The Effect Of Function On Dendritic Cells By Intestinal Epithelial Cell-derived Integrin αVβ6

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.