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目的 观察大鼠来源的间充质干细胞(MSC)外泌体对实验性自身免疫性葡萄膜炎(EAU)大鼠模型的治疗作用.方法 将12只Lewis大鼠采用随机数字表法随机分为实验组和对照组,每组6只大鼠.实验组大鼠建立EAU模型,并于建模后第9天单次球周注射外泌体100μl(含量50 μg);对照组大鼠注射相同体积的磷酸盐缓冲液.建模后不同时间点,采用苏木精伊红(HE)染色观察大鼠视网膜结构,并对其临床和病理进行评分;免疫组织化学染色观察巨噬细胞表面标志物CD68的表达;T细胞增生实验观察在1、10、30 μg/mlR16刺激下MSC外泌体对T细胞增生的影响;流式细胞技术检测Th1、Th17及调节性T细胞的变化;视网膜电图(ERG)评估大鼠视网膜功能;两组间数据比较采用t检验.结果 HE染色观察发现,实验组大鼠于建模后第15天视网膜结构较对照组更完整.免疫组织化学染色观察发现,实验组大鼠视网膜CD68阳性表达明显少于对照组.建模后第15天,实验组大鼠视网膜病理评分较对照组更低,差异有统计学意义(P＜0.05);建模后第9～13天,实验组大鼠视网膜临床评分均低于对照组,差异有统计学意义(t=3.665、3.210、3.181、4.121、3.227,P＜0.01).T细胞增生实验结果显示,在1、10、30 μg/ml R16刺激下1.0、10.0 μg/ml外泌体对T细胞的增生有抑制作用,差异有统计学意义(F=1 1.630、4.188、6.011,P＜0.05).流式细胞技术检测结果显示,实验组大鼠眼部浸润的Th1、Th17细胞和调节性T细胞亚群数量较对照组减少,差异有统计学意义(t=7.374、4.525、6.910,P＜0.01);两组淋巴结中的细胞比例无差异(t=1.126、0.493、0.178,P=0.286、0.632、0.862).ERG检测结果显示,建模后第15天暗适应0.01、3.0 cd/m2 a波(t=3.604、4.178)和b波(t=4.551、2.566)振幅均较对照组增高,差异有统计学意义(P＜0.05).结论 大鼠来源的MSC外泌体可以减轻EAU的临床及病理表现,保护视网膜功能,减少眼部巨噬细胞浸润,下调眼部致炎性T细胞的比例,抑制T细胞增生.

Objective To observe the effects of exosomes derived from rat mesenchymal stem cells (MSC-exosomes) on the rat experimental autoimmune uveitis (EAU) model.Methods Twelve Lewis rats were randomly divided into experimental group and control group by random number table,with 6 rats in each group.Rats in the experimental group were established with EAU model,100 μl of MSC-exosomes (50 μg) were periocular injected on the 9th day after modeling while the control rats were injected with the same volume of phosphate buffer.At different time points after modeling,the retinal structure was observed by hematoxylin and eosin (HE) staining,and the clinical and pathological manifestations were evaluated.T cells from the two groups were analyzed by flow cytometry,Immunohistochemical staining was used to observe the expression of macrophage surface marker CD68.The effect of MSC-exosomes on T cells was measured by lymphocyte proliferation assays.And flow cytometry was used to detect Th 1,Th 17 and regulatory T cells Variety.Electroretinogram (ERG) was used to evaluate the retinal function.Data were compared between the two groups using the t test.Results HE staining showed that the retina structure of the experimental group was more complete than that of the control group on the 15th day after modeling.Immunohistochemical staining showed that the positive expression of CD68 in the experimental group was significantly less than that in the control group.On the 15th day after modeling,the retinal pathological score of the experimental group was lower than that of the control group.On the 9th to 13th day after modeling,compared to the control group,the average clinical scores of the retina in the experimental group were lower,and the difference was statistically significant (t=3.665,3.21,3.181,4.121,3.227;P＜0.01).The results of T cell proliferation assay showed that exosomes (1.0,10.0 μg/ml) inhibited the proliferation of T cells under different concentrations of R16 (1,10,30 μg/ml),and the difference was statistically significant (F=1 1.630,4.188,6.011;P＜0.05).The results of flow cytometry showed that the number ofThl,Th17 and Treg cell subsets in the experimental group was decreased compared with the control group,and the difference was statistically significant (t=7.374,4.525,6.910;P＜0.01).There was no difference in the proportion of cells in the T cells and lymph nodes (t=1.126,0.493,0.178;P=0.286,0.632,0.862).The results of ERG showed that,compared with the control group,the amplitudes of 0.01,3.0 cd/m2 a wave and b wave of the experiment group were all increased on the 15th day after modeling,and the differences were statistically significant (t=3.604,4.178,4.551,2.566,P＜0.05).Conclusions MSC-exosomes can reduce the clinical and pathological manifestations of EAU,protect retinal function,reduce ocular macrophage infiltration,down-regulate the proportion of inflammatory cells in the eye,and inhibit T cell proliferation.