本文選題：記憶T細胞　切入點：支氣管哮喘　出處：《吉林大學》2013年博士論文　論文類型：學位論文

【摘要】：研究背景： 支氣管哮喘目前被認為是一種Th2優(yōu)勢的疾病，記憶T細胞處于其發(fā)病機制的上游環(huán)節(jié)，它能夠在哮喘機體長期存在，并維持Th2細胞的形態(tài)和功能，當機體再次接觸同種過敏原時，記憶T細胞能夠迅速增殖、活化為Th2細胞，分泌Th2細胞因子，引發(fā)氣道炎癥。因此，了解記憶T細胞在哮喘機體中的作用機制和規(guī)律，尋找可以調(diào)控記憶T細胞增殖與活化的物質(zhì)，將有利于我們以記憶T細胞為靶點，探尋有效防治哮喘的新措施。IL-23能夠促進正常機體記憶T細胞的增殖與活化，同時IL-23又能夠促進哮喘小鼠Th2氣道炎癥，那么，IL-23是不是通過促進哮喘小鼠記憶T細胞增殖、從而促進哮喘Th2氣道炎癥的呢？本研究試圖解答該問題，為治療哮喘提供新思路。 研究目的： 1.了解哮喘小鼠肺臟、脾臟記憶T細胞比例在哮喘不同時期的變化規(guī)律，確定觀察記憶T細胞增殖功能的最佳時間點，并為深入研究記憶T細胞在哮喘中的功能奠定基礎(chǔ)； 2.明確抑制IL-23是否能夠減少哮喘小鼠肺臟記憶T細胞的數(shù)量，試圖揭示IL-23參與哮喘Th2氣道炎癥的作用機理。 研究方法： 1.應(yīng)用OVA致敏、激發(fā)小鼠，構(gòu)建哮喘小鼠動物模型，于激發(fā)前1天始至激發(fā)后30天，按規(guī)定時間點處死小鼠，分離脾臟、肺臟，制成單個核細胞懸液，應(yīng)用流式抗體、流式細胞儀檢測小鼠脾臟、肺臟記憶T細胞比例，確定觀察記憶T細胞增殖功能的最佳時機； 2.根據(jù)小鼠IL-23p19基因序列設(shè)計干涉靶序列，慢病毒包裝，體外感染小鼠細胞系，通過實時-PCR法檢測細胞內(nèi)IL-23p19mRNA表達水平，篩選出能夠沉默小鼠IL-23p19基因的有效序列； 3.將小鼠分為正常組、哮喘組、空載體組、哮喘IL-23p19基因沉默組，后2組模型致敏、激發(fā)方法與哮喘組相同，在第1次激發(fā)前1天經(jīng)鼻滴入攜帶有空載體或有效干涉靶序列的慢病毒液。于前期實驗確定的最佳時機處死小鼠，進行相關(guān)指標的檢測，包括實時-PCR、免疫組化法檢測IL-23p19基因體內(nèi)沉默效果，流式細胞儀分析肺組織中記憶T細胞比例，實時-PCR法檢測肺組織轉(zhuǎn)錄因子T-bet、GATA-3、RORC表達水平，HE染色、剛果紅染色觀察肺組織病理學改變以及PCR檢測Th2細胞因子表達水平等哮喘炎癥指標。 結(jié)果： 1.記憶T細胞檢測結(jié)果 ⑴哮喘小鼠脾臟、肺臟CD4~+、CD4~-記憶T細胞比值均比正常小鼠明顯增高（p0.0001）； ⑵CD4~+記憶T細胞變化規(guī)律：哮喘小鼠脾臟、肺臟CD4~+記憶T細胞在應(yīng)用OVA激發(fā)后持續(xù)上升，激發(fā)停止后仍繼續(xù)上升，肺臟CD4~+記憶T細胞在末次激發(fā)后2~9天達到高峰，峰值超過50%，后逐漸下降；脾臟CD4~+記憶T細胞在末次激發(fā)后7天達到高峰，峰值超過40%，后逐漸下降。至末次激發(fā)后30天，脾臟、肺臟記憶T細胞均回降至激發(fā)前水平； ⑶CD4~-記憶T細胞變化規(guī)律：哮喘小鼠肺臟CD4~-記憶T細胞在應(yīng)用OVA激發(fā)后持續(xù)上升，末次激發(fā)后2天達到峰值，，然后迅速下降，至末次激發(fā)后7天時下降至激發(fā)前水平；脾臟CD4~-記憶T細胞于激發(fā)7天后始上升，維持在30%左右，于末次激發(fā)后14天下降至激發(fā)前水平； ⑷在本模型的構(gòu)建條件下，觀察小鼠肺臟記憶T細胞增殖功能的最佳時機為末次激發(fā)后2天； 2.沉默哮喘小鼠肺臟IL-23p19基因表達對小鼠的影響 ⑴記憶T細胞：哮喘基因沉默組小鼠肺臟CD4~+記憶T細胞占CD4~+T細胞比值較哮喘組降低（p 0.0001），CD4~-記憶T細胞占CD4~-T細胞比值無變化（p0.05）； ⑵Th2氣道炎癥：基因沉默組小鼠肺組織Th2細胞因子IL-4、IL-13mRNA表達較哮喘組降低（p 0.001，p 0.0001），肺組織炎癥病變程度、嗜酸性粒細胞氣道浸潤均較哮喘組減輕（p 0.05，p 0.0001）； ⑶轉(zhuǎn)錄因子：哮喘基因沉默組小鼠肺組織Th2轉(zhuǎn)錄因子GATA-3、Th1轉(zhuǎn)錄因子T-bet mRNA表達較哮喘組減低（p 0.0001）；Th17轉(zhuǎn)錄因子Rocr mRNA表達無變化（p0.05）。 結(jié)論： 1.哮喘小鼠體內(nèi)的記憶T細胞在哮喘不同時期呈現(xiàn)出一定的規(guī)律：⑴無論CD4~+還是CD4~-記憶T細胞，肺臟總是先于脾臟升高，且在哮喘急性期，肺臟記憶T細胞的比例顯著高于脾臟；⑵在哮喘效應(yīng)器官肺臟，CD4~+與CD4~-記憶T細胞均參與了哮喘的發(fā)病，其中CD4~+記憶T細胞是引起哮喘氣道炎癥持續(xù)的主要細胞，CD4~-記憶T細胞可能在哮喘急性發(fā)作方面發(fā)揮了作用； 2.抑制IL-23表達能夠減少哮喘小鼠肺臟CD4~+記憶T細胞的增殖，抑制Th2轉(zhuǎn)錄因子GATA-3的表達，同時能夠減輕哮喘氣道炎癥，提示IL-23有可能通過調(diào)控CD4~+Tm的增殖來參與哮喘Th2氣道炎癥，IL-23主要對CD4~+Tm發(fā)揮作用。
[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錛

本文編號：1587068