新生儿坏死性小肠结肠炎患者体内粘膜相关恒定T细胞亚群的检测及其临床意义

发布时间：2018-08-23 19:55

【摘要】：背景:新生儿坏死性小肠结肠炎(NEC)是新生儿重症监护室中一个常见的威胁生命的胃肠道急性炎症性疾病。近年来新生儿重症监护和治疗水平逐步提升,但NEC在早产儿中依旧具有较高患病率和死亡率。目前针对NEC的危险因素研究较多,但NEC的发病机制并不明确,且NEC一个或多个危险因素也并不能完全解释NEC的发生,进展速度和严重程度在不同个体间的差异,这也限制了对NEC更有效的预防和治疗方法的应用,因此对于NEC发病机制进行多方面多角度的研究具有重要的临床意义。粘膜相关恒定T(mucosal associated invariant T,MAIT)细胞是近年来发现的一种天然样T细胞,主要通过IL-17等炎症因子的分泌和细胞毒性作用在多种疾病的免疫调节过程中发挥重要作用。MAIT细胞可分为CD8+,CD4+,和CD8-CD4-(double-negative,DN)三种细胞亚群,CD8+MAIT细胞可根据表达CD8同源二聚体的不同分为CD8αα和CD8αβ两个亚群。已有研究表明,MAIT细胞参与炎症性肠病的疾病进程,且MAIT细胞主要炎症因子IL-17的基因多态性也被证实和炎症性肠病易感性相关。但MAIT细胞及其亚群,和IL-17基因多态性与NEC的关系尚无研究报道。因此,探究MAIT细胞及其亚群在NEC早产儿体内的变化以及其与临床指标的相关性,为研究NEC免疫学发病机制奠定理论基础;对IL-17基因多态性与NEC易感性和疾病严重程度关系的研究,从基因层面为NEC发病机制的研究提供新方向。目的:研究MAIT细胞及其亚群在NEC早产儿体内分布的变化,及其变化和临床指标的相关性;探究MAIT细胞主要炎症因子IL-17基因多态性和NEC易感性,严重程度的相关性。为早产儿NEC发病机制提供新的研究思路。方法:1.通过流式细胞术检测NEC早产儿和对照组研究对象外周血单个核细胞(Peripheral blood mononuclear cells,PBMC)和肠组织的固有层单个核细胞(Lamina propria mononuclear cells,LPMC)中MAIT细胞及其各亚群的表达情况。并通过Bell分级对NEC早产儿进行分层分析,探究MAIT细胞及其亚群和NEC疾病严重程度的关系。2.通过流式多重蛋白定量(Cytometric bead array,CBA)技术检测NEC早产儿和对照组研究对象血清炎症因子IL-2,IL-4,IL-17A,IL-6,IL-10,TNFα,和IFNγ的水平,并对NEC早产儿血清炎症因子和MAIT细胞及其亚群的相关性进行分析。3.对治疗后痊愈的NEC患儿外周血MAIT细胞及其亚群进行流式细胞术检测,通过比较治疗前后MAIT细胞及其亚群的变化,进一步探究MAIT细胞及其亚群在NEC疾病中的作用。4.通过聚合酶链式反应(Polymerse chain reaction,PCR)和Sanger测序法探究IL-17A(rs2275913)和IL-17F(rs763780)SNP位点多态性和早产儿NEC易感性及严重程度的相关性。结果:1.NEC早产儿外周血MAIT细胞百分比和对照组相比显著降低,肠组织中MAIT细胞百分比则显著升高,且二者呈负相关;NEC早产儿无论是外周血还是肠组织中,CD8αα+MAIT细胞亚群占MAIT细胞的百分比都较对照组显著降低,且NEC早产儿外周血CD8αα+MAIT细胞亚群百分比与疾病的严重程度相关;2.IL-2,IL-4,TNFα,IFNγ,和IL-17A炎症因子水平在NEC患儿血清中较低,IL-6在NEC患儿血清中的水平较对照组高。但是和外周血CD8αα+MAIT细胞亚群和总的MAIT细胞百分比之间没有相关性;3.经随访,NEC患儿治疗痊愈后,外周血MAIT细胞百分比较治疗前显著上升,且上升至对照组水平;NEC患儿治疗后,外周血CD8αα+MAIT细胞亚群百分比较治疗前无显著差异,仍然低于对照组水平;4.IL-17A(rs2275913)SNP位点无论是基因型还是等位基因频率在NEC和对照组之间没有显著差异;IL-17F(rs763780)SNP位点TC+CC基因型和C等位基因的分布频率在NEC组比对照组明显升高;IL-17F(rs763780)SNP位点TC+CC基因型在Bell分级Ⅲ级的NEC患儿中分布频率明显高于Bell分级Ⅱ级的NEC患儿;IL-17F(rs763780)SNP位点TC+CC基因型在发生NEC相关性气腹的患儿中分布频率明显高于未发生NEC相关性气腹的患儿。结论:本研究结果证明,MAIT细胞在NEC疾病过程中,可能从外周血向肠道炎症组织中迁移,发挥免疫调节作用;早产儿体内CD8αα+MAIT细胞亚群百分比降低可能导致MAIT细胞抵御微生物的能力下降,参与NEC的发生和疾病进程。提示了CD8αα+MAIT细胞是潜在的评估NEC疾病严重程度的生物学标志;IL-17A(rs2275913)SNP位点的基因多态性可能和NEC的易感性无关;IL-17F(rs763780)SNP位点的突变可能和NEC的易感性及疾病严重程度相关。本研究为进一步探究NEC的免疫学和基因学发病机制奠定了基础,并提供了新的研究方向。
[Abstract]:BACKGROUND: Neonatal necrotizing enterocolitis (NEC) is a common life-threatening gastrointestinal acute inflammatory disease in neonatal intensive care unit (NICU). In recent years, the level of neonatal intensive care and treatment has gradually improved, but NEC still has a high morbidity and mortality in premature infants. However, the pathogenesis of NEC is not clear, and one or more risk factors of NEC can not fully explain the occurrence of NEC, progress rate and severity of the difference between individuals, which also limits the use of more effective prevention and treatment of NEC, so for the pathogenesis of NEC has important multi-angle research. Mucosal associated invariant T (MAIT) cells are a kind of natural T cells discovered in recent years, which play an important role in the immune regulation of many diseases mainly through the secretion of inflammatory factors such as IL-17 and cytotoxicity. MAIT cells can be divided into CD8+, CD4+, and CD8-CD4-(double-negative, DN) CD8+MAIT cells can be divided into two subgroups according to the expression of CD8 homologous dimers. Previous studies have shown that MAIT cells are involved in the pathogenesis of inflammatory bowel disease, and the gene polymorphism of IL-17, the main inflammatory factor of MAIT cells, has been confirmed to be associated with the susceptibility to inflammatory bowel disease. Therefore, to explore the changes of MAIT cells and their subsets in premature infants with NEC and their correlation with clinical indicators, and to lay a theoretical foundation for the study of the pathogenesis of NEC immunology; to study the relationship between IL-17 gene polymorphism and susceptibility to NEC and disease severity, from the gene perspective. Objective: To study the distribution of MAIT cells and their subsets in premature neonates with NEC, and to explore the correlation between the IL-17 gene polymorphism of MAIT cells and the susceptibility and severity of NEC. Methods: 1. The expression of MAIT cells and its subsets in peripheral blood mononuclear cells (PBMC) and intestinal lamina propria mononuclear cells (LPMC) of premature neonates with NEC and control subjects were detected by flow cytometry, and early NEC was detected by Bell classification. Levels of serum inflammatory cytokines IL-2, IL-4, IL-17A, IL-6, IL-10, TNFa and IFN-gamma in premature neonates with NEC were measured by Cytometric Bead array (CBA) technique. The correlation between MAIT cells and their subsets was analyzed. 3. The peripheral blood MAIT cells and their subsets were detected by flow cytometry. The changes of MAIT cells and their subsets were compared before and after treatment to further explore the role of MAIT cells and their subsets in NEC diseases. 4. Polymerase chain reaction (PCR) Sequence chain reaction, PCR and Sarger sequencing were used to explore the correlation between IL-17A (rs2275913) and IL-17F (rs763780) SNP polymorphisms and the susceptibility and severity of NEC in premature infants. The percentage of CD8 alpha + MAIT cell subsets in peripheral blood and intestinal tissue of NEC preterm infants was significantly lower than that of the control group, and the percentage of CD8 alpha + MAIT cell subsets in peripheral blood of NEC preterm infants was related to the severity of the disease. 2. The levels of IL-2, IL-4, TNF-alpha, IFN-gamma and IL-17A inflammatory factors were lower in serum of NEC infants. The level of - 6 in serum of NEC patients was higher than that of control group, but there was no correlation between CD8alpha + MAIT cell subsets and the percentage of total MAIT cells in peripheral blood. There was no significant difference in the percentage of cell subsets between the two groups. 4. There was no significant difference in the genotype and allele frequencies of IL-17A (rs2275913) SNP loci between the NEC and the control group. The distribution frequencies of TC+CC genotype and C allele of IL-17F (rs763780) SNP loci in the NEC group were significantly higher than those in the control group. Distribution frequency of TC+CC genotype in F(rs763780) SNP locus was significantly higher in NEC patients with Bell grade III than in NEC patients with Bell grade II. Distribution frequency of TC+CC genotype in IL-17F(rs763780) SNP locus was significantly higher in children with NEC-related pneumoperitoneum than in children without NEC-related pneumoperitoneum. In the course of NEC disease, it may migrate from peripheral blood to intestinal inflammation tissues and play an immunoregulatory role. The decrease of the percentage of CD8alpha+MAIT cell subsets in premature infants may lead to the decrease of the ability of MAIT cells to resist microorganisms and participate in the occurrence and progression of NEC. The genetic polymorphism of IL-17A (rs2275913) SNP locus may not be related to the susceptibility of NEC, and the mutation of IL-17F (rs763780) SNP locus may be related to the susceptibility and severity of NEC.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R722.1

【相似文献】