【摘要】：背景:新生兒壞死性小腸結(jié)腸炎(NEC)是新生兒重癥監(jiān)護(hù)室中一個(gè)常見(jiàn)的威脅生命的胃腸道急性炎癥性疾病。近年來(lái)新生兒重癥監(jiān)護(hù)和治療水平逐步提升,但NEC在早產(chǎn)兒中依舊具有較高患病率和死亡率。目前針對(duì)NEC的危險(xiǎn)因素研究較多,但NEC的發(fā)病機(jī)制并不明確,且NEC一個(gè)或多個(gè)危險(xiǎn)因素也并不能完全解釋NEC的發(fā)生,進(jìn)展速度和嚴(yán)重程度在不同個(gè)體間的差異,這也限制了對(duì)NEC更有效的預(yù)防和治療方法的應(yīng)用,因此對(duì)于NEC發(fā)病機(jī)制進(jìn)行多方面多角度的研究具有重要的臨床意義。粘膜相關(guān)恒定T(mucosal associated invariant T,MAIT)細(xì)胞是近年來(lái)發(fā)現(xiàn)的一種天然樣T細(xì)胞,主要通過(guò)IL-17等炎癥因子的分泌和細(xì)胞毒性作用在多種疾病的免疫調(diào)節(jié)過(guò)程中發(fā)揮重要作用。MAIT細(xì)胞可分為CD8+,CD4+,和CD8-CD4-(double-negative,DN)三種細(xì)胞亞群,CD8+MAIT細(xì)胞可根據(jù)表達(dá)CD8同源二聚體的不同分為CD8αα和CD8αβ兩個(gè)亞群。已有研究表明,MAIT細(xì)胞參與炎癥性腸病的疾病進(jìn)程,且MAIT細(xì)胞主要炎癥因子IL-17的基因多態(tài)性也被證實(shí)和炎癥性腸病易感性相關(guān)。但MAIT細(xì)胞及其亞群,和IL-17基因多態(tài)性與NEC的關(guān)系尚無(wú)研究報(bào)道。因此,探究MAIT細(xì)胞及其亞群在NEC早產(chǎn)兒體內(nèi)的變化以及其與臨床指標(biāo)的相關(guān)性,為研究NEC免疫學(xué)發(fā)病機(jī)制奠定理論基礎(chǔ);對(duì)IL-17基因多態(tài)性與NEC易感性和疾病嚴(yán)重程度關(guān)系的研究,從基因?qū)用鏋镹EC發(fā)病機(jī)制的研究提供新方向。目的:研究MAIT細(xì)胞及其亞群在NEC早產(chǎn)兒體內(nèi)分布的變化,及其變化和臨床指標(biāo)的相關(guān)性;探究MAIT細(xì)胞主要炎癥因子IL-17基因多態(tài)性和NEC易感性,嚴(yán)重程度的相關(guān)性。為早產(chǎn)兒NEC發(fā)病機(jī)制提供新的研究思路。方法:1.通過(guò)流式細(xì)胞術(shù)檢測(cè)NEC早產(chǎn)兒和對(duì)照組研究對(duì)象外周血單個(gè)核細(xì)胞(Peripheral blood mononuclear cells,PBMC)和腸組織的固有層單個(gè)核細(xì)胞(Lamina propria mononuclear cells,LPMC)中MAIT細(xì)胞及其各亞群的表達(dá)情況。并通過(guò)Bell分級(jí)對(duì)NEC早產(chǎn)兒進(jìn)行分層分析,探究MAIT細(xì)胞及其亞群和NEC疾病嚴(yán)重程度的關(guān)系。2.通過(guò)流式多重蛋白定量(Cytometric bead array,CBA)技術(shù)檢測(cè)NEC早產(chǎn)兒和對(duì)照組研究對(duì)象血清炎癥因子IL-2,IL-4,IL-17A,IL-6,IL-10,TNFα,和IFNγ的水平,并對(duì)NEC早產(chǎn)兒血清炎癥因子和MAIT細(xì)胞及其亞群的相關(guān)性進(jìn)行分析。3.對(duì)治療后痊愈的NEC患兒外周血MAIT細(xì)胞及其亞群進(jìn)行流式細(xì)胞術(shù)檢測(cè),通過(guò)比較治療前后MAIT細(xì)胞及其亞群的變化,進(jìn)一步探究MAIT細(xì)胞及其亞群在NEC疾病中的作用。4.通過(guò)聚合酶鏈?zhǔn)椒磻?yīng)(Polymerse chain reaction,PCR)和Sanger測(cè)序法探究IL-17A(rs2275913)和IL-17F(rs763780)SNP位點(diǎn)多態(tài)性和早產(chǎn)兒NEC易感性及嚴(yán)重程度的相關(guān)性。結(jié)果:1.NEC早產(chǎn)兒外周血MAIT細(xì)胞百分比和對(duì)照組相比顯著降低,腸組織中MAIT細(xì)胞百分比則顯著升高,且二者呈負(fù)相關(guān);NEC早產(chǎn)兒無(wú)論是外周血還是腸組織中,CD8αα+MAIT細(xì)胞亞群占MAIT細(xì)胞的百分比都較對(duì)照組顯著降低,且NEC早產(chǎn)兒外周血CD8αα+MAIT細(xì)胞亞群百分比與疾病的嚴(yán)重程度相關(guān);2.IL-2,IL-4,TNFα,IFNγ,和IL-17A炎癥因子水平在NEC患兒血清中較低,IL-6在NEC患兒血清中的水平較對(duì)照組高。但是和外周血CD8αα+MAIT細(xì)胞亞群和總的MAIT細(xì)胞百分比之間沒(méi)有相關(guān)性;3.經(jīng)隨訪,NEC患兒治療痊愈后,外周血MAIT細(xì)胞百分比較治療前顯著上升,且上升至對(duì)照組水平;NEC患兒治療后,外周血CD8αα+MAIT細(xì)胞亞群百分比較治療前無(wú)顯著差異,仍然低于對(duì)照組水平;4.IL-17A(rs2275913)SNP位點(diǎn)無(wú)論是基因型還是等位基因頻率在NEC和對(duì)照組之間沒(méi)有顯著差異;IL-17F(rs763780)SNP位點(diǎn)TC+CC基因型和C等位基因的分布頻率在NEC組比對(duì)照組明顯升高;IL-17F(rs763780)SNP位點(diǎn)TC+CC基因型在Bell分級(jí)Ⅲ級(jí)的NEC患兒中分布頻率明顯高于Bell分級(jí)Ⅱ級(jí)的NEC患兒;IL-17F(rs763780)SNP位點(diǎn)TC+CC基因型在發(fā)生NEC相關(guān)性氣腹的患兒中分布頻率明顯高于未發(fā)生NEC相關(guān)性氣腹的患兒。結(jié)論:本研究結(jié)果證明,MAIT細(xì)胞在NEC疾病過(guò)程中,可能從外周血向腸道炎癥組織中遷移,發(fā)揮免疫調(diào)節(jié)作用;早產(chǎn)兒體內(nèi)CD8αα+MAIT細(xì)胞亞群百分比降低可能導(dǎo)致MAIT細(xì)胞抵御微生物的能力下降,參與NEC的發(fā)生和疾病進(jìn)程。提示了CD8αα+MAIT細(xì)胞是潛在的評(píng)估NEC疾病嚴(yán)重程度的生物學(xué)標(biāo)志;IL-17A(rs2275913)SNP位點(diǎn)的基因多態(tài)性可能和NEC的易感性無(wú)關(guān);IL-17F(rs763780)SNP位點(diǎn)的突變可能和NEC的易感性及疾病嚴(yán)重程度相關(guān)。本研究為進(jìn)一步探究NEC的免疫學(xué)和基因?qū)W發(fā)病機(jī)制奠定了基礎(chǔ),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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R722.1

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