背景 视网膜移植是治疗视网膜变性性疾病的新方法,但如何避免或减少视网膜移植后的免疫排斥反应是亟待解决的问题之一.目前的实验研究中常将正常C57 BL/6小鼠视网膜作为供体,视网膜变性(rd)小鼠作为受体.研究表明视网膜中Fas配体(FasL)蛋白通过FasL/Fas途径诱导Fas+炎症细胞凋亡,推测可能与视网膜移植后的免疫排斥反应密切相关. 目的 探讨FasL蛋白在不同鼠龄C57BL/6小鼠和rd小鼠视网膜中的表达特点,揭示小鼠视网膜的免疫特性,为视网膜移植免疫排斥反应的研究提供参考依据.方法 分别将出生后(PN)-0周(出生日)、PN-1周、PN-2周、PN-3周、PN-4周的正常C57BL/6小鼠和rd小鼠处死制备眼球冰冻切片,采用免疫荧光技术摄片,并经激光扫描共焦显微镜采集图片,用图像分析软件对各鼠龄不同品系小鼠间视网膜中FasL蛋白表达的荧光强度(FI)变化进行半定量分析. 结果 PN-1周C57BL/6小鼠视网膜发育尚不完善,FasL蛋白在视网膜各层均呈阳性表达.PN-2、3、4周C57BL/6小鼠已发育成10层的视网膜结构,FasL蛋白在视网膜色素上皮(RPE)、内节段(IS)、外界膜(OLM)、外丛状层(OPL)、内核层(INL)、内丛状层(IPL)及节细胞层(GCL)呈阳性表达.PN-1周rd小鼠视网膜结构与同龄C57BL/6小鼠接近,FasL蛋白在视网膜各层均呈阳性表达.PN-2周至PN-4周rd小鼠视网膜中外核层(ONL)细胞随着鼠龄增大逐渐减少,FasL蛋白在RPE、OPL、INL、IPL及GCL呈阳性表达;PN-2、3、4周rd小鼠RPE中FasL蛋白表达FI值分别为184.199±16.747、186.797±7.904和184.319±18.795,均明显高于同龄C57BL/6小鼠的160.402±22.851、160.995±22.799和105.787±17.676,差异均有统计学意义(t=-3.360,P=0.002;t’=-4.277,P=O.000;t=-12.175,P=0.000);各周龄rd小鼠与C57BL/6小鼠间视网膜IPL中FasL蛋白表达的FI值差异均无统计学意义(均P＞O.05).结论 rd小鼠随着鼠龄增加视网膜ONL细胞逐渐减少,其RPE内免疫赦免相关抗原FasL蛋白的表达强度明显高于同龄C57BL/6小鼠.%Background Retinal transplantation is a new approach to the treatment of retinal degeneration diseases,and how to avoid or reduce the immune rejection after transplantation is a problem.In experimental studies on retinal transplantation,C57BL/6 mice are often used as donors and rd mice serve as recipients.Studies showed that Fas ligand (FasL) protein induces the apoptosis of Fas+ inflammatory cells by FasL/Fas signal pathway,speculating that FasL protein is associated with immune rejection after transplantation.Objective The aim of this study was to investigate FasL protein expression change in retinas of C57BL/6 mice and rd mice with aging and reveal the immune characteristics of the mouse retinas.Methods The frozen sections of eyeball from C57BL/6 mice and rd mice at the age of postnatal (PN)-0 week,PN-1 week,PN-2 week,PN-3 week and PN-4 week were prepared.The expression of FasL protein in the mouse retinas was examined by immnofluorescense technique.Images were acquired by fluorescence microscope and analyzed semi-quantitively by software from laser scanning confocal microscope as the fluorescence intensity (FI).The results were compared among different strains of mice.Results The retina developed imperfectly in PN-1 week C57BL/6 mice and FasL protein was positively expressed in the whole retina.In PN-2,3 and 4 week C57BL/6 mice,retinas finished the development with 10 layers,and retinal pigment epithelium (RPE),inner segment (IS),outer limiting membrane (OLM),outer plexiform layer (OPL),inner nuclear layer (INL),inner plexiform layer (IPL) and ganglion cell layer (GCL).Retinal structure and expression of FasL protein in the whole retina of rd mice in the PN-1 week were similar with C57BL/6 mice,however,ONL cells of rd mice were evidently decreased with aging.The RPE,OPL,INL,IPL and GCL expressed the FasL protein in PN-2,PN-3 and PN-4 week rd mice.The mean FI of FasL protein in the RPE layer was 184.199±16.747,186.797±7.904 and 184.319± 18.795 in rd mice of PN-2 week,PN-3 week and PN-4 week,which were significantly higher than 160.402±22.851,160.995 ±22.799 and 105.787 ± 17.676 in C57BL/6 mice (t =-3.360,P =0.002;t'=-4.277,P =0.000;t =-12.175,P=0.000).There were not significant differences in the mean FI of FasL protein in IPL between C57BL/6 mice and rd mice at ages of PN-2 week,PN-3 week and PN-4 week (all at P＞0.05).Conclusions The cells of retinal ONL are gradually decreased with the development of rd mice,and FasL protein expression intensity in RPE is evidently enhanced in rd mice compared with C57BL/6 mice.
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