本文選題：肺臟基質(zhì)細(xì)胞 + 成熟樹突狀細(xì)胞　； 參考：《泰山醫(yī)學(xué)院》2010年碩士論文

【摘要】：樹突狀細(xì)胞(dendritic cells, DC),是體內(nèi)已知功能最強(qiáng)的專職抗原遞呈細(xì)胞(antigen presenting cells, APC),是免疫應(yīng)答的中心環(huán)節(jié),不僅能活化初始T細(xì)胞啟動免疫應(yīng)答,還能誘導(dǎo)免疫耐受負(fù)向調(diào)節(jié)免疫,有雙重調(diào)節(jié)免疫反應(yīng)維持免疫平衡的作用。DC的最終免疫效應(yīng)取決于其發(fā)育階段及所處的免疫微環(huán)境。免疫微環(huán)境不僅為免疫應(yīng)答提供反應(yīng)場所,還通過調(diào)控免疫細(xì)胞的分化發(fā)育和功能來調(diào)節(jié)免疫應(yīng)答。肺臟通過氣道與外界相通,每天都要接觸大量外界抗原卻能維持免疫平衡,可見肺臟有精確的免疫調(diào)控機(jī)制,我們通過實(shí)驗(yàn)證實(shí)了肺臟基質(zhì)細(xì)胞微環(huán)境對免疫細(xì)胞DC的分化發(fā)育及功能有調(diào)節(jié)作用,這與維持免疫平衡有密切關(guān)系。 目的 觀察小鼠肺臟基質(zhì)細(xì)胞微環(huán)境對骨髓來源成熟樹突狀細(xì)胞分化發(fā)育及功能的影響,并探討其影響機(jī)制。 方法 通過小鼠肺臟組織原代培養(yǎng)建立一株成纖維樣基質(zhì)細(xì)胞系,并從細(xì)胞形態(tài)及波形蛋白表達(dá)上給予鑒定,認(rèn)為原代培養(yǎng)的肺臟基質(zhì)細(xì)胞具備成纖維樣細(xì)胞的特點(diǎn)。通過RT-PCR方法檢測肺臟基質(zhì)細(xì)胞高分泌兩種抑制性細(xì)胞因子：TGF-β、VEGF。此基質(zhì)細(xì)胞培養(yǎng)上清與完全培養(yǎng)基以1：l的比例混合后用于培養(yǎng)成熟樹突狀細(xì)胞,一周后成熟樹突狀細(xì)胞被誘導(dǎo)成具有獨(dú)特生物學(xué)特性的調(diào)節(jié)性樹突狀細(xì)胞(DCreg)。HE染色后觀察細(xì)胞形態(tài)特征,特異性夾心酶聯(lián)免疫法檢測培養(yǎng)上清細(xì)胞因子(IL-10和IL-12p70)的含量,流式細(xì)胞術(shù)檢測細(xì)胞表型表達(dá)水平及吞噬能力,MTT法檢測DCreg誘導(dǎo)同種異體T增殖能力。將上述檢測結(jié)果與成熟樹突狀細(xì)胞進(jìn)行比較,觀察誘導(dǎo)前后樹突狀細(xì)胞的區(qū)別。 結(jié)果 調(diào)節(jié)性樹突狀細(xì)胞(DCreg)分泌的細(xì)胞因子IL-10高于成熟樹突狀細(xì)胞組(P＜0.01),而IL-12p70的分泌水平低于此組(P＜0．01)；細(xì)胞表型與成熟樹突狀細(xì)胞比較,CD11b上調(diào)(P0.01),CD11c、Ⅰa、CD86下調(diào)(P＜0.01)；Dcreg的吞噬能力較未成熟DC低(P＜0.01),與成熟DC無顯著差異(P＞0.05)；而其誘導(dǎo)同種異體T增殖能力明顯低于成熟DC組(mDC)。 結(jié)論 我們首次證實(shí)了肺臟基質(zhì)細(xì)胞微環(huán)境可誘導(dǎo)成熟樹突狀細(xì)胞分化發(fā)育為另一種生物學(xué)特性不同的DC亞群--調(diào)節(jié)性樹突狀細(xì)胞(DCreg),其有抑制同種異體T細(xì)胞增殖進(jìn)而誘導(dǎo)免疫耐受負(fù)向調(diào)節(jié)免疫的作用。明確了解肺臟的免疫調(diào)節(jié)機(jī)制對預(yù)防和治療呼吸系統(tǒng)的過敏性疾病有很大的幫助。
[Abstract]:Dendritic cells (DC) are the most powerful professional antigen presenting cells known in vivo. They are the central link of the immune response. They not only activate the initial T cells to initiate the immune response, but also induce the immune tolerance to negative regulatory immunity. The final immune effect of DC depends on its developmental stage and its immune microenvironment. Immune microenvironment not only provides a response site for immune response, but also regulates immune response by regulating the differentiation, development and function of immune cells. The lungs communicate with the outside world through the airway. They are exposed to a large number of external antigens every day, but they can maintain the immune balance. It can be seen that the lungs have precise immunomodulation mechanisms. We have confirmed that the microenvironment of pulmonary stromal cells can regulate the differentiation, development and function of immune cells. Objective to observe the effects of microenvironment of mouse lung stromal cells on the differentiation, development and function of mature dendritic cells derived from bone marrow. Methods A fibroblast-like stromal cell line was established by primary culture of mouse lung tissue and identified from cell morphology and vimentin expression. It is believed that the primary cultured pulmonary stromal cells have the characteristics of fibroblast. RT-PCR was used to detect the hypersecretion of two inhibitory cytokines: TGF- 尾 and VEGF in lung stromal cells. The culture supernatant of the stromal cells was mixed with a complete medium of 1: l and used for the culture of mature dendritic cells. After one week, mature dendritic cells were induced into regulatory dendritic cells with unique biological characteristics. The morphological characteristics of the cells were observed after staining, and the levels of IL-10 and IL-12p70 in the supernatants were detected by specific sandwich enzyme-linked immunosorbent assay (Elisa). The phenotypic expression and phagocytosis of T cells were detected by flow cytometry. MTT assay was used to detect the ability of DCreg to induce allogeneic T proliferation. The results were compared with mature dendritic cells. Results IL-10 secreted by regulatory dendritic cells was higher than that of mature dendritic cells (P < 0.01), but the secretion level of IL-12p70 was lower than that of mature dendritic cells (P < 0.01). The phagocytic ability of CD11b was lower than that of immature DC (P < 0.01), and there was no significant difference between CD11b and mature DC (P > 0.05), but its ability to induce proliferation of allogeneic T was significantly lower than that of mature DC. Conclusion it is the first time that we have confirmed that the ability of lung stroma to proliferate is significantly lower than that of mature DC. Cellular microenvironment can induce the differentiation and development of mature dendritic cells into another kind of DC subsets with different biological characteristics-regulatory dendritic cells, which can inhibit the proliferation of allogeneic T cells and induce immune tolerance to negative regulatory immunity. A clear understanding of the immunomodulatory mechanism of the lung is of great help in the prevention and treatment of allergic diseases of the respiratory system.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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