【作者】 张敏；

【导师】 王鹏；

【作者基本信息】 郑州大学 ， 免疫学， 2024， 硕士

【摘要】 背景和目的炎症性肠病(Inflammatory bowel diseases,IBD)是主要发生于回肠、直肠和结肠部位的一种特发性慢性肠道炎症性疾病。IBD包括克罗恩病(Crohn’s disease,CD)和溃疡性结肠炎(Ulcerative colitis,UC),CD和UC在某些症状、发病部位和组织病理学特征上有所不同,但它们有共同的胃肠道症状表现。IBD发病机制比较复杂,现有的研究表明,免疫、微生物、环境、营养和遗传因素都与IBD的发病机制和严重程度有关。研究表明,IBD患者肠上皮细胞与免疫细胞失衡,但IBD的发病机制中肠上皮细胞和免疫细胞的关系仍未完全明了。转录因子ELF-1属于Ets家族。Ets家族由20多个转录因子组成,这些转录因子参与调节不同细胞过程的各种基因的表达,包括细胞增殖、分化、凋亡和细胞与细胞、细胞与基质相互作用。一项分析散发性结肠癌附近组织的研究发现,相对于非癌症组织,ELF-1表达降低,这表明ELF-1在结肠癌发生或早期进展中的可能功能。鉴于Ets因子在上皮、基质、血管和免疫、炎症生物学中的重要功能,转录因子ELF-1很可能在肠道非肿瘤性疾病(包括炎症性肠病)中具有重要作用,但目前这些作用与具体机制仍有待确定。为探究ELF-1是否影响炎症性肠病,本研究利用Elf-1-/-鼠构建溃疡性结肠炎模型,分析ELF-1在IBD发病中的作用。方法1.利用PCR和琼脂糖凝胶电泳鉴定小鼠基因型。2.利用DSS构建溃疡性结肠炎模型,通过体重检测、DAI评分、生存率分析、结肠长度、结肠组织H&E染色评分、脾脏大小、qRT-PCR探究转录因子ELF-1缺失是否影响溃疡性结肠炎。3.利用单细胞测序技术,对DSS处理后的WT鼠和Elf1-/-鼠的结肠组织进行分析,通过UMAP降维分析、KEGG通路富集分析、GO基因功能富集分析、GSVA基因集变异分析、GSEA基因集富集分析来探究转录因子ELF-1缺失在细胞层面如何影响溃疡性结肠炎。4.利用完全骨髓嵌合体小鼠构建溃疡性结肠炎模型,通过体重检测、DAI评分、结肠长度、结肠组织H&E染色评分、脾脏大小探究转录因子ELF-1缺失主要是通过免疫细胞还是非造血细胞影响溃疡性结肠炎。5.利用单细胞测序技术,对DSS处理的WT鼠和Elf-/-鼠的结肠上皮细胞进行分析,通过UMAP降维分析、KEGG通路富集分析、GO基因功能富集分析,以及qRT-PCR检测来探究转录因子ELF-1缺失在肠上皮细胞层面如何影响溃疡性结肠炎。6.利用ChIP-Seq测序和qRT-PCR检测,对DSS处理和未处理的WT鼠结肠组织进行分析,探究转录因子ELF-1可能结合的DNA序列。结果1.通过PCR和琼脂糖凝胶电泳,根据电泳条带的差异分别鉴定出WT鼠和Elf-1-/-鼠。2.溃疡性结肠炎模型结果显示,与WT鼠相比,Elf-1-/-鼠的肠炎症状更严重。3.单细胞测序分析结果显示,与WT鼠相比,Elf-1-/-鼠结肠的中性粒细胞显著增多,且Elf-1-/-小鼠炎症性肠组织中特异表达两个中性粒细胞亚群,即转录因子ELF-1缺失导致的肠炎加重以中性粒细胞浸润为主。单细胞测序分析结果显示,与WT鼠相比,Elf-1-/-鼠结肠的IL-17信号通路高表达。4.完全骨髓嵌合体小鼠构建的溃疡性结肠炎模型结果显示,与WT鼠为受体的两组小鼠相比,以Elf-1-/-鼠为受体的两组小鼠肠炎症状更严重,说明转录因子ELF-1缺失主要通过非造血细胞即肠上皮细胞影响溃疡性结肠炎。5.单细胞测序分析结果显示,与WT鼠相比,Elf-1-/-鼠肠上皮中的杯状细胞显著减少,Elf-1-/-鼠肠上皮细胞中IL-17信号通路显著上调。qRT-PCR结果显示,Elf-1-/-鼠肠上皮细胞中关于趋化中性粒细胞等免疫细胞的趋化因子显著高表达。6.ChIP-Seq测序分析和qRT-PCR结果显示,转录因子ELF-1直接调控Rack1基因。结论转录因子ELF-1缺失加重DSS诱导的溃疡性结肠炎;转录因子ELF-1缺失导致的肠炎加重以中性粒细胞浸润为主;转录因子ELF-1缺失通过影响肠上皮细胞对免疫细胞的募集进而影响溃疡性结肠炎;在溃疡性结肠炎中,转录因子ELF-1直接调控Rack1基因表达。更多还原

【Abstract】 Background and ObjectiveInflammatory bowel disease(IBD)is an idiopathic chronic inflammatory disease of the intestinal tract that occurs mainly in the ileum,rectum and colon.IBD includes Crohn’s disease(CD)and ulcerative colitis(UC).IBD includes Crohn’s disease(CD)and ulcerative colitis(UC).CD and UC differ in some symptoms,sites of onset,and histopathologic features,but they share common gastrointestinal symptoms.The pathogenesis of IBD is complex,and available research suggests that immunologic,microbiologic,environmental,nutritional,and genetic factors are all involved in the pathogenesis and severity of IBD.Studies have shown that IBD patients have an imbalance between intestinal epithelial cells and immune cells,but the relationship between intestinal epithelial cells and immune cells in the pathogenesis of IBD is still not fully understood.The transcription factor ELF-1 belongs to the Ets family.The Ets family consists of more than 20 transcription factors that are involved in regulating the expression of a variety of genes involved in different cellular processes,including cell proliferation,differentiation,apoptosis,cell-cell and cell-matrix interactions.A study analyzing tissues near sporadic colon cancers found reduced ELF-1 expression relative to non-cancerous tissues,suggesting a possible function for ELF-1 in colon carcinogenesis or early progression.Given the important functions of Ets factors in epithelial,stromal,vascular,immune and inflammatory biology,it is likely that the transcription factor ELF-1 has important roles in intestinal non-tumorigenic disorders,including inflammatory bowel disease,but at present these roles along with the specific mechanisms remain to be determined.To investigate whether ELF-1 affects inflammatory bowel disease,this study was conducted to construct an ulcerative colitis model using Elf-1-/-mice to analyze the role of ELF-1 in the pathogenesis of IBD.Methods1.PCR and agarose gel electrophoresis were used to identify the mouse genotype.2.An ulcerative colitis model was constructed using the DSS,and whether the absence of the transcription factor ELF-1 affects UC was explored by body weight test,DAI score,survival analysis,colon length,H&E staining score of colon tissue,spleen size,and qRT-PCR.3.Colonic tissues from DSS-treated WT mice and Elf-1-/-mice were analyzed using single-cell sequencing to explore how transcription factor ELF-1 deletion affects UC at the cellular level by UMAP downscaling analysis,KEGG pathway enrichment analysis,GO gene function enrichment analysis,GSVA gene set variant analysis,and GSEA gene set enrichment analysis.4.A model of ulcerative colitis was constructed using complete bone marrow chimeric mice,and the body weight test,DAI score,colon length,H&E staining score of colon tissues,and spleen size were used to investigate the absence of the transcription factor ELF-1 affects UC mainly through immune cells or non-hematopoietic cells.5.Colonic epithelial cells from DSS-treated WT mice and Elf-1-/-mice were analyzed by single-cell sequencing to investigate how transcription factor ELF-1 deletion affects UC at the level of the intestinal epithelial cells by UMAP downscaling analysis,KEGG pathway enrichment analysis,GO gene function enrichment analysis,as well as qRT-PCR assay.6.DSS-treated and untreated WT mouse colon tissues were analyzed using ChIP-Seq sequencing and qRT-PCR assays to explore the DNA sequences to which the transcription factor ELF-1 may bind.Results1.WT mice and Elf-1-/-mice were identified by PCR and agarose gel electrophoresis according to the difference in electrophoresis bands.2.The results of the ulcerative colitis model showed that the symptoms of enteritis were more severe in Elf-1-/-mice compared to WT mice.3.Single-cell sequencing analysis showed a significant increase in neutrophils in the colon of Elf-1-/-mice compared with WT mice,and two neutrophil subpopulations were specifically expressed in the inflammatory intestinal tissues of mice,which means that exacerbation of intestinal inflammation caused by the absence of the transcription factor ELF-1 is predom inantly characterized by neutrophil infiltration.Single-cell sequencing analysis showed high expression of the IL-17 signaling pathway in the colon of Elf-1-/-mice compared with WT mice.4.The results of the ulcerative colitis model constructed from complete bone marrow chimeric mice showed that the symptoms of enteritis were more severe in the two groups of mice with Elf-1-/-mice as recipients compared to the two groups of mice with WT mice as recipients,suggesting that the absence of the transcription factor ELF-1 affects the UC mainly through the non-hematopoietic cells,namely the intestinal epithelial cells.5.Single-cell sequencing analysis showed a significant reduction of cup cells in the intestinal epithelium of Elf-1-/-mice and a significant up-regulation of the IL-17 signaling pathway in the intestinal epithelial cells of mice compared with that of WT mice.qRT-PCR showed a significant increase in the expression of chemokines in the intestinal epithelium of Elf-1-/-mice regarding chemotaxis of immune cells such as neutrophils.6.ChIP-Seq sequencing analysis and qRT-PCR results showed that the transcription factor ELF-1 directly regulates the Rack1 gene.ConclusionTranscription factor ELF-1 deficiency exacerbates DSS-induced ulcerative colitis.Exacerbation of enteritis due to transcription factor ELF-1 deletion is dominated by neutrophilic infiltration.Transcription factor ELF-1 deficiency affects ulcerative colitis by influencing the recruitment of intestinal epithelial cells to immune cells.In ulcerative colitis,the transcription factor ELF-1 directly modulates Rack 1 gene expression.更多还原