發(fā)布時(shí)間：2018-11-18 10:52

【摘要】：背景： 變應(yīng)性鼻炎藥物治療效果不理想，近年來(lái)，圍繞增強(qiáng)機(jī)體免疫調(diào)節(jié)、誘發(fā)免疫耐受的非特異性免疫治療逐漸引起了人們的重視。胸腺基質(zhì)淋巴細(xì)胞生成素（TSLP）可以通過(guò)刺激樹(shù)突狀細(xì)胞（DC）表面的TSLP受體促進(jìn)DC的活化及成熟，進(jìn)一步促進(jìn)TH2免疫反應(yīng)的發(fā)生。那么，阻斷TSLP的作用是否可以阻斷或抑制TH2反應(yīng)的進(jìn)程，，進(jìn)而誘發(fā)免疫耐受而改變變應(yīng)性疾病的轉(zhuǎn)歸呢？我們?cè)隗w外模擬其進(jìn)程進(jìn)行了研究。 目的： 體外建立未成熟樹(shù)突狀細(xì)胞（imDC）培養(yǎng)模型，分離總T淋巴細(xì)胞，對(duì)imDC表面的TSLP受體進(jìn)行干預(yù)后檢測(cè)共培養(yǎng)上清液細(xì)胞因子的變化，試圖探討封閉TSLP受體后的DC在TH1/TH2免疫平衡中，是否可以阻斷或抑制TH2反應(yīng)的進(jìn)程，進(jìn)而改變變應(yīng)性疾病的轉(zhuǎn)歸。建立小鼠變應(yīng)性鼻炎模型，對(duì)其進(jìn)行形態(tài)學(xué)觀察及IL-4檢測(cè)，為下一步體內(nèi)實(shí)驗(yàn)奠定部分基礎(chǔ)。 方法： 取6-8周齡雌性BALB/c小鼠骨髓作為DC來(lái)源，粒細(xì)胞-巨噬細(xì)胞集落刺激因子（GM-CSF），白細(xì)胞介素4（IL-4）進(jìn)行體外增殖，采用半量換液的方法，于培養(yǎng)第六天獲得未成熟樹(shù)突狀細(xì)胞（imDC），第八天獲得成熟樹(shù)突狀細(xì)胞（mDC），透射電鏡、顯微電鏡進(jìn)行形態(tài)學(xué)檢查，流式細(xì)胞儀檢測(cè)表面標(biāo)記物驗(yàn)證其成熟度。 培養(yǎng)至第六天的imDC分為兩組，其中一組加入TSLPR中和抗體進(jìn)行受體封閉，繼續(xù)培養(yǎng)兩天后分別加入FITC-MHC-II、PE-CD80進(jìn)行熒光雙標(biāo)，流式細(xì)胞儀檢測(cè)表型。 取6-8周齡雌性BALB/c小鼠脾臟作為T(mén)淋巴細(xì)胞來(lái)源，利用免疫磁珠進(jìn)行CD3+陽(yáng)選分離，流式細(xì)胞儀檢測(cè)純度。 imDC與T淋巴細(xì)胞體外共培養(yǎng)，將其分為4組，TSLP刺激組，TSLP刺激并TSLP受體阻斷組，卵清白蛋白（OVA）刺激組，卵清白蛋白刺激并TSLPR阻斷組，應(yīng)用ELISA法于共培養(yǎng)2天后分別檢測(cè)各組細(xì)胞培養(yǎng)上清中IL-4、IL-8水平。 20只BALB/c小鼠被隨機(jī)分為致敏組和對(duì)照組，使用卵清蛋白（OVA）誘導(dǎo)建立小鼠變應(yīng)性鼻炎模型。通過(guò)HE染色觀察小鼠鼻黏膜的大體重塑情況，吉姆薩染色觀察嗜酸性粒細(xì)胞，阿辛藍(lán)-過(guò)碘酸-希夫染色觀察杯狀細(xì)胞；酶聯(lián)免疫吸附（ELISA）法檢測(cè)小鼠血清中白細(xì)胞介素-4（IL-4）的水平。 結(jié)果： 透射電鏡、顯微電鏡下見(jiàn)細(xì)胞形態(tài)不規(guī)則，表面有較多短小突起，符合未成熟樹(shù)突狀細(xì)胞的形態(tài)學(xué)特征；流式細(xì)胞儀檢測(cè)FITC-CD11c熒光強(qiáng)度為76.83%±1.42%，推斷培養(yǎng)的細(xì)胞多數(shù)為髓樣樹(shù)突狀細(xì)胞；MHC-II、CD80及MHC-II、CD86雙標(biāo)熒光強(qiáng)度分別為2.61%±0.11%和10.7%±0.23%，推斷培養(yǎng)的樹(shù)突狀細(xì)胞大部分處于未成熟狀態(tài)。 流式細(xì)胞儀檢測(cè)兩組的FITC-MHC-II、PE-CD80雙標(biāo)熒光強(qiáng)度，兩組間差異有統(tǒng)計(jì)學(xué)意義，推斷封閉imDC表面的TSLP受體可以有效的維持其未成熟狀態(tài)。 利用免疫磁珠進(jìn)行T淋巴細(xì)胞陽(yáng)選分離后PE-CD3為96.1%±2.8%，推斷通過(guò)免疫磁珠法可以獲得高純度的總T淋巴細(xì)胞。 共培養(yǎng)上清液的ELISA結(jié)果顯示，TSLP受體阻斷組IL-4水平明顯低于TSLP刺激組及OVA刺激組，而TSLP受體阻斷對(duì)細(xì)胞培養(yǎng)上清中IL-8表達(dá)水平無(wú)顯著影響。體外阻斷TSLP受體后IL-4水平顯著降低，說(shuō)明阻斷TSLP受體可一定程度上抑制TH2反應(yīng)。 小鼠變應(yīng)性鼻炎模型的生物學(xué)行為評(píng)分為6.5±1.3，提示造模成功。與對(duì)照組相比，致敏組鼻腔黏膜出現(xiàn)上皮細(xì)胞脫落、壞死，杯狀細(xì)胞增生，鱗狀上皮化生，固有層和黏膜下層腺體增生、血管擴(kuò)張，組織水腫，固有層內(nèi)可見(jiàn)特征性的嗜酸性粒細(xì)胞浸潤(rùn)。致敏組小鼠鼻黏膜嗜酸性粒細(xì)胞計(jì)數(shù)及杯狀細(xì)胞計(jì)數(shù)分別為（26.4±5.72）和（24.14±3.12），而對(duì)照組分別是（8.31±2.42）和（9.41±1.22），兩組比較均具有統(tǒng)計(jì)學(xué)意義；致敏組血清中白細(xì)胞介素4（IL-4）水平為（18.9±3.1）pg/ml，對(duì)照組為（8.3±1.4）pg/ml，致敏組顯著高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論： 體外行TSLP受體阻斷可抑制T淋巴細(xì)胞向TH2方向轉(zhuǎn)化，這可能為臨床變應(yīng)性鼻炎及哮喘等TH2優(yōu)勢(shì)的疾病提供新的治療策略。
[Abstract]:Background: The treatment effect of allergic rhinitis is not ideal. In recent years, the non-specific immunotherapy around the enhancement of the immune regulation of the body and inducing the immune tolerance gradually causes people The TSLP receptor can stimulate the activation and maturation of the DC by stimulating the TSLP receptor on the surface of the dendritic cell (DC), and further promote the immune response of the TH2. It happens that blocking the TSLP action can block or inhibit the process of the TH2 reaction, thereby inducing immune tolerance to change the rotation of the allergic disease. What's the matter? We're simulating the process in vitro. Research. Objective: To establish an immature dendritic cell (imDC) culture model in vitro, to isolate the total T-lymphocytes and to detect the changes of the cytokines in coculture after the TSLP receptors on the imDC surface. In the TH2 immune equilibrium, it is possible to block or inhibit the process of the TH2 reaction and then to change The model of allergic rhinitis in mice was established, and the morphological observation and IL-4 detection were carried out to the next. experimental base The bone marrow of the 6-8-week-old female BALB/ c mice was used as the DC source, the granulocyte-macrophage colony-stimulating factor (GM-CSF) and the interleukin-4 (IL-4) were used for the in vitro proliferation. Immature dendritic cells (imDC), mature dendritic cells (mDC), transmission electron microscopy (TEM) and micro-electron microscope (TEM) were obtained by flow cytometry. The instrument detects the maturity of the surface marker. The imDC cultured to the sixth day is divided into two groups, one group is added to the TSLPR and the antibody is closed, and the FITC-MHC-II and the PE-CD8 are respectively added after two days of culture. 0. The fluorescent double-label and flow cytometry were used to detect the phenotype. The spleen of the 6-8-week-old female BALB/ c mice was used as the source of T-lymphocytes and the immunomagnetic beads were used. The samples were co-cultured with T-lymphocytes and divided into 4 groups, TSLP-stimulated group, TSLP-stimulated and TSLP-receptor blocking group, ovalbumin (OVA) stimulation group, ovalbumin-stimulated and TSLPR blocking group. 2 days after co-culture The levels of IL-4 and IL-8 in the supernatant of each group were not detected. 20 BALB/ c mice were randomly divided into the group-sensitive group and the control group. A model of mouse allergic rhinitis was established by using ovalbumin (OVA). The general remodeling of the nasal mucosa of mice was observed by HE staining. A) Act The level of interleukin-4 (IL-4) in the serum of the mice was detected. The results showed that the morphology of the cells was irregular under the microscope and the microscopic electron microscope, and the surface had more short protrusions, which met the morphological characteristics of the immature dendritic cells. The fluorescence intensity of FITC-CD11c was detected by flow cytometry. The fluorescence intensity of MHC-II, CD80 and MHC-II and CD86 was 2.61% and 0.11%, respectively. The fluorescence intensity of FITC-MHC-II and PE-CD80 in both groups was detected by flow cytometry. To study the significance of the study, it is concluded that the TSLP receptor on the surface of the closed imDC can effectively maintain its immature state. The results of ELISA showed that the level of IL-4 in TSLP receptor blocking group was lower than that of TSLP. The effect of TSLP receptor blockade on the level of IL-8 expression in the supernatant of cell culture was not significantly affected by the stimulation group and the OVA-stimulated group. The level of IL-4 after TSLP receptor is significantly reduced, indicating that the blocking of TSLP receptor can be a certain extent. In response to the inhibition of TH2, the biological behavior of the model of allergic rhinitis in mice was 6. 5-1.3, suggesting that the model was successful. In contrast to the control group, the epithelial cells of the nasal mucosa of the sensitive group were shed, necrosis, the proliferation of the cup-like cells, and the squamous epithelium. The number of eosinophils in the nasal mucosa of the sensitive group and the count of the cup-like cells were (26. 4, 5.72) and (24.14, 3.12), respectively. The levels of interleukin4 (IL-4) in the serum of the sensitive group were (18.9% 3.1). pg/ Results: TSLP receptor in vitro was found to be higher in the control group than in the control group.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R765.21

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 田陽(yáng);饒歡;薛文通;;花生過(guò)敏原檢測(cè)、降敏加工及脫敏治療研究進(jìn)展[J];食品工業(yè)科技;2017年18期

2 姚娟;鄭曉汾;李冰;;胸腺基質(zhì)淋巴細(xì)胞生成素在過(guò)敏性結(jié)膜炎發(fā)病中的作用[J];國(guó)際眼科縱覽;2015年03期

相關(guān)碩士學(xué)位論文 前1條

1 姚娟;TSLP、IL-13在不同類(lèi)型過(guò)敏性結(jié)膜炎患者眼表的表達(dá)[D];山西醫(yī)科大學(xué);2015年

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