摘要 目的研究前房相关性免疫偏离(ACAID)诱导及预防角膜移植排斥反应的作用。
方法采用甘氨酸法制备可溶性角膜分类抗原,分组行兔前房注射诱导ACAID,并观察各组部分耳膨胀试验和穿透角膜移植术(PKP)术后排斥反应。
结果角膜上皮、内皮、基质及全角膜可溶性抗原皆可诱导ACAID,诱导质量浓度为2.0mg/ml。成功率分别为90%,100%,80%和100%。同期角膜移植免疫排斥率降低,植片存活时间延长。高危观察组仍可诱导出ACAID,并可推迟排斥反应的发生。
结论角膜可溶性抗原可以诱导产生ACAID,无严重并发症,对兔角膜移植排斥反应具有一定的预防和治疗作用。
分类号 R772 R446.6
Experimental study on induction of anterior chamber associated immune
deviation and its preventive function against corneal grafts rejection
Yang Chaozhong Jiang Guomin Sun Lixia et al.
(Department of Ophthalmology,the 2nd Affiliated Hospital,Qingdao Medical College,Qingdao266042)
Abstract ObjectiveTo study the induction of anterior chamber associated immune deviation(ACAID) and its preventive function against corneal grafts rejection.MethodsSoluble corneal classified antigens were made by Glycine method and ACAID was induced by injecting glycine into the anterior chamber(AC) of rabbits.The ear swelling was detected to test ACAID induction,and the corneal grafts rejection of each group was observed.ResultsCorneal epithelium,endothelium,stroma and the whole corneal soluble antigens induced ACAID.The concentration for induction was 2.0mg/ml,and the ratio for induction was 90%,100%,80% and 100%,respectively.The rate of rejection was lower than that of the control group and the survival period of donor graft became longer.ACAID was still induced in high risk group and the rejection was delayed.ConclusionCorneal soluble antigen can induce ACAID which prevent from or treat the rejection of corneal grafts.There is no severe complication during the induced process.
Key words anterior chamber associated immune deviation corneal transplantation rejection
角膜盲约占世界盲目的1/3[1],近年的研究证明:同种异体抗原前房注射可在受体内诱导出针对供体抗原的前房相关性免疫偏离(anterior chamber associated-immune deviation,ACAID),使受体的迟发型超敏反应特异性抑制,使角膜移植术后的植片存活、透明时间延长,角膜移植成功率明显提高。本研究首次利用角膜上皮、基质、内皮及全角膜可溶性抗原诱导ACAID,取得较好的效果。现报告如下。
1 材料与方法
1.1 实验动物
1.0~1.5kg新西兰大白兔110只(青岛市实验动物和动物实验中心提供),家兔120只,雌雄不分,随机分为4组:即正常对照组、高危对照组、正常观察组和高危观察组。
1.2 抗原制备:甘氨酸法粗制角膜可溶性抗原,比色法(752紫外分光光度计,OD为280nm)计算抗原含量,使最终抗原质量浓度为2.0mg/ml,-18℃保存备用。
1.3 角膜酸、碱烧伤后血管化模型制作:分别用1mol/L NaOH和HCl烧伤角膜,自然愈合后获得角膜深层及浅层血管化动物模型。
1.4 前房注射诱导ACAID:麻醉,固定动物,显微镜下30号针头角膜缘部穿刺入前房,注入0.10ml抗原和0.05ml(0.2mg)BCG-PPD佐剂混合液。对照组仅注入等量生理盐水和BCG-PPD佐剂混合液。
1.5 ACAID检测
1.5.1 耳膨胀试验:前房注射7天后各组兔腹部及四肢腋窝皮下多处注射相应诱导抗原及BCG-PPD佐剂的混合液(1∶ 1比例混合)。再7天后,于右耳廓皮内注射相应抗原0.2ml。24h后,游标卡尺测耳膨胀结果,公式为:迟发型超敏反应耳膨胀数值=(24h右耳厚度值-0h右耳厚度值)-(24h左耳厚度值-0h左耳厚度值)。厚度值≥1.50mm,范围≥5mm者为迟发型超敏反应阳性,厚度值<1.40mm,范围<5mm者为阴性。
1.5.2 角膜和脾脏病理学检查:术后1周,1月和4月取角膜和脾,病理及HE染色观察。
1.6 部分穿透角膜移植术:局部麻醉后,6.25mm环钻钻切植床,滴用肝素防凝抗渗出,移植6.75mm的角膜植片,用10/0尼龙线连续缝合,术毕,红霉素眼膏涂于结膜囊,缝合眼睑。
1.7 术后观察记录:术后全身及局部不使用任何免疫抑制剂。定期观察并记录角膜植片情况,分为上皮型、基质型、内皮型和混合型排斥。试验结果分别用方差分析和χ2检验进行处理。
2 结果
2.1 对照组角膜移植免疫排斥率与时间关系(图1)
图1 对照组免疫排斥率(%)
Fig.1 The rate of immune rejection of normal group(%)
图2 高危观察组免疫排斥率
Fig.2 The rate of immune rejection of high-risk control group
2.2 观察组角膜移植排斥率与时间的关系
2.2.1 高危观察组免疫排斥率与时间的关系(图2)
2.2.2 正常观察组免疫排斥率与时间的关系(图3)
2.3 ACAID诱导免疫学及病理学检查结果
2.3.1 ACAID诱导后正常观察组、高危观察组、正常对照组和高危对照组耳膨胀数值分别为(0.51±0.11)mm,(1.02±0.31)mm,(3.34±0.65)mm和(4.54±0.97)mm。耳膨胀试验阴性率正常观察组、高危观察组、正常对照组和高危对照组分别是7.5%,45%,100%和100%。
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