本文關(guān)鍵詞：肺臟基質(zhì)細胞分泌的VEGF對樹突狀細胞分化發(fā)育的影響　出處：《泰山醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肺臟基質(zhì)細胞 調(diào)節(jié)性樹突狀細胞 血管內(nèi)皮生長因子 免疫耐受

【摘要】：樹突狀細胞(Dendritic cells,DC)不僅是最強的抗原遞呈細胞(antigen presenting cells,APC),而且其本身還具有調(diào)節(jié)免疫反應(yīng)強度、誘導(dǎo)免疫耐受的作用。機體是一個有機復(fù)雜的系統(tǒng),各組分在各層面上相互作用共同維持機體內(nèi)環(huán)境的穩(wěn)態(tài),免疫系統(tǒng)亦是如此。研究證實在脾臟、肝臟微環(huán)境都可誘導(dǎo)DC及造血前體細胞(hematopoietic stem cells,HSC)進一步增殖分化為具有免疫負向調(diào)控功能的調(diào)節(jié)性樹突狀細胞(regulatory DC,DCreg),并對其微環(huán)境中的各細胞、趨化因子對DC的影響機制做了闡述。我們前期研究發(fā)現(xiàn),成熟樹突狀細胞(mature dendritic cells,mDC)在肺臟基質(zhì)微環(huán)境中可被誘導(dǎo)分化發(fā)育為特殊表型的DCreg,且具有較弱的激活T細胞的能力,一定程度上可以降低免疫反應(yīng)強度誘導(dǎo)免疫耐受,但是DCreg的具體誘導(dǎo)機制尚未明確。肺臟基質(zhì)微環(huán)境由多種功能性支架細胞、趨化因子與細胞因子(VEGF,TGF-β,GM-CSF和PGE2等)等組成,它們是協(xié)調(diào)肺臟局部產(chǎn)生先天性免疫應(yīng)答和獲得性免疫應(yīng)答的主要信息傳遞者,具有極其廣泛的生物學(xué)活性;那么肺臟基質(zhì)微環(huán)境的各細胞因子在DCreg的誘導(dǎo)分化過程中發(fā)揮了怎樣的作用呢?在本項研究中,我們通過實驗證實作為肺臟基質(zhì)微環(huán)境中重要的細胞因子---血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)通過影響樹突狀細胞的表型CD86與NO的分泌表達參與了肺臟基質(zhì)微環(huán)境對免疫細胞的誘導(dǎo)分化過程與肺臟免疫穩(wěn)態(tài)維持。 研究目的 建立穩(wěn)定的小鼠肺臟基質(zhì)細胞系(murine pulmonary stmoral cells,MPSC),觀察小鼠肺臟基質(zhì)細胞分泌的細胞因子VEGF對成熟樹突狀細胞(mature dendritic cells,mDC)分化發(fā)育影響,并進一步探討其對樹突狀細胞誘導(dǎo)免疫耐受的影響。 方法 通過小鼠肺臟組織細胞的原代培養(yǎng)建立穩(wěn)定的成纖維樣基質(zhì)細胞系,并從細胞形態(tài)及特異性蛋白表達兩方面給予鑒定,結(jié)論為原代培養(yǎng)的肺臟基質(zhì)細胞具備成纖維樣細胞的特點,可以模擬肺臟局部微環(huán)境;采用RT-PCR方法檢測肺臟成纖維樣基質(zhì)細胞分泌的細胞因子:血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF),轉(zhuǎn)化生長因子-β(transforming growth factor-beta,TGF-β),粒細胞-巨噬細胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF),白介素10(IL-10)的表達水平。建立肺臟基質(zhì)細胞培養(yǎng)上清液/樹突狀細胞(細胞濃度2×10~6)共培養(yǎng)體系作為對照組,向體系中加入含有濃度為(5μg/ml)的VEGF中和性抗體作為試驗組,各自細胞培養(yǎng)一周。通過特異性夾心酶聯(lián)免疫法(ELISA)檢測兩組細胞培養(yǎng)上清中細胞因子(IL-10和IL-12p70)的含量,流式細胞術(shù)檢測DC細胞表型(CD86,CD11c,Ia)表達水平,Griess試劑檢測體系中NO的分泌量,CCK-8法檢測樹突狀細胞誘導(dǎo)同種異體T細胞的增殖能力,兩組結(jié)果進行比較。 結(jié)果 VEGF中和抗體添加組誘導(dǎo)的DC與未添加抗體組DCreg相比較其表面細胞因子IL-10、IL-12p70表達差別無統(tǒng)計學(xué)意義(P0.05),同樣兩組細胞誘導(dǎo)的T細胞增殖率無明顯差別(P0.05);VEGF中和抗體添加組誘導(dǎo)的DC其細胞表型CD86表達與未添加抗體組DCreg相比明顯上調(diào)(P0.05),而NO的表達卻明顯低于未添加抗體組DCreg(P0.05)。 結(jié)論 我們通過細胞原代培養(yǎng)的的方法建立了穩(wěn)定的肺臟基質(zhì)細胞系;證明成熟樹突狀細胞并非終末細胞,它可以在肺臟基質(zhì)微環(huán)境中分化發(fā)育DC新亞群--調(diào)節(jié)性樹突狀細胞(DCreg);肺臟基質(zhì)細胞分泌的VEGF通過下調(diào)細胞表面共刺激分子CD86的表達與調(diào)節(jié)NO的分泌水平參與了DCreg誘導(dǎo)免疫耐受的過程。
[Abstract]:Dendritic cells (Dendritic, cells, DC) is not only the strongest antigen-presenting cells (antigen presenting cells, APC), but also can regulate the immune response, inducing immune tolerance. It is an organic complex system, the steady-state components interact each other at various levels to maintain internal environment and so is the immune system. The study confirmed that the spleen, liver microenvironment can induce hematopoietic precursor cells (DC and hematopoietic stem cells, HSC) for further proliferation and differentiation with negative immune regulation function of regulatory dendritic cells (regulatory DC, DCreg), and the cells in its micro environment, influence the mechanism of chemokine DC in detail. In our previous study we found that mature dendritic cells (mature dendritic cells, mDC) can be induced to differentiation and development of DCreg special phenotype in pulmonary stromal microenvironment, And with the ability to activate T cells, to a certain extent, can reduce the immune response to induce immune tolerance, but the specific mechanism induced by DCreg is not clear. The lung microenvironment by a variety of functional support cells, chemokines and cytokines (VEGF, TGF-, GM-CSF and PGE2, beta, etc.), they are the main information coordination of locally produced lung innate immune response and acquired immune response transmission, has extensive biological activity; then the cytokine lung stromal microenvironment in DCreg induced differentiation process play a role in what? In this study, we experimentally confirmed as pulmonary stromal micro in the environment of important cytokines, vascular endothelial growth factor (vascular endothelial, growth factor, VEGF CD86 and NO) by influencing the phenotype of dendritic cells in the secretory expression in the lung The induced differentiation process of the immune cells and the immune homeostasis of the lungs were maintained by the dirty matrix microenvironment.
research objective
The establishment of mouse lung stromal cell line (murine pulmonary stmoral cells stable, MPSC), to observe the cytokine VEGF secretion of mouse lung stromal cells of mature dendritic cells (mature dendritic cells, mDC) differentiation and development, and further explore its effect on dendritic cells to induce immune tolerance.
Method
The primary mouse lung tissue cells cultured fibroblast like stromal cell lines to establish a stable, and from the cell morphology and the expression of specific proteins identified two aspects, the conclusion is the primary cultured lung stromal cells have the characteristics of fibroblast like cells, can simulate the lung department of the Ministry of micro environment; RT-PCR method was used to detect cytokine lung fibroblast like stromal cells to secrete vascular endothelial growth factor (vascular endothelial, growth factor, VEGF), transforming growth factor beta (transforming growth factor-beta, TGF-), granulocyte macrophage colony stimulating factor (granulocyte-macrophage colony-stimulating, factor, GM-CSF), interleukin 10 (IL-10) expression level. The establishment of the culture supernatant / dendritic cell lung stromal cells (cell concentration of 2 * 10~6) system as the control group were cultured, added to the system with the density of (5 g/ml) VEGF neutralizing antibody as the experimental group, the cells cultured for one week. By specific sandwich ELISA (ELISA) detection of two groups of cell cytokines in culture supernatants (IL-10 and IL-12p70) in DC cell phenotype was detected by flow cytometry (CD86, CD11c, Ia) expression and secretion detection system Griess reagent NO, CCK-8 detection of dendritic cells induced by allogeneic T cell proliferation. The results of two groups were compared.
Result
VEGF neutralizing antibody induced DC group added and not added DCreg antibody group compared to the surface of cell factor IL-10, IL-12p70 expression had no significant difference (P0.05), T cell proliferation induced by the same cells in two groups was no significant difference (P0.05); VEGF group added neutralizing antibody induced DC cell phenotype compared with expression of CD86 without adding DCreg antibody group was significantly increased (P0.05), and the expression of NO was significantly lower than that without adding DCreg antibody group (P0.05).
conclusion
The primary cell culture method established lung stromal cell lines stably; that of mature dendritic cells is not terminal cell differentiation and development, it can be DC - subset of regulatory dendritic cells in the lung stromal microenvironment (DCreg); lung stromal cells secreted VEGF is involved in the process of inducing immune tolerance by DCreg the secretion level of downregulation of cell surface expression of costimulatory molecule CD86 and regulation of NO.

【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】：2011
【分類號】：R392

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