建立小鼠角膜植片慢性失功模型的初步探讨

The preliminary study of establishing animal model of chronic allograft dysfunction after penetrating keratoplasty

目的探讨建立小鼠角膜植片慢性失功（CCAD）的模型.方法 实验研究.将雄性C57BL/6小鼠与雌性BALB/c小鼠杂交,获得抗原半相合CB6FI代（即F1代）小鼠.分别以C57BL/6（异系）、F1代、BALB/c小鼠（同系）为供体,以BALB/c小鼠为受体,建立免疫背景渐同的小鼠穿透性角膜移植（PK）模型,BALB/c小鼠为正常对照组.PK术后体外对植片进行观察;采用茜素红联合碘化丙啶（PI）/Hoechst双标法观察角膜内皮细胞形态、凋亡与坏死情况;免疫组织化学法检测各组植片CD4＋、CD8＋T淋巴细胞浸润情况;电镜观察各组角膜片超微结构改变,筛选与临床接近的CCAD模型.生存曲线之间比较采用Log-rank检验.结果 （1）角膜植片观察：异系、F1代、同系和对照组小鼠发生混浊时间的中位数分别为17.0 d、85.5 d、＞100 d及＞100 d（F=344.0,p＜0.01）.（2）免疫组织化学检查：异系移植组植片基质中见大量CD4＋T淋巴细胞浸润,较多CD8＋T淋巴细胞浸润;F1代移植组和同系移植组术后偶见CD4＋T淋巴细胞浸润,未见CD8＋T淋巴细胞浸润.（3）茜素红联合PI/Hoechst检查：发现异系移植组有较多坏死和凋亡的内皮细胞;F1代移植组内皮细胞减少,可见散在凋亡内皮细胞,未见坏死内皮细胞;同系移植组角膜内皮细胞较正常减少,偶见凋亡内皮细胞,未见坏死内皮细胞.（4）透射电镜检查：各移植组小鼠角膜植片内皮细胞均存在萎缩性改变,异系移植组角膜基质内可见较多炎性细胞;F1代和同系移植组未见明显炎性细胞浸润.结论 PK术后F1代移植组与同系移植组植片出现与临床慢性失功相似的变化过程,可作为研究CCAD的动物模型.

Objective To establish a murine model of chronic corneal allograft dysfunction （CCAD） after penetrating keratoplasty （PK）. Methods Experimental study. PK model in mice：Semiallogeneic CB6F1 mice were obtained from matching of female BALB/c and male C57BL/6 mice.C57BL/6 （al logeneic group）, CB6F1 （semiallogeneic group） and BALB/c （syngeneic group） grafts were transplanted orthotopically to BALB/c recipients respectively, and BALB/c mice as a control group. The follow-up time was more than 100 d, and graft survival time and corneal opacity score were monitored, and corneal endothelium were examined by alizarin red and PI/Hoechst stain. CD4 ＋ and CD8 ＋ T lymphocytes were examined by immunohistochemistry. Ultrastructure changes of the grafts were examined by electromicroscopy. Log-rank test were used to compare survival curves. Results （ 1 ） Graft examination：Median graft survival times were 17.0 d, 85.5 d, 〉 100 d and 〉 100 d in allogeneic, semiallogeneic,syngeneic and control groups, respectively （ F = 344. 0, P 〈 0. 01 ） . （ 2 ） Immunohistochemistry examination： There were large amount of CD4 ＋ and CD8 ＋ T lymphocyte infiltration in allografts in allogeneic group at 3 weeks after PK; Few CD4 ＋ and CD8 ＋ T lymphocytes were observed in semiallogeneic group and syngeneic goups at 3 weeks after PK; CD4＋ and CD8＋ T lymphocyte infiltration was not observed in the control group. （3） Endothelium examination： The endothelium can not be counted because the blurred image after the alizarin red combined PI/Hoechst stain and apoptotic and necrotic cells can be seen in allogeneic group; the endothelial cell density decreased and few apoptosis can be detected in semiallogeneic and syngeneic groups; no apoptotic and necrotic endothelial cells were found in the control group.（4） Ultrastructural characteristic changes mainly include fibrosis formation and endothelium atrophy and degeneration in failed grafts in all transplanted groups by electron microscopy examination. Inflammation cells can only be found in the allogeneic group. Conclusions Semiallogeneic and syngeneic transplantation groups present the changes similar to CCAD in clinical study, and both can be regarded as the model that permits molecular evaluation of CCAD.