白细胞介素-23对哮喘小鼠记忆T细胞增殖功能的影响

发布时间：2018-03-09 05:01

  本文选题：记忆T细胞　切入点：支气管哮喘　出处：《吉林大学》2013年博士论文　论文类型：学位论文

【摘要】：研究背景： 支气管哮喘目前被认为是一种Th2优势的疾病，记忆T细胞处于其发病机制的上游环节，它能够在哮喘机体长期存在，并维持Th2细胞的形态和功能，当机体再次接触同种过敏原时，记忆T细胞能够迅速增殖、活化为Th2细胞，分泌Th2细胞因子，引发气道炎症。因此，了解记忆T细胞在哮喘机体中的作用机制和规律，寻找可以调控记忆T细胞增殖与活化的物质，将有利于我们以记忆T细胞为靶点，探寻有效防治哮喘的新措施。IL-23能够促进正常机体记忆T细胞的增殖与活化，同时IL-23又能够促进哮喘小鼠Th2气道炎症，那么，IL-23是不是通过促进哮喘小鼠记忆T细胞增殖、从而促进哮喘Th2气道炎症的呢？本研究试图解答该问题，为治疗哮喘提供新思路。 研究目的： 1.了解哮喘小鼠肺脏、脾脏记忆T细胞比例在哮喘不同时期的变化规律，确定观察记忆T细胞增殖功能的最佳时间点，并为深入研究记忆T细胞在哮喘中的功能奠定基础； 2.明确抑制IL-23是否能够减少哮喘小鼠肺脏记忆T细胞的数量，试图揭示IL-23参与哮喘Th2气道炎症的作用机理。 研究方法： 1.应用OVA致敏、激发小鼠，构建哮喘小鼠动物模型，于激发前1天始至激发后30天，按规定时间点处死小鼠，分离脾脏、肺脏，制成单个核细胞悬液，应用流式抗体、流式细胞仪检测小鼠脾脏、肺脏记忆T细胞比例，确定观察记忆T细胞增殖功能的最佳时机； 2.根据小鼠IL-23p19基因序列设计干涉靶序列，慢病毒包装，体外感染小鼠细胞系，通过实时-PCR法检测细胞内IL-23p19mRNA表达水平，筛选出能够沉默小鼠IL-23p19基因的有效序列； 3.将小鼠分为正常组、哮喘组、空载体组、哮喘IL-23p19基因沉默组，后2组模型致敏、激发方法与哮喘组相同，在第1次激发前1天经鼻滴入携带有空载体或有效干涉靶序列的慢病毒液。于前期实验确定的最佳时机处死小鼠，进行相关指标的检测，包括实时-PCR、免疫组化法检测IL-23p19基因体内沉默效果，流式细胞仪分析肺组织中记忆T细胞比例，实时-PCR法检测肺组织转录因子T-bet、GATA-3、RORC表达水平，HE染色、刚果红染色观察肺组织病理学改变以及PCR检测Th2细胞因子表达水平等哮喘炎症指标。 结果： 1.记忆T细胞检测结果 ⑴哮喘小鼠脾脏、肺脏CD4~+、CD4~-记忆T细胞比值均比正常小鼠明显增高（p0.0001）； ⑵CD4~+记忆T细胞变化规律：哮喘小鼠脾脏、肺脏CD4~+记忆T细胞在应用OVA激发后持续上升，激发停止后仍继续上升，肺脏CD4~+记忆T细胞在末次激发后2~9天达到高峰，峰值超过50%，后逐渐下降；脾脏CD4~+记忆T细胞在末次激发后7天达到高峰，峰值超过40%，后逐渐下降。至末次激发后30天，脾脏、肺脏记忆T细胞均回降至激发前水平； ⑶CD4~-记忆T细胞变化规律：哮喘小鼠肺脏CD4~-记忆T细胞在应用OVA激发后持续上升，末次激发后2天达到峰值，，然后迅速下降，至末次激发后7天时下降至激发前水平；脾脏CD4~-记忆T细胞于激发7天后始上升，维持在30%左右，于末次激发后14天下降至激发前水平； ⑷在本模型的构建条件下，观察小鼠肺脏记忆T细胞增殖功能的最佳时机为末次激发后2天； 2.沉默哮喘小鼠肺脏IL-23p19基因表达对小鼠的影响 ⑴记忆T细胞：哮喘基因沉默组小鼠肺脏CD4~+记忆T细胞占CD4~+T细胞比值较哮喘组降低（p 0.0001），CD4~-记忆T细胞占CD4~-T细胞比值无变化（p0.05）； ⑵Th2气道炎症：基因沉默组小鼠肺组织Th2细胞因子IL-4、IL-13mRNA表达较哮喘组降低（p 0.001，p 0.0001），肺组织炎症病变程度、嗜酸性粒细胞气道浸润均较哮喘组减轻（p 0.05，p 0.0001）； ⑶转录因子：哮喘基因沉默组小鼠肺组织Th2转录因子GATA-3、Th1转录因子T-bet mRNA表达较哮喘组减低（p 0.0001）；Th17转录因子Rocr mRNA表达无变化（p0.05）。 结论： 1.哮喘小鼠体内的记忆T细胞在哮喘不同时期呈现出一定的规律：⑴无论CD4~+还是CD4~-记忆T细胞，肺脏总是先于脾脏升高，且在哮喘急性期，肺脏记忆T细胞的比例显著高于脾脏；⑵在哮喘效应器官肺脏，CD4~+与CD4~-记忆T细胞均参与了哮喘的发病，其中CD4~+记忆T细胞是引起哮喘气道炎症持续的主要细胞，CD4~-记忆T细胞可能在哮喘急性发作方面发挥了作用； 2.抑制IL-23表达能够减少哮喘小鼠肺脏CD4~+记忆T细胞的增殖，抑制Th2转录因子GATA-3的表达，同时能够减轻哮喘气道炎症，提示IL-23有可能通过调控CD4~+Tm的增殖来参与哮喘Th2气道炎症，IL-23主要对CD4~+Tm发挥作用。
[Abstract]:Research background:
Bronchial asthma is considered to be a Th2 dominant disease, upstream of memory T cells in the pathogenesis of asthma, it can exist for a long time in the body, and maintain the morphology and function of Th2 cells, when the body is again exposed to the same allergens, memory T cells can rapidly proliferation, activation of Th2 cells and secretion Th2 cytokines, causing airway inflammation. Therefore, understanding the mechanism and regularity of asthma in the body memory T cells, can find the proliferation of memory T cells and activated substances, will help us in memory T cells as the target, to explore new effective measures for the prevention and treatment of asthma.IL-23 can promote the activation and normal body memory T cell proliferation, while IL-23 can promote the airway inflammation of asthmatic mice, then Th2, IL-23 is not by promoting the proliferation of memory T cells in asthmatic mice, so as to promote the airway inflammation of asthma Th2 this study? Trying to answer this problem provides a new way of thinking for the treatment of asthma.
The purpose of the study is:
1., to understand the change rule of the proportion of T cells in the lungs and spleen of asthmatic mice in different periods of asthma, determine the best time to observe the proliferation function of T cells, and lay a foundation for further studying the function of T cells in asthma.
2. whether the inhibition of IL-23 can reduce the number of T cells in lung memory in asthmatic mice and attempts to reveal the role of IL-23 in the airway inflammation of asthmatic Th2.
Research methods:
1. application of OVA sensitized asthmatic mice challenged mice, constructing animal model, to stimulate the 1 day before and 30 days after excitation, the lungs at the specified time point from the spleen of mice were sacrificed, and made into mononuclear cell suspension, using flow cytometry to detect antibody of mouse spleen cells by flow cytometry, the proportion of lung T memory the best time to observe the memory cells, determine the proliferative function of T cells;
2., according to the sequence of mouse IL-23p19 gene, we designed the interference target sequence, lentivirus package, infected the mouse cell line in vitro, detected the expression level of IL-23p19mRNA in the cell by real-time -PCR method, and screened out the effective sequence that could silence mouse IL-23p19 gene.
3. mice were divided into normal group, asthma group, vector group, asthma IL-23p19 gene silencing group, the 2 groups after model sensitized excitation with the same method of asthma group in first shot 1 days before nasal instillation of lentivirus carrying liquid air carrier or effective interference target sequence. The best time to experiment determine the mice were sacrificed, detection of relevant indicators, including real time -PCR, immunohistochemistry was used to detect IL-23p19 gene silencing effect in vivo, analysis of lung tissue in memory T cells by flow cytometry, detection of transcription factor T-bet, lung tissue by real time -PCR and GATA-3, the expression level of RORC, HE stain, Congo red staining of lung the histopathological changes of Th2 and cytokine PCR expression level detection of asthma inflammation index.
Result锛

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