[1]殷学伟,刘滨,魏慧霞,等.rno-miR-30b-5p对葡萄膜炎大鼠Notch1和Dll4基因表达的调控作用[J].眼科新进展,2019,39(1):012-16.[doi:10.13389/j.cnki.rao.2019.0003]
　YIN Xue-Wei,LIU Bin,WEI Hui-Xia,et al.Regulatory role of rno-miR-30b-5p inNotch1 and Dll4 geneexpressions in experimental autoimmune uveitis rats[J].Recent Advances in Ophthalmology,2019,39(1):012-16.[doi:10.13389/j.cnki.rao.2019.0003]
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
39卷
期数:
2019年1期
页码:
012-16
栏目:
实验研究
出版日期:
2019-01-05

文章信息/Info

Title:
Regulatory role of rno-miR-30b-5p inNotch1 and Dll4 geneexpressions in experimental autoimmune uveitis rats
作者:
殷学伟; 刘滨; 魏慧霞; 唐凯; 毕宏生; 郭大东
250014　山东省济南市，山东中医药大学［殷学伟（2017级硕士研究生），刘滨（2016级硕士研究生），魏慧霞（2018级硕士研究生）］；250002　山东省济南市，山东省中西医结合眼病防治重点实验室，山东中医药大学眼科研究所（唐凯，毕宏生，郭大东）
Author(s):
YIN Xue-Wei; LIU Bin; WEI Hui-Xia; TANG Kai; BI Hong-Sheng; GUO Da-Dong
Shandong University of Traditional Chinese Medicine （YIN Xue-Wei，LIU Bin，WEI Hui-Xia），Jinan 250014，Shandong Province，China；Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases，Eye Institute of Shandong University of Traditional Chinese Medicine （TANG Kai，BI Hong-Sheng，GUO Da-Dong），Jinan 250002，Shandong Province，China
关键词:
rno-miR-30b-5p; Notch1; Dll4; 实验性自身免疫性葡萄膜炎
Keywords:
rno-miR-30b-5p; Notch1; Dll4; experimental autoimmune uveitis
分类号:
R773
DOI:
10.13389/j.cnki.rao.2019.0003
文献标志码:
A
摘要:
目的　探讨rno-miR-30b-5p对Notch1和Dll4基因表达的调控作用及其在实验性自身免疫性葡萄膜炎（experimental autoimmune uveitis，EAU）中的表达变化。方法　应用双荧光素酶检测rno-miR-30b-5p对Notch1和Dll4基因表达的调控作用。6~8周龄健康Lewis大鼠12只，随机分为对照组和EAU模型组，EAU模型组大鼠建立EAU模型。于免疫后12 d分离大鼠的脾脏、淋巴结和眼组织，Q-PCR检测rno-miR-30b-5p、Notch1和Dll4基因的表达水平，ELISA检测Notch1和Dll4蛋白的表达变化；流式细胞仪检测两组大鼠脾脏和淋巴结中Th17和Treg细胞的表达水平。结果　双荧光素酶检测结果表明，Notch1和Dll4为rno-miR-30b-5p调控的靶基因。Q-PCR分析结果显示，相比于正常对照组（1.00），免疫后12 d，rno-miR-30b-5p基因在EAU模型组大鼠脾脏、淋巴结和眼组织中的相对表达水平分别为0.41±0.12、0.37±0.09和0.25±0.07，均呈显著下调表达，而Notch1和Dll4基因呈显著上调表达，且差异均有统计学意义（均为P<0.05）。ELISA检测结果显示，免疫后12 d，EAU模型组大鼠脾脏、淋巴结和眼组织中Notch1和Dll4蛋白的表达水平均明显高于对照组（均为P<0.05）。流式细胞仪检测结果显示，免疫后12 d，EAU模型组大鼠脾脏和淋巴结中Th17细胞因子的表达水平明显升高，而Treg细胞因子的表达水平明显降低。结论　rno-miR-30b-5p可负调控Notch1和Dll4基因的表达。在EAU模型大鼠的脾脏、淋巴结和眼组织中，rno-miR-30b-5p的下调表达可使Notch1和Dll4基因的表达水平显著升高，并使Th17/Treg比例发生紊乱，从而影响葡萄膜炎的发生发展。
Abstract:
Objective　To investigate the regulatory role of rno-miR-30b-5p in Notch1 and Dll4 gene and their expression changes in experimental autoimmune uveitis （EAU） rats.Methods　Using dual luciferase report system，we investigated the regulatory role of rno-miR-30b-5p in Notch1 and Dll4 gene expressions.Twelve healthy Lewis rats （6-8 weeks） were randomly divided into control group and EAU model group.EAU model group established EAU model.On day 12 after immunization，spleen，lymph nodes and eye tissues were isolated.Subsequently，quantitative PCR （Q-PCR） was performed to monitor the expressions of rno-miR-30b-5p，Notch1 and Dll4 genes.Moreover，the changes of both Notch1 and Dll4 protein levels were determined by ELISA technique.Th17 and Treg cell levels were analyzed by using flow cytometry.Results　Dual luciferase report gene expression assay showed that Notch1 and Dll4 are target genes of rno-miR-30b-5p.The Q-PCR analysis showed that compared with the normal control group （1.00），the relative expression level of rno-miR-30b-5p in spleen，lymph nodes and ocular tissues of the EAU model group was 0.41±0.12，0.37±0.09，and 0.25±0.07 at 12 d after immunization，accompanied by significantly down-regulated expression，while Notch1 and Dll4 were significantly up-regulated，and the difference was statistically significant （all P<0.05）；meanwhile，ELISA results showed that Notch1 and Dll4 proteins were also markedly elevated in spleen，lymph nodes and eye tissues in EAU rats compared with those in control groups （all P<0.05）.Flow cytometry determination revealed that Th17 cell levels in spleen and lymph nodes in EAU model group were up-regulated，whereas Treg cell levels were significantly down-regulated.Conclusion　rno-miR-30b-5p can negatively regulate Notch1 and Dll4 gene expressions.The down-regulation of rno-miR-30b-5p expressions in spleen，lymph nodes and eye tissues in EAU model group rats can significantly enhance the expressions of Notch1 and Dll4 genes，and the ratio of Th17/Treg was out of balance，thereby regulating the pathogenesis of uveitis.

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备注/Memo

备注/Memo:
国家自然科学基金（编号：81873163、81674032）；山东省自然科学基金（编号：ZR2017LH042、ZR2016HP27）；山东中医药科技发展计划（编号：2015-145）
更新日期/Last Update: 2019-01-04