Ad-siICOS阻断ICOS共刺激通路抑制大鼠角膜移植排斥反应的实验研究

The effect of blockade ICOS co-stimulator pathway with Ad-siICOS on cornea transplantation immunology

目的观察携带报告基因GFP且表达ICOS-siRNA的重组腺病毒(Ad-siICOS-EGFP)阻断可诱导共刺激分子(in-ducible costimulator,ICOS)共刺激通路对角膜移植急性免疫排斥的抑制作用。设计实验性研究。研究对象Wistar大鼠(供体)及SD(受体)大鼠。方法取Wistar大鼠(供体)及SD(受体)大鼠,建立大鼠同种异体穿透性角膜移植模型,将其随机分为A、B、C、D4组,每组20只鼠:A组为单纯角膜移植对照组;B组为Ad-siICOS-EGFP注射组;C组为Ad-EGFP注射组;D组为生理盐水注射组。术后每日在裂隙灯显微镜下观察角膜植片以及眼前段组织的变化。对角膜混浊、水肿、新生血管3项指标进行评分,观察排斥反应指数(RI)的变化并记录植片的存活时间。各移植组分别于术后5天随机取10只大鼠,麻醉处死后,取植片行免疫组织化学及RT-PCR检测。各组其余大鼠进行角膜植片存活时间统计。主要指标角膜植片平均存活时间I;COS蛋白表达情况I;COSmRNA表达情况。结果各组角膜植片平均存活时间:A组(9.800±0.632)天;B组(14.700±1.418)天;C组(10.000±1.054)天;D组(10.100±0.876)天(F=52.383,P=0.000)。ICOS蛋白表达情况:各组植片均可见ICOS蛋白阳性表达,各组植片组织ICOS蛋白免疫组化染色阳性细胞评分:A组(1.900±0.568);B组(1.100±0.316);C组(1.800±0.632);D组(1.900±0.568)(F=5.214,P=0.004)I。COSmRNA表达情况:各组大鼠植片组织ICOS的PCR产物凝胶电泳条带辉度值比值:A组(1.004±0.164);B组(0.621±0.096);C组(0.929±0.134);D组(0.918±0.120)(F=16.717,P=0.000)。结论 Ad-siICOS-EGFP可有效抑制大鼠植片组织ICOSmRNA及ICOS蛋白的表达,从而阻断ICOS共刺激通路;移植术后即刻受体鼠球结膜下注射Ad-siICOS-EGFP,可抑制大鼠角膜移植急性免疫排斥反应,延长角膜植片存活时间。

Objective To observe the inhibitory action of Ad-siICOS-EGFP to the cornea transplantation acute immunological rejection with the co-stimulator passageway blockade. Design Experimental study. Participants Wistar rat and SD rat. Methods Build homogeneity penetrability corneal transplantation rat model with Wistar rat （donor） and SD （acceptor） rat. Divide the SD rats into four group with 20 rats in each group： penetrability corneal transplantation group as the control group （group A）; Ad-siICOS-EGFP injection group （group B）; Ad-EGFP injection group （group C）; saline injection group （group D）. The implanted cornea situation was observed with slit lamp microscope everyday after operation. The change of rejection index （RI） and the implant survival time were observed. 10 rats in each group were selected to test the implant ICOS mRNA expression with RT-PCR and ICOS albumen level in implant lymphocyte with immunohistochemical method on the 5th day after operation. Main Outcome Measures The average survival time of implanted cornea, expression of ICOS protein, and expression of ICOS mRNA. Results The average survival time of implanted cornea in each group was （9.800~0.632） days in group A, （14.700±1.418） days in group B, （10.000±1.054） days in group C, and （10.100±0.876） days in group D （F=52.383, P=0.000）. The score of ICOS protein immunohistochemistry dying positive cell was 1.900±0.568 in group A, 1.100±0.316 in group B, 1.800±0.632 in group C, and 1.900±0.568 in group D （F=5.214, P=0.004）. The ratio of electrophoresis strip brightness of the ICOS PCR product in cornea implant was 1.004±0.164 in group A, 0.621±0.096 in group B, 0.929±0.134 in group C, and 0.918±0.120 in group D （F=16.717, P=0.000）. Conclusion Ad-siICOS-EGFP can effectively inhibit the lymphocyte ICOSm-RNA and ICOS protein expression in rat cornea implant therefore blockade the ICOS co-stimulator passage. Injecting Ad-siICOS-EGFP in bulbar conjunctiva can inhibit the acute immunological rejection in rat cornea transplantation so as to extend the survival time of cornea implant. （Ophthalmol CHN, 2011： 20： 161-164）