间充质干细胞对实验性自身免疫性葡萄膜炎眼部CD4＋T细胞亚群浸润的影响

Effects of mensenchymal stem cell on infiltration of CD4＋ T cell subsets in the eyes of experimental autoimmune uveitis

目的观察大鼠骨髓间充质干细胞（MsCs）对大鼠实验性自身免疫性葡萄膜炎（EAU）的治疗效果。方法Lewis大鼠12只随机分为对照组与治疗组，均为6只大鼠。大鼠单后足及背部6点注射含30μg光感受器间维生素A类结合蛋白多肽片段R16的乳剂0．2m1，建立EAU模型，观察各组临床表现。贴壁培养法纯化Wistar大鼠骨髓MSCs。建模后第9～11天，治疗组每日行尾静脉注射浓度为5×10^6／ml的MSCs悬液1ml，对照组注射等体积磷酸盐缓冲液。建模后第15天，分离各组大鼠眼部细胞，通过流式细胞仪检测干扰素-7、白细胞介素-17和叉状头螺旋转录因子3阳性细胞的比例。采用混合线性模型进行重复测量资料的方差分析统计临床评分；独立样本t检验统计流式细胞检测结果。结果建模后第6天，虹膜血管轻度扩张充血；第9天前房轻度混浊，瞳孔缩小，对光反射消失；第12天前房混浊，瞳孔膜闭，眼底红光反射变暗甚至消失；之后炎症反应逐渐减轻。第11～15天，MSCs治疗组临床评分明显低于对照组，差异有统计学意义（t=2．42、2．21、4．16、5．24、4．03，P〈0．05）。流式细胞仪检测结果显示，MSCs治疗可以显著减少眼部CD4＋T细胞、Th1细胞和Th17细胞比例以及增加调节性T细胞比例。结论MSCs治疗可以减轻EAu的临床表现；减少CD4＋ T细胞向眼部浸润，下调眼部效应性Th1细胞、Th17细胞以及上调调节性T细胞。

Objective To observe the therapeutic effect of mensenchymal stem cells （MSCs） for experimental autoimmune uveitis （EAU）. Methods MSCs were obtained from Wistar rats and selected by plastic adherence. Lewis rats were divided into treatment group and control group, six rats in each group. EAU models were induced by immunization with an emulsion （0.2 ml） containing 30μg interphotoreceptor retinoid-binding protein derived peptide R16 and complete Freundts adjuvant. The clinical manifestations of two groups were observed. Nine to 11 day after modeling, 1 ml MSCs suspension, which contained 5）~ 106 MSCs, were injected into the rats in treatment group via tail vein, and the rats in control group were given equal volume of phosphate buffer solution. Fifteen day after modeling, the eyes were collected to test the proportion of interferon-y, interleukin-17 and Foxp3 positive cells by flow cytometry. The clinical scores were analyzed by mixed linear model and statistical analysis of variance of repeated measurement data. The results of flow cytometry were analyzed using independent-sample t-test. Results Six days after immunization, mild dilatation and congestion of iris vascular was observed. Nine days after immunization, mild muddy anterior chamber, myosis and absent pupillary reaction to light were observed. Twelve days after immunization, muddy anterior chamber, occlusion of pupil and dimmed or disappeared red reflex were observed, and then inflammation was slowly reduced. From 11 to 15 days after immunization, the clinical score of treatment group was lower than that in control group, the difference was statistically significant （t=2.42, 2.21, 4.16, 5.24, 4.03, P〈0.05）. The results of flow eytometry showed that MSCs treatment could decrease the proportion of CD4＋ T cells, Thl cells and Th17 cells, increase the proportion of Treg cells. Conelusion MSCs treatment can ameliorate EAU, up-regulate the expression of Treg cells and down- regulate the expression of CD4＋ T cells, Th1 cells and Th17 cells.