【作者】 高华；

【导师】 谢立信；

【作者基本信息】 青岛大学 ， 眼科学， 2007， 博士

【摘要】 目的采用抗原半相合小鼠建立免疫因素逐渐减弱的小鼠角膜移植模型，从中筛选与临床角膜植片慢性失功（chronic corneal allograft dysfunction，CCAD）接近的模型。利用角膜植片慢性失功模型研究免疫与非免疫因素在CCAD发生及发展过程中的作用，揭示CCAD的发生机理。方法第一部分：小鼠角膜植片慢性失功模型（CCAD模型）的建立1)小鼠角膜移植模型建立：雄性C57BL／6（H-2b）小鼠与雌性BALB／c（H-2d）小鼠杂交，获得抗原半相合CB6F1代（简称F1代）小鼠（H-2b／d）。分别以C57BL／6、F1代、BALB／c小鼠为供体，以BALB／c小鼠为受体，建立免疫因素逐渐减弱的小鼠穿透性角膜移植（penetrating keratoplasty，PK）模型。2)实验分组：共分4组，每组18只小鼠。A组为异系移植组，供体来自C57BL／6小鼠；B组为F1代（抗原半相合）移植组，供体来自F1代小鼠；C组为同系移植组，供体来自BALB／c小鼠。D组为正常对照组，正常BALB／c小鼠不作任何处理。3)术后观察与实验室检查：术后观察＞100天，每周2次采用裂隙灯显微镜观察各组角膜植片情况并进行评分，记录植片发生混浊（失功）的时间；术后1个月采用茜素红联合PI／Hoechst双标法观察角膜内皮细胞形态、凋亡与坏死情况：移植术后3周、2个月、3个月，免疫组化法检测各组植片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。第二部分：免疫与非免疫因素在CCAD发生过程中作用的研究1)实验动物分组：同实验第一部分。2)实验室检查：移植术后3周、2个月、3个月，免疫组化法检测植片中F4／80、转化生长因子-β（TGF-β）、碱性成纤维细胞生长因子（bFGF）、α平滑肌肌动蛋白（α-SMA）表达情况；RT-PCR法检测各组小鼠角膜植片中F4／80、TGF-β表达情况；原位末端标记（TUNEL）法检测各组角膜细胞凋亡情况。结果第一部分：小鼠角膜植片慢性失功模型（CCAD模型）的建立角膜植片观察：各移植组小鼠角膜移植术后均有新生血管长入植床，伴有植片轻度水肿。异系移植组于术后2～4周内植片全部发生混浊；F1代移植组角膜拆线后结膜充血和角膜新生血管逐渐消退，植片恢复透明，之后临床未发现明显的免疫排斥反应，13只小鼠于术后2～3个月逐渐出现角膜混浊；同系移植组角膜拆线后结膜充血和角膜新生血管逐渐消退，植片恢复透明。之后始终无新生血管长入，1只小鼠观察期内出现植片混浊，其余小鼠植片混浊程度始终未超过2分；正常对照组小鼠角膜始终维持透明。四组植片发生混浊时间的中位数分别为17d、85.5d、＞100d、＞100d。免疫组化检查：异系移植组于术后3周发生植片混浊时，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3周时偶见CD4⁺T淋巴细胞浸润，未见CD8⁺T淋巴细胞浸润；正常对照组未见CD4⁺及CD8⁺T淋巴细胞浸润；术后2个月，异系移植组仍可见较多CD4⁺及少量CD8⁺T淋巴细胞浸润，F1代移植组角膜基质内偶见CD4⁺及CD8⁺T淋巴细胞，同系移植组和正常对照组未见CD4⁺及CD8⁺T淋巴细胞浸润。术后3个月，除异系移植组基质内偶见CD4⁺T淋巴细胞外，其余各组均未发现CD4⁺、CD8⁺T淋巴细胞浸润。茜素红联合PI／Hoechst双标法检查：异系移植组角膜植片内皮细胞成像不清，存在较多坏死和凋亡的内皮细胞；F1代移植组内皮细胞减少，失去正常的六边形结构，可见散在凋亡的内皮细胞，未见坏死的内皮细胞；同系移植组角膜内皮细胞较正常减少，六边形结构规则，偶见凋亡的内皮细胞，未见坏死的内皮细胞；正常对照组内皮细胞六边形结构规则，未见坏死和凋亡的内皮细胞。透射电镜检查：各移植组小鼠角膜植片内皮细胞存在萎缩性改变，异系移植组角膜基质内可以见到较多炎性细胞浸润，内皮细胞层发生萎缩性改变；F1代移植组和同系移植组未见明显炎性细胞浸润；异系移植组和F1代移植组在植片发生混浊时可见纤维增殖。第二部分：免疫与非免疫因素在CCAD发生过程中作用的研究1)免疫组化检查：异系移植组、F1代移植组及同系移植组术后3周、2个月和3个月时，免疫组化检查角膜基质内均可见F4／80、TGF-β、bFGF阳性表达。异系移植组和F1代移植组植片发生混浊时，角膜基质内可见α-SMA阳性表达，同系移植组呈弱阳性表达，正常对照组未见阳性表达。2)RT-PCR检查：F4／80在移植组均有表达，术后早期表达量低，晚期表达量增高，正常对照组F4／80未见表达；TGF-β四组不同时间点均有表达，移植组早期表达量较多，晚期表达量下降，正常对照组各时间点均为低表达。3)TUNEL检查：术后3周，各移植组植片上皮基底层柱状细胞和基质层内均可见阳性细胞，内皮细胞未见明显阳性表达；术后2个月，各移植组上皮基底细胞仍可见阳性细胞，数量较3周时少；术后3个月，各移植组上皮层和内皮层均未见明显阳性细胞，基质层内偶见阳性细胞：正常对照组角膜各层组织在各时间点均未见阳性细胞。结论1)F1代移植组与同系移植组角膜移植术后植片出现与临床慢性失功相似的变化，可以作为研究CCAD的动物模型；前者主要用于研究免疫和非免疫因素共同作用导致慢性植片失功发生的模型，后者主要用于研究排除免疫因素，在非免疫因素作用下植片发生慢性失功的模型。2)抗原依赖的致敏淋巴细胞浸润以与非抗原依赖的炎性细胞浸润及相关细胞因子异常表达在CCAD发生和发展过程中发挥重要的作用。更多还原

【Abstract】 PurposeTo establish a murine model of chronic corneal allograft dysfunction (CCAD) thatpermits molecular evaluation of alloantigen specific factors and non-alloantigenspecific factors in the process of chronic allograft dysfunction after cornealtransplantation.MethodsPartⅠ: Establishment of murine model of CCAD1) Penetrating keratoplasty (PK) model in mice: Semiallogeneic CB6F1 (BALB/c×C57BL/6) mice (H-b/d) were obtained from matching of female BALB/c (H-2d)mice and male C57BL/6 mice. C57BL/6, CB6F1 and BALB/c corneal grafts weretransplanted orthotopically to BALB/c recipients respectively. 2)Groups: Seventy-twoBLAB/c mice were randomly divided into four groups (18 mice per group): Group A,allogenic transplantion group, C57BL/6 corneal grafts were transplanted to BALB/crecipients; Goups B, semiallogeneic transplantation group, CB6F 1 corneal grafts weretransplanted to BALB/c recipients; Group C, syngeneic transplantation group,BALB/c corneal grafts were transplanted to BALB/c recipients. Group D, controlgroup, BALB/c mice without corneal transplantation. 3) Postoperative examination:The follow-up time was more than 100 days, and the graft was evaluated by slit-lampbiomicroscopy two times every week. Graft survival time and corneal opacity scorewere monitored, and corneal endothelium were examined by alizarin red andPI/Hoechst stain. CD4~+ and CD8~+ T lymphocytes were examined byimmunohistochemistry. Ultrastructure changes of the grafts were examined byelectromicroscopy. PartⅡ: Effect of ailoantigen specific and non-alloantigen specific factors inCCAD1) Animal model: Animals were divided into 4 groups and consult partⅠinexperiment. 2) Lab examination: F4/80, TGF-βand bFGF were examined byimmunohistochemistry, F4/80 and TGF-βwere examined by RT-PCR, and cellapoptosis was examined by TUNEL analysis at 3 weeks, 2 and 3 monthspostoperation.ResultsPartⅠ: Establishment of murine model of CCADAllograft examination: Mild corneal edema and opacity occurred in the grafts in allgroups immediately after PK, and then disappeared after removal of corneal sutures.Corneal grafts opacity combined with angiogenesis reoccurred in group A, andresulted in grafts dysfunction within 12 to 28 days postoperation ultimately. In groupB, the grafts became transparent completely at 1 to 2 months, and then mostly becameopacity gradually without corneal angiogenesis at 2 to 3 months. In group C, thegrafts remained transparent and survived over 100 days. All the corneas remainedtransparent in group D. Median graft survival times were 17d, 85.5d,＞100d and＞100d in 4 groups, respectively.Immunohistochemistry examination: A large amount of CD4~+ and CD8~+ Tlymphocyte infiltration was present in allografts in group A at 3 weeks after PK, withmore expressions at 2 months, and hardly seen at 3 months; Few CD4~+ and CD8~+ Tlymphocytes were observed in allografts in group B and C at 3 weeks after PK, andno can be seen at 2 and 3 months; CD4~+ and CD8~+ T lymphocyte infiltration was notobserved in group D.The endothelium can not be counted because the blurred image after the alizarin redcombined PI/Hoechst stain and apoptotic and necrotic cells can be seen in group A;The endothelial cell density decreased and few apoptosis can be detected in group Band C. No apoptotic and necrotic endothelial cells were found in group D. Ultrastructural characteristic changes mainly include fibrosis formation andendothelium atrophy and degeneration in failed grafts in group A, B and C by electronmicroscopy examination.PartⅡ: Effect of alloantigen specific and non-alloantigen specific factors inCCAD1) Immunohistochemistry examination: F4/80, TGF-β, bFGF were detected at 3weeks, 2 and 3 months in group A, B and C. Alpha-SMA could only be detected at thelate stage in group A, B and C. 2) RT-PCR examination: F4/80 was positiveexpression in group A, B and C, and the relative expression amount was low at earlystage and increased at the late stage; Positive expression of TGF-βwas present in allgroups, and the relative expression amount was high at early stage and decreased atthe late stage. 3) TUNEL analysis: TUNEL positive cells could be seen in thecolumnar epithelium and in the stroma at 3 weeks and 2 months in group A, B and C,but not in group D. No TUNEL positive endothelial cells could be found in all groups.Conclusions1) Semiallogeneic and syngeneic transplantation groups present the changessimilar to CCAD in clinical study, and both can be regarded as the model that permitsmolecular evaluation of CCAD. The former model is applicable for the research ofalloantigen specific and non-alloantigen specific factors, and the latter is applicablefor study of non-alloantigen specific factors.2) Early infiltration of immune and inflammatory cells and production ofnon-immune related cytokines might take part in the pathogenesis of chronicdysfunction of corneal allograft.更多还原