房水微环境对树突细胞成熟状态的影响

发布时间：2018-06-30 08:57

  本文选题：房水 + 树突细胞　； 参考：《河北医科大学》2011年硕士论文

【摘要】：目的:眼处于免疫赦免状态和很多因素有关,其中包括解剖因素,如血眼屏障、缺少直接淋巴引流通路等,此外,房水中还存在大量起免疫抑制作用的因子对免疫细胞活性有广泛的调节作用。树突细胞(dendritic cells, DCs)是体内功能最强的抗原递呈细胞(antigen-presenting cell, APC),广泛存在于虹膜、睫状体和房水流出通道却没有得到应有的关注。本实验通过在体外模拟炎症反应环境,来观察房水微环境对脂多糖(lipopolysaccharide, LPS)介导的DCs成熟的抑制作用。 方法:取新鲜处死的猪的眼睛,保证球壁完整以防房水被污染和渗漏,用含800U/ml庆大霉素的50%酒精浸泡15min以消毒。于角膜缘处用27G针头刺入前房,使房水自动流入连接的硅化微量离心管,保存于-80°C;应用ELISA法测定房水中PGE_2含量;选用6-8周龄雄性C57BL/6小鼠,培养骨髓源DCs,培养基培养7天,半量换液后,1μg/ml的LPS刺激48h,收集悬浮和半贴壁细胞即为成熟的DCs。经流式细胞术鉴定DCs表面特异性分子标记物CD11c以观察细胞纯度。为了研究房水对DCs成熟状态的影响,将未成熟DCs分为三组:对照组(与1μg/ml LPS共培养48h),实验1组(与1μg/ml LPS+25%AH共培养48h),实验2组(与1μg/ml LPS+50%AH共培养48h),并补足rmGM-CSF、培养液,48h后收集细胞,通过流式细胞术做表型和内吞功能的鉴定。尼龙毛柱法分离得到雄性6-8周龄BALB/c小鼠脾脏的T细胞,应用于混合淋巴细胞反应来观察DCs刺激T细胞增殖的能力;根据文献结果于房水中加入10000倍于TGF-β_2浓度的抗TGF-β_2中和抗体(20μg/ml),与DCs作用48h后检测拮抗TGF-β_2后房水对DCs成熟状态的影响;第7天于DCs中加入AH6809(EP2受体拮抗剂)至终浓度为100μM,作用30min,以阻断PGE_2的作用的受体,半量换液加入房水,继续培养48h,观察DCs的成熟状态。实验数据应用SPSS13.0软件包进行统计学分析。 结果:1 DCs培养7天后,可见较多细胞呈半悬浮生长,大量细胞集落形成,集落部分细胞表面出现长短不一毛刺样突起,并可见少量单个悬浮的典型DCs,经流式细胞术鉴定DCs表面特异性分子标记物CD11c细胞纯度达到70%。 2房水抑制DCs表型成熟:对照组呈现出表型成熟状态,表现为高表达MHC-II、CD80、CD86;实验组可见依AH浓度呈剂量依赖性抑制DCs成熟,MHC-II、CD80、CD86相应的MFI明显下降;各组的散点图表明不同处理组并没有影响DCs数量。 3房水抑制DCs功能成熟:流式细胞术检测DCs吞噬FITC-dextran的能力发现,房水呈剂量依赖性抑制LPS介导内吞功能;尼龙毛柱法分离得到BALB/c小鼠脾脏T细胞,经流式细胞术鉴定CD3~+T细胞纯度达到90%;混合淋巴细胞反应中对照组DCs经LPS刺激后表现为强的刺激T细胞增殖能力,实验2组较实验1组刺激T细胞能力则减低,并在1:10比例组可观察到实验2组的刺激能力是对照组1/3。 4阻断细胞因子拮抗房水的抑制作用:经ELISA测得房水中PGE_2的浓度为2603.919±771.1536pg/ml,远远小于10~(-6)mol/L,因此选用EP2受体特异性拮抗剂AH6809预处理DCs1h以阻断PGE_2细胞膜作用通路。培养液中加入房水、抗TGF-β_2中和抗体、AH6809和DMSO并没有影响CD11c的表达,也即各组DCs具有均一性。房水能够显著抑制LPS诱导的DCs MHC-II的高表达,而抗TGF-β_2中和抗体预处理房水后则完全中和房水的抑制作用。尽管封闭EP2受体对房水的抑制作用没有影响,但是内吞功能测定,同时拮抗EP2受体和中和TGF-β_2可以协同提高DCs的内吞功能。 结论:1房水呈剂量依赖性抑制LPS介导的DCs表型和功能的成熟,表现为低表达MHC-II和共刺激分子CD80、CD86,高的内吞功能以及弱的刺激T细胞增殖的能力。 2房水中TGF-β_2在抑制LPS介导的DCs成熟过程中起主导作用。 3房水中基础量PGE_2对LPS介导的DCs表型成熟没有抑制作用,但是可以协同TGF-β_2提高DCs的内吞功能。
[Abstract]:Objective: the immune pardon is related to many factors, including anatomical factors, such as the blood barrier, the lack of direct lymphatic drainage and so on. In addition, there are a large number of immunosuppressive factors that have extensive regulation on immune cell activity in aqueous humor. Dendritic cells (DCs) is the strongest resistant body in the body. Antigen-presenting cell (APC), which is widely present in the iris, ciliary body and aqueous efflux channel, has not received due attention. This experiment was conducted to observe the inhibitory effect of aqueous microenvironment on the maturation of lipopolysaccharide (LPS) mediated by lipopolysaccharide (LPS) by simulating the inflammatory response environment in vitro.
Methods: take the eyes of fresh and dead pigs, ensure that the wall of the ball is intact to prevent the water from being polluted and leaked, and soaked in 50% alcohol containing gentamicin 800U/ml to disinfect it with 15min. At the edge of the cornea, the anterior chamber is inserted into the anterior chamber with 27G needle, so that the aqueous humor automatically flows into the connected silicified micro centrifuge tube and is stored in -80 degree C; PGE_2 content in aqueous humor is determined by ELISA; selection of PGE_2 in aqueous humor is used. With 6-8 weeks male C57BL/6 mice, bone marrow source DCs was cultured, culture medium was cultured for 7 days. After half volume, 1 u g/ml LPS stimulated 48h, and suspended and half adherent cells were collected to identify the specific molecular marker of DCs surface to observe the purity of DCs surface specific molecular marker, which was mature DCs. flow cytometry. In order to study the effect of aqueous humor on the mature state of DCs, it would not be possible. The mature DCs is divided into three groups: the control group (co culture 48h with 1 mu LPS), the experiment 1 groups (1 mu g/ml LPS+25%AH co culture 48h), the experiment 2 groups (1 mu g/ml LPS+50%AH co culture 48h), and fill the rmGM-CSF, culture liquid, collect the cells after 48h, and identify the phenotypic and endocytosis by flow cytometry. The nylon wool column method was used to separate the male 6-8 weeks old age The T cells of mouse spleen were used in mixed lymphocyte reaction to observe the ability of DCs to stimulate the proliferation of T cells. According to the results, the anti TGF- beta _2 neutralization antibody (20 u g/ml) was added to the aqueous humor of 10000 times the concentration of TGF- beta _2 in aqueous humor, and the effect of the antagonism of the aqueous humor on the mature state after TGF- beta _2 was detected after DCs action 48h. EP2 receptor antagonist) to the final concentration of 100 mu M, the action of 30min, to block the effect of PGE_2 receptor, half of the liquid into the aqueous humor, continue to cultivate 48h, observe the mature state of DCs. The experimental data were statistically analyzed by the SPSS13.0 package.
Results: after 7 days of 1 DCs culture, more cells were found to be semi suspended, a large number of cell colonies were formed, and the surface of the colonies appeared on the surface of the colony. A small number of single suspended typical DCs were seen, and the purity of the DCs surface specific molecular marker CD11c cells was identified as 70%. by flow cytometry.
2 aqueous humor inhibited DCs phenotypic maturation: the control group showed a phenotypic maturity, showing a high expression of MHC-II, CD80, CD86; the experimental group showed a dose dependent inhibition of DCs maturity with AH concentration, MHC-II, CD80, and CD86 corresponding MFI significantly decreased; the scatter plot of each group showed that the number of different groups did not affect DCs.
3 aqueous humor inhibited DCs function maturation: the ability to detect DCs phagocytosis by flow cytometry found that the aqueous humor showed a dose dependent inhibitory LPS mediated endocytosis, the BALB/c mouse spleen T cells were isolated by nylon hairy column method, and the purity of CD3~+T cells was 90% by flow cytometry, and the control group of the control group was stimulated by LPS stimulation in the control group of mixed lymphocyte reaction. The ability to stimulate the proliferation of T cells was strongly stimulated, and the ability of stimulating T cells in the 2 groups was lower than that in the experimental 1 groups, and the stimulation ability of the 2 groups was observed at 1:10 in the control group as the control group of 1/3..
4 blocking the inhibitory effect of cytokine on aqueous humor: the concentration of PGE_2 in aqueous humor was measured by ELISA, which was 2603.919 + 771.1536pg/ml, far less than 10~ (-6) mol/L, so EP2 receptor specific antagonist AH6809 pretreated DCs1h to block the pathway of PGE_2 cell membrane. The expression of CD11c was also affected by the homogeneity of DCs in each group. Aqueous humor could significantly inhibit the high expression of DCs MHC-II induced by LPS, while the anti TGF- beta _2 neutralization antibody pretreated aqueous humor was completely neutralized with aqueous humor. Although the inhibitory effect of the enclosed EP2 receptor on aqueous humor was not affected, the endocytosis was measured and the EP2 receptor was antagonized. Neutralization of TGF- beta _2 can enhance the endocytosis function of DCs.
Conclusion: 1 aqueous humor shows a dose dependent inhibition of LPS mediated DCs phenotype and function maturity, which is characterized by low expression of MHC-II and co stimulatory molecules CD80, CD86, high endocytosis and weak stimulation of T cell proliferation.
2 TGF- _2 in aqueous humor plays a leading role in inhibiting LPS mediated DCs maturation.
3 PGE_2 in aqueous humor has no inhibitory effect on LPS mediated DCs phenotype maturation, but it can synergism with TGF- beta _2 to enhance endocytosis of DCs.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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