重组法国梧桐花粉过敏原（rPla a2）对小鼠ORMDL3基因转录调控机制初步研究

发布时间：2018-01-04 15:39

本文关键词：重组法国梧桐花粉过敏原（rPla a2）对小鼠ORMDL3基因转录调控机制初步研究　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:分析转录因子GATA3对小鼠ORMDL3基因转录的影响机制,探索重组法国梧桐花粉过敏原rPla a2对小鼠ORMDL3基因转录表达的影响及相关机制,为植物吸入性过敏原所致过敏性哮喘的防治寻找新的靶点提供思路。方法:(1)染色质免疫沉淀、RNA干扰、转录因子过表达、双荧光素酶活性分析等实验,鉴定GATA3与小鼠ORMD3启动子结合和启动子活性的调控作用;(2)通过实时定量PCR和Western Blot实验,观察GATA3对小鼠ORMDL3表达水平的影响;(3)利用法国梧桐花粉重组过敏原rPla a2探索对小鼠ORMDL3启动子活性、mRNA及蛋白表达的影响,用ChIP-qPCR观察rPla a2刺激后GATA3与小鼠ORMDL3启动子区结合的影响。结果:(1)ChIP实验表明GATA3与小鼠ORMDL3核心启动子-74bp至+141 bp结合,但不能与小鼠ORMD3基因转录起始位点上游-921至-835 bp序列结合。(2)RNA干扰、转录因子过表达、实时荧光定量PCR、Western Blot实验证明GATA3能正向调控ORMDL3的表达。(3)rPla a2上调小鼠ORMDL3启动子活性,突变GATA3和STAT6结合序列后该上调作用受抑制,rPla a2促进ORMDL3和GATA3表达,干扰GATA3表达可抑制rPla a2对ORMDL3的上调作用。ChIP-qPCR实验发现rPlaa2增强GATA3与ORMDL3启动子区的结合。结论:(1)GATA3通过直接结合小鼠ORMDL3核心启动子区域正向调控ORMDL3基因表达,为以GATA3为靶点的哮喘治疗提供理论依据。(2)rPla a2通过上调内源性GATA3表达和增强GATA3与核心启动子区域的结合上调ORMDL3表达,为植物过敏原诱发的过敏性哮喘治疗提供新的思路。
[Abstract]:Objective: to analyze the influence mechanism of transcription factor GATA3 on mouse ORMDL3 gene transcription, to explore the effect of recombinant Platanus pollen allergen rPla A2 expression of mouse ORMDL3 gene transcription and related mechanisms to control the plant inhaled allergen induced allergic asthma to find new targets to provide ideas. Methods: (1) chromatinimmune precipitation, RNA interference and overexpression of the transcription factor, dual luciferase activity analysis and experimental identification, GATA3 and mouse ORMD3 promoter binding and promoter activity regulation; (2) by real-time quantitative PCR and Western Blot experiment, the level of the effect of GATA3 on the expression of ORMDL3 in mice; (3) using recombinant Platanus pollen allergen rPla A2 to explore the promoter activity of ORMDL3 in mice, the expressions of mRNA and protein, GATA3 and mouse ORMDL3 promoter region with rPla after A2 stimulation was observed by ChIP-qPCR. Results: (1) ChIP. The results show that GATA3 and mouse ORMDL3 core promoter -74bp to +141 with BP, but not with the mouse ORMD3 gene upstream of the transcription initiation site of -921 to -835. (2) BP sequence combined with RNA interference and overexpression of the transcription factor Western, real-time PCR, Blot experiments show that GATA3 can positively regulate the expression of ORMDL3 (3) rPla. Upregulation of A2 mouse ORMDL3 promoter activity, GATA3 mutations and STAT6 binding sequence after the upregulation of rPla was inhibited, A2 promotes the expression of ORMDL3 and GATA3, GATA3 interference can inhibit the expression of A2 on rPla ORMDL3.ChIP-qPCR induced test showed that rPlaa2 enhanced GATA3 and ORMDL3 binds promoter region. Conclusion: (1) GATA3 by directly binding mouse ORMDL3 core promoter region of the positive regulation of ORMDL3 gene expression, and provide a theoretical basis for the treatment of asthma by targeting GATA3. (2) rPla A2 by upregulating the expression of endogenous GATA3 and enhanced GATA3 and core promoter The combination of subregions up-regulated the expression of ORMDL3 provides a new way of thinking for the treatment of allergic asthma induced by plant allergen.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.1

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