方法：用双荧光素酶表达报告系统探讨miR-223-3p对Rbpj基因表达的调控作用。将24只雌性Lewis大鼠随机分为EAU模型组、正常对照组(NC组)和空白对照组(BC组)，每组8只。EAU模型组注射含光感受器间维生素A结合蛋白(IRBP)、结核菌素和完全弗氏佐剂的乳糜液以诱导葡萄膜炎，BC组注射等体积的不含IRBP多肽的乳糜液，NC组注射等体积的无菌生理盐水。免疫后12d，无菌分离三组大鼠的脾脏、淋巴结和眼组织，实时荧光定量PCR(Q-PCR)检测miR-223-3p和Rbpj基因的表达水平； 酶联免疫吸附测定(ELISA)方法检测Rbpj、IFN-γ、IL-17蛋白的表达水平； 流式细胞仪检测三组大鼠不同组织中Th1和Th17细胞的表达水平。

结果：双荧光素酶检测结果证实Rbpj是miR-223-3p调控的靶基因。免疫后12d，相比于NC组，EAU模型组大鼠脾脏、淋巴结和眼组织中miR-223-3p相对表达水平分别为0.33±0.29、0.11±0.12、0.18±0.11，均呈下调表达(均P<0.05)； Rbpj mRNA水平均呈上调表达，分别为3.00±0.06、1.52±0.12、3.01±0.34(均P<0.05)； BC组大鼠脾脏、淋巴结和眼组织中miR-223-3p和Rbpj mRNA水平与NC组相比均无差异(P>0.05)。ELISA检测结果发现，免疫后12d的EAU模型组大鼠各组织中Rbpj、IFN-γ、IL-17蛋白表达水平均明显高于NC组(均P<0.05)，BC组大鼠各组织中Rbpj、IFN-γ、IL-17蛋白表达水平与NC组相比均无差异(P>0.05)。流式细胞仪检测结果显示，免疫后12d，EAU模型组各组织的Th1和Th17细胞比例均显著高于NC组(均P<0.05)。BC组与NC组各组织Th1和Th17细胞比例无明显变化(均P>0.05)。

结论：miR-223-3p可负调控Notch信号通路转录因子Rbpj的表达。EAU大鼠中miR-223-3p的下调表达可升高Rbpj基因和蛋白的表达水平，促进Th1和Th17细胞的分化以及提高相关分子IFN-γ和IL-17的表达水平，进而影响葡萄膜炎的发生发展。

METHODS: The regulatory role of miR-223-3p in Rbpj gene expression was investigated by a dual luciferase expression reporter system. In the present study, 24 female Lewis rats were randomly divided into EAU model group, normal control(NC)group and blank control(BC)group, and each group included 8 rats. The EAU model group was injected with interphotoreceptor retinoid-binding protein(IRBP)emulsion containing Mycobacterium tuberculin H37RA and complete Freund's adjuvant to induce uveitis, while the NC group was injected with an equal volume of emulsion without IRBP peptide. The rats in the BC group received the same volume of sterile saline solution. At 12d after immunization, the spleen, lymph node and eye tissues in both groups were aseptically isolated, and the expression levels of miR-223-3p and Rbpj RNAs were detected by real-time quantitative PCR(Q-PCR); Meanwhile, the expression levels of Rbpj, IFN-γ and IL-17 proteins were detected by ELISA, and the levels of Th1 and Th17 cell lineages in each tissue from each groups were detected by flow cytometry.

RESULTS: The results of dual fluorescein assay indicated that Rbpj was the target gene which regulated by miR-223-3p. At 12d after immunization, compared with NC group, the relative expression levels of miR-223-3p in spleen, lymph node and eye tissues from EAU model rats were 0.33±0.29, 0.11±0.12 and 0.18±0.11, respectively, accompanied by the down-regulated expression, and the differences were statistically significant(all P<0.05); Rbpj mRNA levels were 3.00±0.06, 1.52±0.12 and 3.01±0.34, respectively, and were all up-regulated, while the differences were statistically significant(all P<0.05). Moreover, the differences in miR-223-3p and Rbpj mRNA levels in spleen, lymph node and eye tissues of rats in the blank control group were not statistically significant compared with those in the NC group(P>0.05); ELISA results revealed that the expression levels of RBPJ, IFN-γ and IL-17 proteins in all tissues from EAU rats at 12d after immunization were significantly higher than those in the NC group( all P<0.05), and there was no statistically significant difference in the expression levels of Rbpj, IFN-γ and IL-17 protein in all tissues of rats in the blank control group compared with the NC group(P>0.05); Meanwhile, flow cytometry results showed that the proportions of Th1 and Th17 cell lineages in all tissues from EAU model group were significantly higher than those from the NC group at 12d after immunization, and the differences were statistically significant(all P<0.05). Furthermore,there was no significant change in the proportion of Th1 and Th17 cells in each tissue in the BC and NC groups(all P >0.05).

CONCLUSION: The miR-223-3p can negatively regulate the expression of the transcription factor Rbpj of Notch signaling pathway. The down-regulated miR-223-3p expression in EAU rats can increase the expression levels of Rbpj gene and protein, and aggravate the differentiation of Th1 and Th17 cells and the expression levels of related molecules IFN-γ and IL-17, which in turn affect the development of uveitis.