腸上皮細(xì)胞來源的整合素αVβ6對(duì)樹突狀細(xì)胞功能的影響
發(fā)布時(shí)間：2018-09-07 08:07
【摘要】：研究背景 在臨床上新型抗生素被廣泛推廣應(yīng)用,新型的疫苗的研制也獲取了長足的發(fā)展,我們?cè)谥委煾腥拘约膊〉姆矫嬉讶〉镁薮筮M(jìn)步,但是在過去的幾十年中,過敏性疾病的上升趨勢(shì)已明顯呈現(xiàn),食物過敏(food allergy, FA)以及相關(guān)疾病在全球范圍內(nèi)迅速增加,大約4%—8%的兒童和1%-2%的成年人對(duì)食物抗原具有IgE介導(dǎo)的高反應(yīng)性。食物過敏是以腸道口服耐受受損和Th2極化為特征的免疫反應(yīng)。許多免疫活性細(xì)胞參與其中如：調(diào)節(jié)性T細(xì)胞(T regulatory cell, Treg)、樹突狀細(xì)胞(DC)、效應(yīng)性CD4+T和CD8+T細(xì)胞在腸道固有層聚集。Treg功能失常將導(dǎo)致過敏性疾病的發(fā)生,導(dǎo)致口服耐受(oral tolerance)受損和以Th2細(xì)胞為主的免疫反應(yīng),同時(shí)產(chǎn)生食物抗原特異性的IgE抗體,IgE與肥大細(xì)胞結(jié)合,并導(dǎo)致肥大細(xì)胞脫顆粒釋放致炎因子,啟動(dòng)針對(duì)特異性抗原的過敏反應(yīng)。耐受型樹突狀細(xì)胞(tolerogenic dendritic cell, TolDC)是一類未成熟樹突狀細(xì)胞亞型,高表達(dá)TGF-β或／和IL-10,但表達(dá)低水平共刺激分子,TolDC在Treg產(chǎn)生和口服耐受中起關(guān)鍵作用。在生物醫(yī)學(xué)研究領(lǐng)域,如腫瘤治療、抗微生物感染研究顯示,過繼轉(zhuǎn)移功能性修飾后的DC細(xì)胞能夠起到有效的免疫調(diào)節(jié)作用。腸道粘膜細(xì)胞特別是腸上皮細(xì)胞(Intestinal epithelial cells, IEC),能夠促進(jìn)TolDC的分化。正常腸上皮細(xì)胞能夠增加DC細(xì)胞表達(dá)TGF-p,而TGF-p在維持DC耐受表型中起重要作用TGF-p主要通過與TGF-p受體結(jié)合來調(diào)節(jié)免疫功能,可通過凋亡的方式誘導(dǎo)細(xì)胞死亡 整合素αvβ6可結(jié)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,從而使TGF-β更易于與其受體結(jié)合并進(jìn)一步形成活化TGF-β。外泌體可作為細(xì)胞間信息傳遞、轉(zhuǎn)運(yùn)的載體,外泌體是由一些細(xì)胞分泌的直徑在30-100納米的小囊泡,由多囊泡體膜與細(xì)胞膜融合而生成。腸上皮細(xì)胞來源的外泌體能夠攜帶細(xì)胞成分和攝入的蛋白質(zhì)成分,從而誘導(dǎo)腸道免疫反應(yīng)。卵清蛋白(ovalbvmin, OVA)抗原性穩(wěn)定可靠,是一種理想食物抗原模型。脂多糖是內(nèi)毒素,可引起了強(qiáng)烈免疫反應(yīng),促進(jìn)炎性細(xì)胞分泌多種細(xì)胞因子。IL-12p70是啟動(dòng)Th1反應(yīng)的關(guān)鍵細(xì)胞因子,DC在成熟的晚期和向T細(xì)胞遞呈抗原的早期,DC可分泌具有生物活性的IL-12p70,IL-12p70可誘導(dǎo)Th0向Thl分化,啟動(dòng)細(xì)胞免疫,決定免疫激活還是免疫耐受。有研究顯示,腸上皮細(xì)胞來源的外泌體能夠被DC捕獲并調(diào)節(jié)其自身功能一些物質(zhì)如整合素αvβ6可能具有使DC產(chǎn)生耐受表型的能力,但這些分子是否能夠增加DC表達(dá)TGF-β仍有待進(jìn)一步研究。 本研究是通過體外培養(yǎng)小鼠腸上皮細(xì)胞和骨髓來源樹突狀細(xì)胞,用卵清蛋白刺激腸上皮細(xì)胞后來源的外泌體與樹突狀細(xì)胞共同培養(yǎng),用來研究整合素αvβ6對(duì)樹突狀細(xì)胞的影響,探討整合素avβ6是否能夠增加活化TGF-p在DC中的表達(dá)并使之分化為To1DC,從而了解整合素αvβ6在To1DC發(fā)生過程中的作用,并為后期研究提供理論依據(jù)。 目的 通過分離培養(yǎng)BALB/c小鼠腸上皮細(xì)胞和骨髓來源樹突狀細(xì)胞,OVA刺激腸上皮細(xì)胞,獲取外泌體,檢測整合素αvβ6的表達(dá),DC表面分子CDllc表達(dá),和DC在不同條件下進(jìn)行共同培養(yǎng),檢測DC細(xì)胞上清液中活化的TGF-β1和總TGF-β1細(xì)胞因子的水平和脂多糖刺激前后IL-12p70細(xì)胞因子的水平。探討腸上皮細(xì)胞來源整合素αvβ6對(duì)樹突狀細(xì)胞功能的影響。 方法 培養(yǎng)BALB/c小鼠的腸上皮細(xì)胞(intestinal epithelial cell, IEC)和骨髓來源的樹突狀細(xì)胞(bone marrow-derived DC, BMDC),腸上皮細(xì)胞在卵清蛋白刺激后,獲取外泌體,通過免疫磁珠分離出DC細(xì)胞。和DC細(xì)胞共同進(jìn)行培養(yǎng)分為5組：空白對(duì)照組、OVA組、外泌體組、外泌體+抗整合素αvβ6抗體組及外泌體+羊抗小鼠IgG抗體組。進(jìn)行相關(guān)指標(biāo)的檢測。 1.腸上皮細(xì)胞的病理檢測和CK-18的免疫組化檢測。 2.免疫膠體金檢測外泌體的整合素αvβ6的存在。 3.流式細(xì)胞儀檢測DC表面CDllc的表達(dá)。 4. ELISA法測定不同干預(yù)條件下DC細(xì)胞上清液活化TGF-β1和總TGF-β1的分泌變化與脂多糖刺激前后的IL-12p70的分泌變化。 實(shí)驗(yàn)數(shù)據(jù)均錄入SPSS17.0統(tǒng)計(jì)軟件包分析,比較各組間均值采用單因素方差分析,以a=0.05為假設(shè)檢驗(yàn)標(biāo)準(zhǔn)。 結(jié)果 1.在體外的環(huán)境下,將小鼠骨髓細(xì)胞分離出來,使用集落刺激因子和白介素4誘導(dǎo)BMDC分化為樹突狀細(xì)胞,通過免疫磁珠的分離,使用流式細(xì)胞儀檢測樹突狀細(xì)胞特征性標(biāo)志CDllc,分離的細(xì)胞純度可達(dá)90%以上,并符合其特征。 2.使用OVA刺激DC后,與空白實(shí)驗(yàn)組相比,總TGF-β1量明顯增加(226.636±40.355vs176.947±23.072,P0.05),活化TGF-p1(43.322±13.479vs35.930土10.108,P0.05)無明顯變化。外泌體組與空白對(duì)照組相比,活化TGF-β1的量(80.532±26.167vs35.930±10.108,P0.05)和總TGF-β1(210.749±31.509vs176.947±23.072,P0.05)量明顯增加,而抗整合素aVβ6抗體組可以阻止這一現(xiàn)象,作為對(duì)照抗體羊抗小鼠IgG組并不能改變此現(xiàn)象。 3.細(xì)胞因子IL-12p70量的變化,在LPS刺激前,各組的分泌量無明顯的變化。在LPS刺激24小時(shí)后,OVA組和空白對(duì)照組相比較,分泌量(327.388±25.005vs348.015±17.272,P0.05)無明顯變化。外泌體組和空白對(duì)照組相比,分泌IL-12p70的能力明顯降低(145.804±11.690vs327.388±25.005,P0.05),抗整合素aVβ6抗體組可以阻止這一現(xiàn)象,作為對(duì)照抗體羊抗小鼠IgG組并不能改變此現(xiàn)象。 結(jié)論 1.OVA和DC共培養(yǎng)后,總TGF-β1量的表達(dá)增加,而活化TGF-β1量無明顯變化。 2.外泌體和DC共培養(yǎng)后,促進(jìn)總TGF-β1量和活化TGF-β1的表達(dá)上調(diào),抗整合素αVβ6抗體組可以阻止活化TGF-β1的表達(dá)上調(diào)這種變化,作為對(duì)照抗體羊抗小鼠IgG組并不能阻止此變化。 3.脂多糖刺激DC前,各組間IL-12p70的表達(dá)無明顯變化。脂多糖刺激DC后,外泌體組與空白對(duì)照組和OVA組相比,IL-12p70的表達(dá)無明顯上調(diào),抗整合素αVβ6抗體組可以阻止這種變化,作為對(duì)照抗體羊抗小鼠IgG組并不能阻止此變化,外泌體組可以有效的抵抗了脂多糖(lipopolysaccharide, LPS)對(duì)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
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