肺脏基质细胞分泌的VEGF对树突状细胞分化发育的影响

发布时间：2018-01-04 12:27

本文关键词：肺脏基质细胞分泌的VEGF对树突状细胞分化发育的影响　出处：《泰山医学院》2011年硕士论文　论文类型：学位论文

【摘要】：树突状细胞(Dendritic cells,DC)不仅是最强的抗原递呈细胞(antigen presenting cells,APC),而且其本身还具有调节免疫反应强度、诱导免疫耐受的作用。机体是一个有机复杂的系统,各组分在各层面上相互作用共同维持机体内环境的稳态,免疫系统亦是如此。研究证实在脾脏、肝脏微环境都可诱导DC及造血前体细胞(hematopoietic stem cells,HSC)进一步增殖分化为具有免疫负向调控功能的调节性树突状细胞(regulatory DC,DCreg),并对其微环境中的各细胞、趋化因子对DC的影响机制做了阐述。我们前期研究发现,成熟树突状细胞(mature dendritic cells,mDC)在肺脏基质微环境中可被诱导分化发育为特殊表型的DCreg,且具有较弱的激活T细胞的能力,一定程度上可以降低免疫反应强度诱导免疫耐受,但是DCreg的具体诱导机制尚未明确。肺脏基质微环境由多种功能性支架细胞、趋化因子与细胞因子(VEGF,TGF-β,GM-CSF和PGE2等)等组成,它们是协调肺脏局部产生先天性免疫应答和获得性免疫应答的主要信息传递者,具有极其广泛的生物学活性;那么肺脏基质微环境的各细胞因子在DCreg的诱导分化过程中发挥了怎样的作用呢?在本项研究中,我们通过实验证实作为肺脏基质微环境中重要的细胞因子---血管内皮生长因子(vascular endothelial growth factor,VEGF)通过影响树突状细胞的表型CD86与NO的分泌表达参与了肺脏基质微环境对免疫细胞的诱导分化过程与肺脏免疫稳态维持。研究目的建立稳定的小鼠肺脏基质细胞系(murine pulmonary stmoral cells,MPSC),观察小鼠肺脏基质细胞分泌的细胞因子VEGF对成熟树突状细胞(mature dendritic cells,mDC)分化发育影响,并进一步探讨其对树突状细胞诱导免疫耐受的影响。方法通过小鼠肺脏组织细胞的原代培养建立稳定的成纤维样基质细胞系,并从细胞形态及特异性蛋白表达两方面给予鉴定,结论为原代培养的肺脏基质细胞具备成纤维样细胞的特点,可以模拟肺脏局部微环境;采用RT-PCR方法检测肺脏成纤维样基质细胞分泌的细胞因子:血管内皮生长因子(vascular endothelial growth factor,VEGF),转化生长因子-β(transforming growth factor-beta,TGF-β),粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF),白介素10(IL-10)的表达水平。建立肺脏基质细胞培养上清液/树突状细胞(细胞浓度2×10~6)共培养体系作为对照组,向体系中加入含有浓度为(5μg/ml)的VEGF中和性抗体作为试验组,各自细胞培养一周。通过特异性夹心酶联免疫法(ELISA)检测两组细胞培养上清中细胞因子(IL-10和IL-12p70)的含量,流式细胞术检测DC细胞表型(CD86,CD11c,Ia)表达水平,Griess试剂检测体系中NO的分泌量,CCK-8法检测树突状细胞诱导同种异体T细胞的增殖能力,两组结果进行比较。结果 VEGF中和抗体添加组诱导的DC与未添加抗体组DCreg相比较其表面细胞因子IL-10、IL-12p70表达差别无统计学意义(P0.05),同样两组细胞诱导的T细胞增殖率无明显差别(P0.05);VEGF中和抗体添加组诱导的DC其细胞表型CD86表达与未添加抗体组DCreg相比明显上调(P0.05),而NO的表达却明显低于未添加抗体组DCreg(P0.05)。结论我们通过细胞原代培养的的方法建立了稳定的肺脏基质细胞系;证明成熟树突状细胞并非终末细胞,它可以在肺脏基质微环境中分化发育DC新亚群--调节性树突状细胞(DCreg);肺脏基质细胞分泌的VEGF通过下调细胞表面共刺激分子CD86的表达与调节NO的分泌水平参与了DCreg诱导免疫耐受的过程。
[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.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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