declined in comparison with the alkali burning group (Z =2.373,P =0.018;Z =3.936,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.729,P =0.000 ).The survival times of the grafts were ( 17.9±2.0 ) days,( 13.4 ±2.4) days and ( 15.5 ±2.0 ) days in these three groups with a significant difference among them ( F =9.474,P =0.001 ).The neovascularization area in the xenotransplantation group was smaller than the suturing group and alkali burning group (P＜ 0.05 ).Histological examination revealed a large number of inflammatory cells infiltration in the grafts 2 and 4 weeks after transplantation in the suturing group and alkali burning group,but less inflammatory cells were seen in the xenotransplantaion group.Immunofluorescence staining showed abundant CD4+ T positive cells in the grafts in the three groups. Conclusions The cat-rabbit corneal xenotransplantation can induce stable and moderate immune rejection.This animal model has milder inflammatory response and less corneal neovascularization than the suture and alkali burn models.This method therefore is an ideal model for high-risk corneal transplantation.