器官培养法保存大鼠角膜移植术后Th1/Th2细胞因子和T细胞亚群表达的研究

摘要

Background Allogenic immunological rejection is still the common cause of keratoplasty failure.Organ culture is a good choice for gradually attenuating corneal antigeniciy.However,the complicated regulatory mechanism of such allogenic rejection with organ culture gratts is unclear until now. Objective To investigate the changes of several Thl/Th2 cytokines and T lymphocyte subsets in the rat corneal allogenic transplantation rejection with organ culture grafts. Methods Thirty-six SPF Wistar rats served as donors,and 72 SPF SD rats served as recipients in this study.Seventy-two SD rats were divided into two groups randomly.Organ culture and fresh grafts from Wistar rats were performed on 36 recipients SD rats,respectively.Six normal SD rats were served as the normal control.After transplantation,graft survival time was observed ; Aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-α were measured by enzyme linked immunosorbent assay.Immunohistochemistry was performed to examine CD25 expressions in grafts.Levels of TNF-αmRNA,IFN-γmRNA,IL-4mRNA and CD25mRNA in grafts were detected by using reverse transcription polymerase chain reagction.The levels of CD28 subsets in peripheral blood were determined by Flow Cytometry.The use of experimental animals followed the Statement of ARVO. Results The mean rat graft survival time was longer in the organ culture grafts group( 13.78 d) than that with fresh gratts( 10.56 d)(t=14.945,P =0.000 ).The aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-α were higher in two allograft groups at day 6,day 13 and day 24 after transplantation than that in the normal control,which peaked at day 13,and the fresh grafts group had the highest level( F=324.891,416.416,240.661,364.533,P=0.000).At day 13,CD25 was weakly expressed in frozen section of two operation groups.The expression of TNF-α and IFN-γmRNA in organ culture grafts group was markedly less than in fresh grafts group at day 13 after surgeries (t =2.464,P =0.039;t=5.438,P=0.001 ),whereas there was no significient difference in IL-4 and CD25 mRNA levels between them (t=-0.782,P =0.457,t =0.712,P =0.497).The percentages of CD28 of the fresh graft group increased significantly and more weakly expressed in organ culture grafts group (P =0.016 ). Conclusions Th1/Th2 cytokines and CD28 lymphocyte subsets may play important roles during corneal allograft rejection with organ culture grafts.%背景 免疫排斥反应是导致角膜移植手术失败的重要原因.器官培养法保存角膜可以逐渐降低植片的免疫原性,有利于减轻排斥反应.但目前对于器官培养法保存角膜植片行角膜移植术后确切的免疫排斥机制研究较少.目的 探讨器官培养法保存大鼠角膜植片行角膜移植后,部分Th1/Th2细胞因子和T细胞亚群在眼局部和/或全身的表达变化.方法 以36只Wistar大鼠为供体,72只SD大鼠为受体,行穿透角膜移植术.受体大鼠按随机数字表法分为器官培养植片组和新鲜植片组,每组36只；另选6只SD大鼠作为正常对照组.术后裂隙灯下观察植片存活时间和排斥情况,ELISA法检测房水中白细胞介素2(IL-2)、干扰素γ(IFN-γ)、IL-4和肿瘤坏死因子α(TNF-α)质量浓度的变化,免疫组织化学法检测植片内CD25的表达,逆转录聚合酶链反应(RT-PCR)检测植片内TNF-α mRNA、IFN-γ mRNA、IL-4 mRNA和CD25 mRNA的表达,流式细胞术检测外周血中CD28亚群的表达.结果 器官培养植片组的术后植片存活时间达13.78 d,长于新鲜植片组的10.56 d,差异有统计学意义(t=14.945,P=0.000).术后6、13、24 d时,两手术组房水中IL-2、IFN-γ、IL-4和TNF-α的质量浓度均高于正常对照组,于13 d时达最高水平,新鲜植片组质量浓度最高(F=324.891、416.416、240.661、364.533,P=0.000).术后13d时,植片内CD25表达较弱,两组间区别不明显；新鲜植片组中TNF-α mRNA和IFN-γ mRNA的表达强于器官培养植片组(t=2.464,P=0.039；t=5.438,P=0.001),两组IL-4 mRNA和CD25 mRNA的表达水平比较差异均无统计学意义(t=-0.782,P=0.457；t=0.712,P=0.497),正常角膜则无表达；3组大鼠外周血淋巴细胞中CD28亚群百分比差异有统计学意义( F=70.489,P=0.000),两手术组显著升高,但器官培养植片组水平低于新鲜植片组(P=0.016). 结论 Th1/Th2因子平衡调节机制和CD28亚群可能在器官培养法保存角膜植片行角膜移植术后的排斥反应中发挥重要的调节作用.