重组腺病毒载体RNA干扰阻断ICOS共刺激通路抑制大鼠角膜移植免疫排斥反应的研究  

Inhibitory effect of recombination adenovirus vector RNA on corneal transplantation immunological rejection by blocking ICOS co-stimulator passageway in rats

作　　者：袁伟[1] 陆晓和[2] 宫玉波[2] 周瑾[2] 

机构地区：[1]济南军区青岛第一疗养院体检科,山东省青岛市266071 [2]南方医科大学珠江医院眼科,广东省广州市510280

出　　处：《眼科新进展》2011年第8期721-724,共4页Recent Advances in Ophthalmology

基　　金：广东省科技计划项目基金资助(编号:2006B36003008)~~

摘　　要：目的观察携带有报告基因绿色荧光蛋白且表达ICOS-siRNA的重组腺病毒载体Ad-siICOS-EGFP阻断ICOS共刺激通路对角膜移植急性免疫排斥反应的抑制作用。方法取Wistar大鼠(供体)及SD大鼠(受体),建立大鼠同种异体穿透性角膜移植模型,将SD大鼠随机分为A、B、C、D4组,每组各20只:A组为单纯角膜移植对照组;B组为Ad-siICOS-EGFP注射组,术后即刻于球结膜下注射Ad-siICOS-EGFP;C组为Ad-EGFP注射组,术后即刻于球结膜下注射Ad-EGFP;D组为生理盐水注射组,术后即刻于球结膜下注射生理盐水;每眼选择角膜缘1200位、600位两个注射点,每个注射点注射剂量为15μL。术后每天在裂隙灯显微镜下观察排斥反应指数的变化并记录植片的存活时间。各组分别于术后5d随机取10只大鼠,麻醉后取角膜,部分植片行RT-PCR检测,部分角膜冰冻切片行共聚焦显微镜观察。各组其余大鼠进行角膜植片存活时间统计。结果 A组、B组、C组、D组角膜植片平均存活时间分别为(9.800±0.632)d、(14.700±1.418)d、(10.000±1.054)d、(10.100±0.876)d,其中B组存活时间最长,与其余各组相比差异均有统计学意义(均为P<0.05);A组、C组、D组各组间差异均无统计学意义(均为P>0.05)。B组及C组角膜植片共聚焦显微镜下可见GFP绿色荧光表达,表达位于角膜植片上皮层及整个角膜基质层;A组、D组角膜植片未见绿色荧光表达。A组、B组、C组、D组植片组织ICOS的PCR产物凝胶电泳条带灰度值比值分别为1.004±0.164、0.621±0.096、0.929±0.134、0.918±0.120,其中B组最低,与其余各组相比差异均有统计学意义(均为P<0.05);A组、C组、D组各组间差异均无统计学意义(均为P>0.05)。结论 Ad-siICOS-EGFP可以有效地抑制大鼠植片组织ICOSmRNA及蛋白的表达,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,可以抑制大鼠角膜移植急性免疫排斥反应,延长角膜植片存Objective To observe the inhibitory effect of Ad-siICOS-EGFP on the corneal transplantation acute immunological rejection by blocking the ICOS co-stimulator passageway in rats.Methods The homogeneity penetrability corneal transplantation rat model with Wistar rat（donor） and SD rat（acceptor） was established.The SD rats were divided 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）,15 μL Ad-siICOS-EGFP was immediately injected below the bulbar conjunctiva after operation;Ad-EGFP injection group（group C）,15 μL Ad-EGFP was immediately injected below the bulbar conjunctiva after operation;Saline injection group（group D）,15 μL normal sodium was immediately injected below the bulbar conjunctiva after operation.The change of rejection index（RI） was observed with slit lamp microscope everyday after operation and the implant survival time was recorded.Ten rats in each group were randomly selected on the 5th day after operation,one part of the cornea was tested with RT-PCR,and the other part was made into frozen action and observed with confocal microscopy.The corneal implant survival time of the rest rats were recorded in each group.Results The average survival time of corneal implant in group A,B,C,D were（9.800±0.632） d,（14.700±1.418） d,（10.100±1.054） d,（10.100±0.876） d,that in group B was the longest,compared with other groups,there were statistical differences（all P0.05）;There was no statistical difference among group A,C,D（all P0.05）.Green fluorescence expression could be found in cornea implant in group B and C,which were located on the cornea epithelium and in all the stroma.There was no green fluorescence expression in cornea implant of group A and D.The ratio of electrophoresis strip brightness of the ICOS PCR product of implant tissue in group A,B,C,D were 1.004±0.164,0.621±0.096,0.929±0.134,0.918±0.120,that in group B was the lowest,compared wi

关 键 词：角膜移植 RNA干扰 ICOS共刺激通路 免疫排斥反应 

分 类 号：Q959.836[生物学—动物学] R772.2[医药卫生—眼科]