肠上皮细胞来源的整合素αVβ6对树突状细胞功能的影响
发布时间：2018-09-07 08:07
【摘要】：研究背景 在临床上新型抗生素被广泛推广应用,新型的疫苗的研制也获取了长足的发展,我们在治疗感染性疾病的方面已取得巨大进步,但是在过去的几十年中,过敏性疾病的上升趋势已明显呈现,食物过敏(food allergy, FA)以及相关疾病在全球范围内迅速增加,大约4%—8%的儿童和1%-2%的成年人对食物抗原具有IgE介导的高反应性。食物过敏是以肠道口服耐受受损和Th2极化为特征的免疫反应。许多免疫活性细胞参与其中如：调节性T细胞(T regulatory cell, Treg)、树突状细胞(DC)、效应性CD4+T和CD8+T细胞在肠道固有层聚集。Treg功能失常将导致过敏性疾病的发生,导致口服耐受(oral tolerance)受损和以Th2细胞为主的免疫反应,同时产生食物抗原特异性的IgE抗体,IgE与肥大细胞结合,并导致肥大细胞脱颗粒释放致炎因子,启动针对特异性抗原的过敏反应。耐受型树突状细胞(tolerogenic dendritic cell, TolDC)是一类未成熟树突状细胞亚型,高表达TGF-β或／和IL-10,但表达低水平共刺激分子,TolDC在Treg产生和口服耐受中起关键作用。在生物医学研究领域,如肿瘤治疗、抗微生物感染研究显示,过继转移功能性修饰后的DC细胞能够起到有效的免疫调节作用。肠道粘膜细胞特别是肠上皮细胞(Intestinal epithelial cells, IEC),能够促进TolDC的分化。正常肠上皮细胞能够增加DC细胞表达TGF-p,而TGF-p在维持DC耐受表型中起重要作用TGF-p主要通过与TGF-p受体结合来调节免疫功能,可通过凋亡的方式诱导细胞死亡 整合素αvβ6可结合在非活性(latent form) TGF-β的羧基末端的精氨酸-甘氨酸-天冬氨酸(arginine-glycine-aspartic acid, RGD)(?)序列上,从而使TGF-β更易于与其受体结合并进一步形成活化TGF-β。外泌体可作为细胞间信息传递、转运的载体,外泌体是由一些细胞分泌的直径在30-100纳米的小囊泡,由多囊泡体膜与细胞膜融合而生成。肠上皮细胞来源的外泌体能够携带细胞成分和摄入的蛋白质成分,从而诱导肠道免疫反应。卵清蛋白(ovalbvmin, OVA)抗原性稳定可靠,是一种理想食物抗原模型。脂多糖是内毒素,可引起了强烈免疫反应,促进炎性细胞分泌多种细胞因子。IL-12p70是启动Th1反应的关键细胞因子,DC在成熟的晚期和向T细胞递呈抗原的早期,DC可分泌具有生物活性的IL-12p70,IL-12p70可诱导Th0向Thl分化,启动细胞免疫,决定免疫激活还是免疫耐受。有研究显示,肠上皮细胞来源的外泌体能够被DC捕获并调节其自身功能一些物质如整合素αvβ6可能具有使DC产生耐受表型的能力,但这些分子是否能够增加DC表达TGF-β仍有待进一步研究。 本研究是通过体外培养小鼠肠上皮细胞和骨髓来源树突状细胞,用卵清蛋白刺激肠上皮细胞后来源的外泌体与树突状细胞共同培养,用来研究整合素αvβ6对树突状细胞的影响,探讨整合素avβ6是否能够增加活化TGF-p在DC中的表达并使之分化为To1DC,从而了解整合素αvβ6在To1DC发生过程中的作用,并为后期研究提供理论依据。 目的 通过分离培养BALB/c小鼠肠上皮细胞和骨髓来源树突状细胞,OVA刺激肠上皮细胞,获取外泌体,检测整合素αvβ6的表达,DC表面分子CDllc表达,和DC在不同条件下进行共同培养,检测DC细胞上清液中活化的TGF-β1和总TGF-β1细胞因子的水平和脂多糖刺激前后IL-12p70细胞因子的水平。探讨肠上皮细胞来源整合素αvβ6对树突状细胞功能的影响。 方法 培养BALB/c小鼠的肠上皮细胞(intestinal epithelial cell, IEC)和骨髓来源的树突状细胞(bone marrow-derived DC, BMDC),肠上皮细胞在卵清蛋白刺激后,获取外泌体,通过免疫磁珠分离出DC细胞。和DC细胞共同进行培养分为5组：空白对照组、OVA组、外泌体组、外泌体+抗整合素αvβ6抗体组及外泌体+羊抗小鼠IgG抗体组。进行相关指标的检测。 1.肠上皮细胞的病理检测和CK-18的免疫组化检测。 2.免疫胶体金检测外泌体的整合素αvβ6的存在。 3.流式细胞仪检测DC表面CDllc的表达。 4. ELISA法测定不同干预条件下DC细胞上清液活化TGF-β1和总TGF-β1的分泌变化与脂多糖刺激前后的IL-12p70的分泌变化。 实验数据均录入SPSS17.0统计软件包分析,比较各组间均值采用单因素方差分析,以a=0.05为假设检验标准。 结果 1.在体外的环境下,将小鼠骨髓细胞分离出来,使用集落刺激因子和白介素4诱导BMDC分化为树突状细胞,通过免疫磁珠的分离,使用流式细胞仪检测树突状细胞特征性标志CDllc,分离的细胞纯度可达90%以上,并符合其特征。 2.使用OVA刺激DC后,与空白实验组相比,总TGF-β1量明显增加(226.636±40.355vs176.947±23.072,P0.05),活化TGF-p1(43.322±13.479vs35.930土10.108,P0.05)无明显变化。外泌体组与空白对照组相比,活化TGF-β1的量(80.532±26.167vs35.930±10.108,P0.05)和总TGF-β1(210.749±31.509vs176.947±23.072,P0.05)量明显增加,而抗整合素aVβ6抗体组可以阻止这一现象,作为对照抗体羊抗小鼠IgG组并不能改变此现象。 3.细胞因子IL-12p70量的变化,在LPS刺激前,各组的分泌量无明显的变化。在LPS刺激24小时后,OVA组和空白对照组相比较,分泌量(327.388±25.005vs348.015±17.272,P0.05)无明显变化。外泌体组和空白对照组相比,分泌IL-12p70的能力明显降低(145.804±11.690vs327.388±25.005,P0.05),抗整合素aVβ6抗体组可以阻止这一现象,作为对照抗体羊抗小鼠IgG组并不能改变此现象。 结论 1.OVA和DC共培养后,总TGF-β1量的表达增加,而活化TGF-β1量无明显变化。 2.外泌体和DC共培养后,促进总TGF-β1量和活化TGF-β1的表达上调,抗整合素αVβ6抗体组可以阻止活化TGF-β1的表达上调这种变化,作为对照抗体羊抗小鼠IgG组并不能阻止此变化。 3.脂多糖刺激DC前,各组间IL-12p70的表达无明显变化。脂多糖刺激DC后,外泌体组与空白对照组和OVA组相比,IL-12p70的表达无明显上调,抗整合素αVβ6抗体组可以阻止这种变化,作为对照抗体羊抗小鼠IgG组并不能阻止此变化,外泌体组可以有效的抵抗了脂多糖(lipopolysaccharide, LPS)对DC的促成熟作用。
[Abstract]:Research background
New antibiotics have been widely used in clinic, and new vaccines have been developed. We have made great progress in treating infectious diseases. But in the past few decades, the rising trend of allergic diseases has been obvious. Food allergy (FA) and related diseases are all over the world. Food allergy is an immune response characterized by impaired oral tolerance and Th2 polarization of the intestine. Many immunocompetent cells are involved in this response, such as regulatory T cells (Treg), dendritic cells (DC), and effectivity. CD4+T and CD8+T cells aggregate in the lamina propria of the intestine. Treg dysfunction can lead to allergic diseases, impaired oral tolerance and immune responses dominated by Th2 cells, and produce food antigen-specific IgE antibodies. IgE binds to mast cells and causes mast cell degranulation to release inflammatory factors and initiate Tolerogenic dendritic cells (TolDC) are a class of immature dendritic cell subtypes that overexpress TGF-beta or/and IL-10, but express low levels of costimulatory molecules. TolDC plays a key role in Treg production and oral tolerance. In biomedical research fields, such as cancer therapy, anti-tumor therapy Microbial infection studies have shown that DC cells modified by adoptive metastasis can play an effective immunomodulatory role. Intestinal mucosal cells, especially intestinal epithelial cells (IEC), can promote the differentiation of TolDC. Normal intestinal epithelial cells can increase the expression of TGF-p in DC cells, while TGF-p can maintain the tolerance phenotype of DC. TGF-p plays an important role in regulating immune function by binding to TGF-p receptor and inducing cell death through apoptosis.
Integrin alpha v beta 6 binds to the arginine-glycine-aspartic acid (RGD) (?) sequence at the carboxyl terminal of inactive (latent form) TGF-beta, making it easier for TGF-beta to bind to its receptor and further form activated TGF-beta. The exosome acts as a carrier for intercellular information transmission and transport. The exosome is Some cells secrete vesicles with a diameter of 30-100 nm, which are formed by the fusion of the polyvesicular membrane with the cell membrane. The secretions from the intestinal epithelial cells can carry cell components and protein components, thus inducing intestinal immune response. OVA is an ideal food antigen with stable antigenicity. Lipopolysaccharide (LPS) is endotoxin, which can induce strong immune response and stimulate inflammatory cells to secrete a variety of cytokines. IL-12p70 is the key cytokine to initiate Th1 reaction. DC can secrete bioactive IL-12p70 in the late maturation and early antigen presentation to T cells. IL-12p70 can induce Th0 to differentiate into Thl and initiate cellular immunity. Studies have shown that intestinal epithelial cell-derived exosomes can be captured by DC and regulate their own functions. Some substances, such as integrin alpha v beta 6, may be able to induce DC to produce tolerant phenotypes, but whether these molecules can increase the expression of TGF-beta in DC remains to be further studied.
The aim of this study was to investigate the effect of integrin alpha v beta 6 on dendritic cells (DCs) and whether integrin AV beta 6 could increase the expression of activated TGF-p in DC by in vitro culture of murine intestinal epithelial cells and bone marrow-derived dendritic cells (BMDCs) stimulated by ovalbumin and co-cultured with dendritic cells (DCs). It differentiates into To1DC, so as to understand the role of integrin alpha v beta 6 in the development of To1DC, and provide a theoretical basis for later research.
objective
By isolating and culturing BALB/c mice intestinal epithelial cells and bone marrow-derived dendritic cells, OVA stimulated intestinal epithelial cells, obtained exosomes, detected the expression of integrin alpha v beta 6, DC surface molecule CDllc, and co-cultured with DC under different conditions, detected the levels of activated TGF-beta 1 and total TGF-beta 1 cytokines in the supernatant of DC cells and lipids. To investigate the effect of integrin alpha v beta 6 on the function of dendritic cells.
Method
Intestinal epithelial cell (IEC) and bone marrow-derived DC (BMDC) were cultured in BALB/c mice. After stimulation with ovalbumin, the exosomes were obtained and DC cells were isolated by immunomagnetic beads. DC cells and DC cells were cultured together and divided into five groups: blank control group, OVA group, exosome. Group A, exosome + anti-integrin alpha v beta 6 antibody group and exosome + sheep anti-mouse IgG antibody group were used to detect the related indexes.
1. pathological examination of intestinal epithelial cells and immunohistochemical detection of CK-18.
2. immunogold gold detected the presence of integrin alpha v beta 6 in exocrine bodies.
3. the expression of CDllc on DC surface was detected by flow cytometry.
4. The secretion of TGF-beta 1 and total TGF-beta 1 activated by DC cell supernatant and the secretion of IL-12p70 stimulated by LPS were measured by ELISA.
The experimental data were analyzed by SPSS17.0 statistical software package. The mean values of each group were analyzed by one-way ANOVA and a=0.05 as the test criterion.
Result
1. BMDC was induced to differentiate into dendritic cells by colony-stimulating factor and interleukin-4 (IL-4) in vitro. After isolation of immunomagnetic beads, CDllc, the characteristic marker of dendritic cells, was detected by flow cytometry. The purity of the isolated cells was over 90% and accorded with the characteristics.
2. Compared with the blank control group, the total TGF-beta 1 level increased significantly (226.636 65 1.509 vs 176.947 (+ 23.072, P 0.05) increased significantly, but the anti-integrin aV beta 6 antibody group could prevent this phenomenon. As a control antibody, sheep anti-mouse IgG group could not change this phenomenon.
3. The secretion of cytokine IL-12p70 did not change significantly before LPS stimulation. After 24 hours of LPS stimulation, the secretion of IL-12p70 in OVA group and blank control group (327.388+25.005 vs 348.015+17.272, P 0.05) did not change significantly. Compared with blank control group, the secretion of IL-12p70 in exosome decreased significantly (145.804+11.690). Anti-integrin aV beta 6 antibody group could prevent this phenomenon, but sheep anti-mouse IgG group could not change this phenomenon.
conclusion
After co culture with 1.OVA and DC, the expression of total TGF- beta 1 increased, while the amount of activated TGF- 1 did not change significantly.
2. After co-culture of exosome and DC, the expression of total TGF-beta 1 and activated TGF-beta 1 were up-regulated. Anti-integrin alpha V-beta 6 antibody group could prevent the up-regulated expression of activated TGF-beta 1. As a control antibody, sheep anti-mouse IgG group could not prevent this change.
3. The expression of IL-12p70 did not change significantly before LPS stimulated DC. After LPS stimulated DC, the expression of IL-12p70 in the exosome was not significantly increased compared with the blank control group and OVA group. The anti-integrin alpha V beta 6 antibody group could prevent this change. As a control antibody, the sheep anti-mouse IgG group could not prevent this change, but the exosome could. Lipopolysaccharide LPS inhibited DC maturation.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2227696
[Abstract]:Research background
New antibiotics have been widely used in clinic, and new vaccines have been developed. We have made great progress in treating infectious diseases. But in the past few decades, the rising trend of allergic diseases has been obvious. Food allergy (FA) and related diseases are all over the world. Food allergy is an immune response characterized by impaired oral tolerance and Th2 polarization of the intestine. Many immunocompetent cells are involved in this response, such as regulatory T cells (Treg), dendritic cells (DC), and effectivity. CD4+T and CD8+T cells aggregate in the lamina propria of the intestine. Treg dysfunction can lead to allergic diseases, impaired oral tolerance and immune responses dominated by Th2 cells, and produce food antigen-specific IgE antibodies. IgE binds to mast cells and causes mast cell degranulation to release inflammatory factors and initiate Tolerogenic dendritic cells (TolDC) are a class of immature dendritic cell subtypes that overexpress TGF-beta or/and IL-10, but express low levels of costimulatory molecules. TolDC plays a key role in Treg production and oral tolerance. In biomedical research fields, such as cancer therapy, anti-tumor therapy Microbial infection studies have shown that DC cells modified by adoptive metastasis can play an effective immunomodulatory role. Intestinal mucosal cells, especially intestinal epithelial cells (IEC), can promote the differentiation of TolDC. Normal intestinal epithelial cells can increase the expression of TGF-p in DC cells, while TGF-p can maintain the tolerance phenotype of DC. TGF-p plays an important role in regulating immune function by binding to TGF-p receptor and inducing cell death through apoptosis.
Integrin alpha v beta 6 binds to the arginine-glycine-aspartic acid (RGD) (?) sequence at the carboxyl terminal of inactive (latent form) TGF-beta, making it easier for TGF-beta to bind to its receptor and further form activated TGF-beta. The exosome acts as a carrier for intercellular information transmission and transport. The exosome is Some cells secrete vesicles with a diameter of 30-100 nm, which are formed by the fusion of the polyvesicular membrane with the cell membrane. The secretions from the intestinal epithelial cells can carry cell components and protein components, thus inducing intestinal immune response. OVA is an ideal food antigen with stable antigenicity. Lipopolysaccharide (LPS) is endotoxin, which can induce strong immune response and stimulate inflammatory cells to secrete a variety of cytokines. IL-12p70 is the key cytokine to initiate Th1 reaction. DC can secrete bioactive IL-12p70 in the late maturation and early antigen presentation to T cells. IL-12p70 can induce Th0 to differentiate into Thl and initiate cellular immunity. Studies have shown that intestinal epithelial cell-derived exosomes can be captured by DC and regulate their own functions. Some substances, such as integrin alpha v beta 6, may be able to induce DC to produce tolerant phenotypes, but whether these molecules can increase the expression of TGF-beta in DC remains to be further studied.
The aim of this study was to investigate the effect of integrin alpha v beta 6 on dendritic cells (DCs) and whether integrin AV beta 6 could increase the expression of activated TGF-p in DC by in vitro culture of murine intestinal epithelial cells and bone marrow-derived dendritic cells (BMDCs) stimulated by ovalbumin and co-cultured with dendritic cells (DCs). It differentiates into To1DC, so as to understand the role of integrin alpha v beta 6 in the development of To1DC, and provide a theoretical basis for later research.
objective
By isolating and culturing BALB/c mice intestinal epithelial cells and bone marrow-derived dendritic cells, OVA stimulated intestinal epithelial cells, obtained exosomes, detected the expression of integrin alpha v beta 6, DC surface molecule CDllc, and co-cultured with DC under different conditions, detected the levels of activated TGF-beta 1 and total TGF-beta 1 cytokines in the supernatant of DC cells and lipids. To investigate the effect of integrin alpha v beta 6 on the function of dendritic cells.
Method
Intestinal epithelial cell (IEC) and bone marrow-derived DC (BMDC) were cultured in BALB/c mice. After stimulation with ovalbumin, the exosomes were obtained and DC cells were isolated by immunomagnetic beads. DC cells and DC cells were cultured together and divided into five groups: blank control group, OVA group, exosome. Group A, exosome + anti-integrin alpha v beta 6 antibody group and exosome + sheep anti-mouse IgG antibody group were used to detect the related indexes.
1. pathological examination of intestinal epithelial cells and immunohistochemical detection of CK-18.
2. immunogold gold detected the presence of integrin alpha v beta 6 in exocrine bodies.
3. the expression of CDllc on DC surface was detected by flow cytometry.
4. The secretion of TGF-beta 1 and total TGF-beta 1 activated by DC cell supernatant and the secretion of IL-12p70 stimulated by LPS were measured by ELISA.
The experimental data were analyzed by SPSS17.0 statistical software package. The mean values of each group were analyzed by one-way ANOVA and a=0.05 as the test criterion.
Result
1. BMDC was induced to differentiate into dendritic cells by colony-stimulating factor and interleukin-4 (IL-4) in vitro. After isolation of immunomagnetic beads, CDllc, the characteristic marker of dendritic cells, was detected by flow cytometry. The purity of the isolated cells was over 90% and accorded with the characteristics.
2. Compared with the blank control group, the total TGF-beta 1 level increased significantly (226.636 65 1.509 vs 176.947 (+ 23.072, P 0.05) increased significantly, but the anti-integrin aV beta 6 antibody group could prevent this phenomenon. As a control antibody, sheep anti-mouse IgG group could not change this phenomenon.
3. The secretion of cytokine IL-12p70 did not change significantly before LPS stimulation. After 24 hours of LPS stimulation, the secretion of IL-12p70 in OVA group and blank control group (327.388+25.005 vs 348.015+17.272, P 0.05) did not change significantly. Compared with blank control group, the secretion of IL-12p70 in exosome decreased significantly (145.804+11.690). Anti-integrin aV beta 6 antibody group could prevent this phenomenon, but sheep anti-mouse IgG group could not change this phenomenon.
conclusion
After co culture with 1.OVA and DC, the expression of total TGF- beta 1 increased, while the amount of activated TGF- 1 did not change significantly.
2. After co-culture of exosome and DC, the expression of total TGF-beta 1 and activated TGF-beta 1 were up-regulated. Anti-integrin alpha V-beta 6 antibody group could prevent the up-regulated expression of activated TGF-beta 1. As a control antibody, sheep anti-mouse IgG group could not prevent this change.
3. The expression of IL-12p70 did not change significantly before LPS stimulated DC. After LPS stimulated DC, the expression of IL-12p70 in the exosome was not significantly increased compared with the blank control group and OVA group. The anti-integrin alpha V beta 6 antibody group could prevent this change. As a control antibody, the sheep anti-mouse IgG group could not prevent this change, but the exosome could. Lipopolysaccharide LPS inhibited DC maturation.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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