白细胞介素1受体相关激酶（IRAK）家族在基于Toll样受体4信号通路介导的急性前葡萄膜炎发病中所起作用及红芪多糖干预机

发布时间：2018-03-29 20:03

本文选题：内毒素诱导葡萄膜炎　切入点：内毒素耐受　出处：《首都医科大学》2017年博士论文

【摘要】：目的:通过脂多糖预处理研究内毒素耐受对于大鼠内毒素诱导葡萄膜炎的作用,并观察包括白细胞介素1受体相关激酶M在内的相关分子的表达改变方法:90只雄性Wistar大鼠被随机分配到三个实验组:正常对照组(NC)、内毒素耐受组(ET)和内毒素诱导葡萄膜炎组(EIU),每组30只。通过皮下注射脂多糖(200μg)诱导Wistar大鼠内毒素诱导葡萄膜炎(EIU、ET)。在诱导模型前连续五天,给予大鼠腹腔注射小剂量脂多糖(0.1 mg/kg)(ET)或其溶剂(EIU、NC)。内毒素诱导葡萄膜炎24小时后,使用裂隙灯检查大鼠眼部炎症后处死大鼠并取眼球。通过组织病理学检查判断大鼠眼内炎症反应强度。通过实时定量逆转录PCR和Western blot研究NF-k B的激活情况以及IRAK-1、IRAK-4和IRAKM的表达。结果:脂多糖的重复预处理显著抑制了眼内炎症(P0.001)及NF-κb p65在细胞核内蛋白表达的表达(P0.01)。内毒素耐受组中IRAK-1和IRAK-4的m RNA及蛋白表达较内毒素诱导葡萄膜炎组(EIU)明显受到抑制(P0.01),而IRAKM的m RNA及蛋白表达则有所上升(P0.01)。结论:内毒素耐受对于内毒素诱导葡萄膜炎有保护作用,IRAK-M通过Toll样受体信号通路的上调可能是内毒素耐受发生的原因。目的:使用包含m RNA及长链非编码RNA(lnc RNA)的高通量基因芯片,研究红芪多糖预处理内毒素诱导葡萄膜炎大鼠后的差异表达m RNA及长链非编码RNA。通过基因功能富集分析(Gene Ontology,GO),信号通路分析(Pathway analysis)和共表达调控网络等筛选出具有研究价值的基因。方法:使用红芪多糖(400mg/kg)或生理盐水于脂多糖注射前24小时及1小时前腹腔注射各预处理5只Wistar大鼠后,使用脂多糖(200μg)诱导大鼠葡萄膜炎。观察大鼠眼内炎症临床表现后提取大鼠虹膜睫状体组织RNA并使用基因表达谱芯片技术检测差异表达的m RNA及lnc RNA基因。运用基因功能富集分析(Gene Ontology,GO)、信号通路分析和共表达调控网络对筛选出的差异基因之间的调控作用进行深入分析并筛选出靶基因。结果:经红芪多糖预处理的大鼠在脂多糖刺激后眼部炎症收到显著抑制(P0.05)。以FC(abs)1.5和P值0.05为标准筛选出254个差异表达的m RNA基因及68个lnc RNA基因。GO分析显示差异基因显著功能主要集中在脂多糖反应、细菌来源分子反应、趋化因子活动、趋化因子受体结合、淋巴细胞迁徙、淋巴细胞迁徙正调控、细胞因子活动、淋巴细胞趋药性和白细胞迁徙等。而差异基因的显著信号通路则包括细胞因子-细胞因子相互作用、Toll样受体信号通路、炎症调控趋化因子及细胞因子信号通路、肿瘤坏死因子(TNF)信号通路和类风湿性关节炎等。通过共表达网络分析筛选出表达于网络核心位置的16个m RNA和7个lnc RNA基因。结论:本研究发现红芪多糖通过多条与炎症及免疫相关的信号传导通路发挥对内毒素诱导葡萄膜炎的抗炎作用。通过一系列筛选过程获得了多个备选的抗炎靶基因,其中通过上调富亮氨酸重复蛋白-1和下调基因集落刺激因子1以及通过lnc RNA调控NF-κB发挥抗炎作用有较大的可能和研究价值。
[Abstract]:Objective: through the study of lipopolysaccharide pretreatment in endotoxin tolerant rats for endotoxin induced uveitis function, and to observe the expression of related molecules including interleukin 1 receptor associated kinase M, the change of methods: 90 male Wistar rats were randomly assigned to three experimental groups: normal control group (NC), endotoxin tolerance group (ET) and endotoxin induced uveitis (EIU group), 30 rats in each group. By subcutaneous injection of LPS (200 g) of Wistar rats induced by endotoxin induced uveitis (EIU, ET). In the former model was induced for five consecutive days, the rats were given small dose of intraperitoneal injection of LPS (0.1 mg/kg) (ET) or its solvent (EIU, NC). 24 hours after endotoxin induced uveitis, ocular inflammation using slit lamp examination in rats after the rats were killed and the eyeballs. By histopathological examination to determine the rat ocular inflammation intensity. By real-time quantitative reverse transcription PCR and West Ern blot of NF-k B activation and IRAK-1, expression of IRAK-4 and IRAKM. Results: repeated LPS pretreatment significantly inhibited the intraocular inflammation (P0.001) and the expression of NF- kappa B p65 in nucleus protein expression (P0.01). The expression of M RNA and IRAK-1 protein and IRAK-4 in endotoxin tolerance group was induced by endotoxin uveitis group (EIU) was significantly inhibited (P0.01), and the expression of M protein and RNA IRAKM increased (P0.01). Conclusion: the endotoxin tolerance for endotoxin induced uveitis has a protective effect, IRAK-M through Toll like receptor signaling pathway upregulation may be the cause of endotoxin tolerance occurred. Objective: using a m RNA and long the chain of non RNA (LNC RNA) encoding the high-throughput gene chip of Hedysari polysaccharide pretreatment in endotoxin induced uveitis in rats after the difference of expression of M RNA and long chain non encoding by RNA. gene function enrichment analysis (Ge NE, Ontology, GO) signal pathway analysis (Pathway analysis) and co expression regulation network of selected valuable genes. Methods: using Radix Hedysari polysaccharide (400mg/kg) or saline injection of lipopolysaccharide in 24 hours before and 1 hours after intraperitoneal injection of the pretreatment of 5 Wistar rats, using lipopolysaccharide (200 g) induced uveitis in rats. Rats were observed the clinical manifestations of ocular inflammation after extraction of rat iris ciliary body tissue RNA and M gene expression of RNA and LNC using RNA gene chip technology to detect the expression of difference spectrum. Using gene function enrichment analysis (Gene, Ontology, GO) signal pathway analysis and co expression network the in-depth analysis of the regulatory role between genes differentially screened and selected target genes. Results: after Radix Hedysari pretreatment in rats after LPS stimulation received significant inhibition of ocular inflammation (P0.05). FC (ABS) 1.5 and P A value of 0.05 M RNA standard screened 254 differentially expressed genes and 68 LNC genes RNA.GO analysis showed significant differential gene function mainly in lipopolysaccharide response from bacteria, molecular reaction, chemokine, chemokine receptor binding, cell migration, lymph cell migration regulation, cytokine activity, lymphocyte chemotaxis and migration of white blood cells. While significant differences in gene signal pathway including interaction of cytokine cytokine, Toll like receptor signaling pathway and regulation of inflammatory chemokines and cytokines signal pathway, tumor necrosis factor (TNF) signaling pathway and rheumatoid arthritis. The co expression network analysis 16 m RNA and 7 LNC RNA gene expression in the core position of network. Conclusion: This study found that Radix Hedysari polysaccharide by multiple signal transduction pathways associated with inflammation and immune to play The anti-inflammatory effect of endotoxin induced uveitis. Through a series of screening process has multiple alternative anti-inflammatory target genes, including through upregulation of -1 and downregulation of leucine rich repeat protein gene colony-stimulating factor 1 and LNC by RNA regulation of NF- K B anti-inflammatory effect can greatly and research value.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R773.9

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