【作者】 李晓蕾；

【导师】 郑鹏远；

【作者基本信息】 郑州大学 ， 内科学， 2012， 硕士

【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,P<0.05),活化TGF-p1(43.322±13.479vs35.930土10.108,P>0.05)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,P<0.05)和总TGF-β1(210.749±31.509vs176.947±23.072,P<0.05)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,P>0.05)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,P<0.05),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。更多还原

【Abstract】 BackgroudIn the clinical application of new antibiotics widely promoted, the new vaccines also got considerable development, the treatment of infectious diseases had achieved a great success. However, in the past a few decades, the prevalence of allergic diseases had grown obviously. Food allergy and related disease in the global range increased rapidly, about4%-8%of children and1%of adults had to food antigen mediated high reaction of the IgE. Food allergy was a immune response, the damage of oral tolerance and Th2activated were the characteristics of it. Many immunity activity cells involved, such as:Treg、DC、CD4+T effector cells and CD8+T effector cells gathered themselves together in the intestinal lamina propria. The function of Treg disordered would cause allergic disease, immune response which caused oral tolerance damaged and the activated of Th2. At the same time produced antigenic specificity IgE of food, IgE and mast cells bonded, and leaded to mast cells degranulation releasing inflammation factors. Started to specific antigen of allergy. To1DC was a subtype of immature dendritic cells. High expression TGF-β or/and IL-10, but the expression of the low level stimulate molecules, TolDC played a key role in the produce Treg and oral tolerance. In the biomedical research field, such as cancer treatment and microbial infections, the research showed adoption of the transfer function after modification DC cells could rise the effective immune adjustment effect. Intestinal mucosa cells especially IEC could promote the differentiation of To1DC. Normal IEC could increase the DC cells expressing TGF-p and TGF-(3plays an important role which could be maintain DC tolerance phenotypes. TGF-P mainly bonded TGF-β receptors to regulate immune function by way of apoptosis induced cell death.Integrin αvβ6could be combined with the activity (latent form) TGF-β at the end of the carboxyl arginine-glycine-aspartic acid (arginine-glycine-aspartic acid, RGD) sequences. So TGF-β bonded its receptor more easily and further becomed activated TGF-β. The exosome could be as a carrier which could transfer information. The exosome vesicle secreted by some cells and the diameter of the secretion in30-1OOnm. By polycystic bubble film and the body and the generated cell membrane fusion. The exosome which was from IEC could be carried composition of cells and intake protein component. Thus induced the immune response of intestinal. The effect of OVA was so reliable that it was a kind of ideal food antigen model. LPS was an endotoxin which could be caused a strong immune response. Promote inflammatory cells secreted a variety of cell factors. IL-12p70was a key cell factors which started the reaction of Thl in the late of the mature DC and in the early of T cells present antigen. DC could secrete bioactive IL-12p70, IL-12p70coild be induced the differentiation of ThO to Thl. Started cellular immune, decided Immune tolerance or Immune activate. A study showed the exosome from IEC could be captured by DC and regulate its own function some substances such as integrin αvβ6May have ability to produced tolerance DC. But whether these molecules could increase the expression of TGF-β of DC still needed further research.This research was through the culture of mice IEC and BMDC in vitro, used the OVA to stimulate IEC, then collected exosome from IEC, after take exosome and DC to coculture. Used to study the influence of dendritic cells by integrin avP6. Explore whether integrin avP6could increase the expression of activation TGF-beta and make the differentiation of DC for TolDC. So as to learn how the function of IEC in process of To1DC, and provided the basis theory for the late-stage study.AimsThrough the separated and cultured mice IEC and BMDC of BALB/c, used OVA stimulate IEC, obtained exosome. Detected the expression of integrin αvβ6, the expression of CD11c, in different conditions to coculture with DC. Detection the level of activated TGF-β1cell factors and total TGF-β1cell factors by liquid supernate of the DC and the level of IL-12p70cell factors before and after stimulated by LPS. Discussed the integrin αvβ6which was from IEC to influence the function of DC.MethodsCultured IEC and BMDC of BALB/c mice, after IEC stimulated by the OVA, obtained exosome, separated DC cells through the immune magnetic beads. And cocultured with DC cells, then divided into5groups:the group of blank control, the group of OVA, the group of exosome, the group of exosome and anti-integrin αvβ6antibody, the group of exosome and IgG, detect the relevant indicators.1. The detection of pathology and CK-18immunohistochemical detection of IEC.2. Used immune colloidal gold to detect the integrin αvβ6expression of exosome.3. Flow cytometric detected the expression of CD11c of DC.4. ELISA method to determine the mixed cultivating cells liquid supernate the expression of activation TGF-β1and total TGF-β1and the expression of IL-12p70before and after stimulated by LPS.All statistical analysis was performed using SPSS statistical version17.0. Differences between groups were determined with one-way ANOVA; a P value of less than0.05was considered to be statistically significant.Results1. Separate BMDC from mice in vitro, differentiation for DC. Separate DC by the immune magnetic beads. Detect CD11c characteristic mark by Flow cytometry, the purity of DC was90%above, with a characteristic form. 2. Use OVA stimulate DC, compared with the blank group, the expression of total TGF-β1increased significantly (226.636±40.355vs176.947±23.072, P<0.05), The expression of activated TGF-β1was not changed (43.322±13.479vs35.930±10.108, P>0.05). The group of exosome compared the blank group, the expression of activated TGF-β1(80.532±26.167vs35.930±10.108, P<0.05) and total TGF-β1(210.749±31.509vs176.947±23.072, P<0.05) increased obviously. But the group of anti-integrin αVβ6could stop this phenomenon. As a compared group, the group of antibody IgG couldn’t change this phenomenon.3. The change of IL-12p70, The cocultured cells were not stimulated by LPS, each of groups was not changed significantly. The cocultured cells were stimulated by LPS in24hours, OVA group compared to the blank control group, the expression of IL-12p70was not changed significantly (327.388±25.005vs348.015±17.272, P>0.05), the group of exosome compared to the blank control group,the expression of IL-12p70was not increased (145.804±11.690vs327.388±25.005, P<0.05) significantly. But the group of anti-integrin αVβ6could stop this phenomenon. As a compaed group, the group of antibody IgG couldn’t change this phenomenon.Conclusions1. After DC and OVA coculture, the expression of total TGF-β1was increased obviously. But the expression of activated TGF-β1was not changed obviously.2. After DC and exosome coculture, the expression of total TGF-β1and activated TGF-β1were increased obviously, but the group of anti-integrin αVβ6could stop this phenomenon. As a compared group, the group of antibody IgG couldn’t change this phenomenon.3. The cocultured cells were not stimulated by LPS, each of groups was not changed significantly, the cocultured cells were stimulated by LPS, the group of exosome compared to the OVA group and blank control group, the expression of IL-12p70was not significantly increased. But the group of anti-integrin αVβ6could stop this phenomenon. As a compared group, the group of antibody IgG couldn’t change this phenomenon. The group of exosome could be resist the DC to be matured by LPS effectively.更多还原