hUMSCs外分泌蛋白治疗实验性自身免疫性葡萄膜炎模型大鼠的机制初探

Mechanism of hUMSCs-derived secretome therapy in an experimental autoimmune uveitis rat model

目的:探讨腹腔注射人脐带间充质干细胞外分泌蛋白(hUMSCs-S)治疗实验性自身免疫性葡萄膜炎(EAU)模型大鼠的可能性及其相关作用机制。方法:6~8周龄Lewis大鼠72只,用随机数字表分方法均分为3组,分别为正常对照(CTRL)组、EAU模型组及hUMSCs-S治疗组,每组24只。EAU组与hUMSCs-S组大鼠采用光感受器间维生素A结合蛋白(IRBP)联合弗氏完全佐剂建立EAU模型,hUMSCs-S组大鼠在建模的同时予以腹腔注射hUMSCs-S,EAU组及CTRL组大鼠均腹腔注射等量PBS。裂隙灯显微镜拍摄记录各组大鼠眼前段情况,根据Caspi评分标准评价眼前段炎症程度;于免疫后第7天(初发期)、14天(高峰期)、21天(恢复期)取眼球组织制成石蜡切片进行HE染色、免疫组化及免疫荧光染色,分别用于眼球组织病理学评分、前房浸润细胞情况评估,观察眼球组织中CD3^(+)T细胞及紧密连接蛋白ZO-1的表达。无菌操作下采腹主动脉血,制备血清,行ELISA法检测各组大鼠血清中白细胞介素10(IL-10)和IL-17的浓度。结果:(1)通过裂隙灯显微镜观察和比较,EAU组大鼠出现明显眼前段炎症反应,在建模后第11~17天,hUMSCs-S治疗有降低眼前段Caspi评分的作用(P<0.05);(2)眼球组织切片HE染色结果显示,建模后第14天,hUMSCs-S组大鼠虹膜睫状体和视网膜结构破坏及前房炎细胞浸润数量均低于EAU组(P<0.05);(3)眼球组织切片免疫组化及免疫荧光染色结果显示,hUMSCs-S组大鼠虹膜睫状体、前房和视网膜中CD3^(+)T细胞浸润较EAU组减少,视网膜及视网膜色素上皮(RPE)层ZO-1表达量较EAU组增加;(4)ELISA法检测大鼠外周血血清IL-17及IL-10浓度结果显示,与EAU组相比,hUMSCs-S组大鼠外周血IL-17表达下降、IL-10表达增加(P<0.05)。结论:hUMSCs外分泌蛋白对EAU模型大鼠治疗有效;腹腔注射hUMSCs外分泌蛋白可影响EAU大鼠外周血中炎症因子表达,减少眼内炎症细胞浸润,减轻RPE层紧密连接蛋白的破坏程度。

AIM:To investigate the potential mechanism of intraperitoneal injection of human umbilical cord mesenchymal stem cells-derived secretome(hUMSCs-S)in treating experimental autoimmune uveitis(EAU)in a rat model.METHODS:72 Lewis rats aged 6~8 weeks were randomly divided into three groups:control(CTRL)group,EAU group and hUMSCs-S treatment group,with 24 rats in each group.The rats in EAU and hUMSCs-S groups were administered with interphotoreceptor retino-binding protein(IRBP)combined with Freund's complete adjuvant.The rats in hUMSCs-S group were injected intraperitoneally with hUMSCs-S,and the rats in EAU and CTRL groups were injected intraperitoneally with equal amount of PBS.After modeling,the anterior segment of rats was observed and photographed under the slit lamp microscope every day,and the degree of anterior segment inflammation was evaluated according to Caspi scoring criteria.On the 7th day(initial stage),14th day(peak stage)and 21st day(recovery stage)after immunization,8 rats in each group were randomly selected.The eyeballs were extracted and fixed to make paraffin sections,and HE staining,immunohistochemical staining and immunofluorescence staining were used for the evaluation of eyeball histopath-ological score and anterior chamber infiltrating cells.The expression of CD3^(+)T cells and ZO-1 tight junction protein in eye tissue was observed.After the eyeball was removed,abdominal aorta blood was collected under aseptic operation,serum was prepared,and serum interleukin 10(IL-10)and IL-17 concentrations were detected by ELISA.RESULTS:(1)Cas-pi scores of the anterior chamber of hUMSCs-S group were lower than those in EAU group on days 11 to 17 after modeling(P<0.05).(2)The histopathological lesion observed in the iris,ciliary body,and retina,as well as the infiltration of in-flammatory cells in anterior chamber of hUMSCs-S group were significantly lower than those in EAU group(P<0.05).(3)In the hUMSCs-S group,there was a lower presence of CD3^(+)T cells in the iris,ciliary body,anterior chamber,and retina of rats compared to the EAU group.Additionally,the expression of ZO-1 in the retina and retinal pigment epithelial(RPE)layer was higher in the hUMSCs-S group.(4)The expression of IL-17 in peripheral blood of rats in hUMSCs-S group was decreased,and IL-10 was increased compared to the EAU group(P<0.05).CONCLUSION:Intraperitoneal injection of hUMSCs-derived secretome intervenes the expression of inflammatory factors in peripheral blood,reduces the infiltration of inflammatory lymphocytes into iris and ciliary body,anterior chamber and retina,ameliorates the destruction of tight junction protein of RPE layer,alleviates damage to the anterior segment and retina of EAU rats.