rno-mir-30b-5p 对葡萄膜炎大鼠IL-10, TLR4 表达的调控作用研究
首发时间:2017-05-24
摘要:目的 探讨rno-miR-30b-5p在葡萄膜炎中对白细胞介素10(interleukin-10, IL-10)和Toll样受体4(toll-like receptor 4, TLR4)的调控作用。方法 制备实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)大鼠模型,在葡萄膜炎炎症高峰期(造模后第12天)观察眼前节炎症反应记录发病程度,并进行病理检查;分离EAU大鼠的脾脏和淋巴结并提取总RNA,通过实时荧光定量PCR检测rno-miR-30b-5p和IL-10、TLR4基因的表达情况, ELISA检测IL-10和TLR4蛋白的表达情况;同时研磨脾脏和淋巴结,用促进和抑制rno-miR-30b-5p表达的miRNA模拟剂和拮抗剂进行T细胞转染,通过PCR检测转染后T细胞中IL-10、TLR4基因的表达情况,通过流式细胞仪、ELISA检测IL-10和TLR4蛋白的表达情况。结果 实时荧光定量PCR检测发现rno-miR-30b-5p在EAU大鼠脾脏和淋巴结组织中的表达水平明显降低,而IL-10、TLR4 mRNA表达明显升高;ELISA检测IL-10、TLR4蛋白表达升高,与对照组相比,表达水平差异均具有统计学意义(均为P<0.05)。体外细胞转染实验发现,加入rno-miR-30b-5p模拟剂转染的脾脏和淋巴结T细胞中IL-10、TLR4的基因和蛋白出现明显的下调表达;rno-miR-30b-5p拮抗剂干预后,TLR4 mRNA在脾脏和淋巴结T细胞中显著上调表达(均为P<0.05),IL-10 mRNA在淋巴结T细胞中上调表达(P<0.05),而 IL-10 mRNA在脾脏T细胞中的表达和IL-10、TLR4蛋白质在脾脏和淋巴结T细胞中的表达与对照组比无统计学意义。结论 动物实验和体外细胞转染实验结果证实,rno-miR-30b-5p在EAU大鼠脾脏和淋巴结的下调表达导致IL-10和TLR4的基因和蛋白表达水平升高,进而调控葡萄膜炎的发展。本研究为进一步探索microRNA在葡萄膜炎发生发展进程中的调控作用、寻找新的葡萄膜炎治疗靶标奠定了基础。
关键词: rno-miR-30b-5p IL-10 TLR4 葡萄膜炎 大鼠
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Regulatory roles of rno-miR-30b-5p in the expressions of IL-10 and TLR4 in rats with experimental autoimmune uveitis
Abstract:ObjectiveTo investigate the regulatoryroleof rno-miR-30b-5p inthe expressions of interleukin-10(IL-10) and toll-like receptor 4(TLR4) in uveitis. Methods Preparation of EAU model, Observe and record of anterior segment inflammation, meanwhile pathological examination. The spleen and lymph nodes of EAU rats were isolated on day 12 after modeling. The expression of rno-miR-30b-5p and IL-10 and TLR4 genes were detected by real-time quantitative PCR., ELISA to detect the expression of IL-10 and TLR4 protein, ; The spleen and lymph nodes were ground and transfected with miRNA mimics and antagonists that promote and inhibit rno-miR-30b-5p expression, The expression of IL-10 and TLR4 gene in T cells after transfection was detected by PCR, and the expression of IL-10 and TLR4 protein were detected by flow cytometry and ELISA.ResultsReal time fluorescent quantitative PCR detection showed that the expression level of rno-miR-30b-5p in the spleen and lymph nodes of EAU rats was significantly reduced, while the expression of IL-10 and TLR4 mRNA was significantly increased;ELISA detection showed that the expression of IL-10 and TLR4 increased,Compared to the control group, the expression level difference was statistically significant (all P < 0.05).In vitro transfection experiments showed that the expression of IL-10 and TLR4 genes and proteins in spleen and lymph node T cells transfected with rno-miR-30b-5psimulant was significantly down regulated;The expression of TLR4 mRNA was significantly up-regulated in spleen and lymph node T cells after intervention with rno-miR-30b-5p antagonist (all P <0.05),IL-10 mRNA was up-regulated in lymph node T cells (P <0.05),The expression of IL-10 mRNA in spleen T cells and the expression of IL-10 and TLR4 proteins in spleen and lymph node T cells were not statistically significant compared with the control group.ConclusionsAnimal experiments and in vitro transfection experiments showed that the down-regulation of rno-miR-30b-5p expression in the spleen and lymph nodes of EAU rats resulted in the increase of IL-10 and TLR4 gene and protein expression, and then the development of uveitis.This study laid a foundation for further study on the regulation of microRNA in the development of uveitis and the treatment of uveitis.
Keywords: rno-miR-30b-5p IL-10 TLR4 Uveitis Rat
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rno-mir-30b-5p 对葡萄膜炎大鼠IL-10, TLR4 表达的调控作用研究
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