阻断树突状细胞TSLP受体对TH2免疫反应的影响
发布时间：2018-11-18 10:52
【摘要】：背景： 变应性鼻炎药物治疗效果不理想，近年来，围绕增强机体免疫调节、诱发免疫耐受的非特异性免疫治疗逐渐引起了人们的重视。胸腺基质淋巴细胞生成素（TSLP）可以通过刺激树突状细胞（DC）表面的TSLP受体促进DC的活化及成熟，进一步促进TH2免疫反应的发生。那么，阻断TSLP的作用是否可以阻断或抑制TH2反应的进程，，进而诱发免疫耐受而改变变应性疾病的转归呢？我们在体外模拟其进程进行了研究。 目的： 体外建立未成熟树突状细胞（imDC）培养模型，分离总T淋巴细胞，对imDC表面的TSLP受体进行干预后检测共培养上清液细胞因子的变化，试图探讨封闭TSLP受体后的DC在TH1/TH2免疫平衡中，是否可以阻断或抑制TH2反应的进程，进而改变变应性疾病的转归。建立小鼠变应性鼻炎模型，对其进行形态学观察及IL-4检测，为下一步体内实验奠定部分基础。 方法： 取6-8周龄雌性BALB/c小鼠骨髓作为DC来源，粒细胞-巨噬细胞集落刺激因子（GM-CSF），白细胞介素4（IL-4）进行体外增殖，采用半量换液的方法，于培养第六天获得未成熟树突状细胞（imDC），第八天获得成熟树突状细胞（mDC），透射电镜、显微电镜进行形态学检查，流式细胞仪检测表面标记物验证其成熟度。 培养至第六天的imDC分为两组，其中一组加入TSLPR中和抗体进行受体封闭，继续培养两天后分别加入FITC-MHC-II、PE-CD80进行荧光双标，流式细胞仪检测表型。 取6-8周龄雌性BALB/c小鼠脾脏作为T淋巴细胞来源，利用免疫磁珠进行CD3+阳选分离，流式细胞仪检测纯度。 imDC与T淋巴细胞体外共培养，将其分为4组，TSLP刺激组，TSLP刺激并TSLP受体阻断组，卵清白蛋白（OVA）刺激组，卵清白蛋白刺激并TSLPR阻断组，应用ELISA法于共培养2天后分别检测各组细胞培养上清中IL-4、IL-8水平。 20只BALB/c小鼠被随机分为致敏组和对照组，使用卵清蛋白（OVA）诱导建立小鼠变应性鼻炎模型。通过HE染色观察小鼠鼻黏膜的大体重塑情况，吉姆萨染色观察嗜酸性粒细胞，阿辛蓝-过碘酸-希夫染色观察杯状细胞；酶联免疫吸附（ELISA）法检测小鼠血清中白细胞介素-4（IL-4）的水平。 结果： 透射电镜、显微电镜下见细胞形态不规则，表面有较多短小突起，符合未成熟树突状细胞的形态学特征；流式细胞仪检测FITC-CD11c荧光强度为76.83%±1.42%，推断培养的细胞多数为髓样树突状细胞；MHC-II、CD80及MHC-II、CD86双标荧光强度分别为2.61%±0.11%和10.7%±0.23%，推断培养的树突状细胞大部分处于未成熟状态。 流式细胞仪检测两组的FITC-MHC-II、PE-CD80双标荧光强度，两组间差异有统计学意义，推断封闭imDC表面的TSLP受体可以有效的维持其未成熟状态。 利用免疫磁珠进行T淋巴细胞阳选分离后PE-CD3为96.1%±2.8%，推断通过免疫磁珠法可以获得高纯度的总T淋巴细胞。 共培养上清液的ELISA结果显示，TSLP受体阻断组IL-4水平明显低于TSLP刺激组及OVA刺激组，而TSLP受体阻断对细胞培养上清中IL-8表达水平无显著影响。体外阻断TSLP受体后IL-4水平显著降低，说明阻断TSLP受体可一定程度上抑制TH2反应。 小鼠变应性鼻炎模型的生物学行为评分为6.5±1.3，提示造模成功。与对照组相比，致敏组鼻腔黏膜出现上皮细胞脱落、坏死，杯状细胞增生，鳞状上皮化生，固有层和黏膜下层腺体增生、血管扩张，组织水肿，固有层内可见特征性的嗜酸性粒细胞浸润。致敏组小鼠鼻黏膜嗜酸性粒细胞计数及杯状细胞计数分别为（26.4±5.72）和（24.14±3.12），而对照组分别是（8.31±2.42）和（9.41±1.22），两组比较均具有统计学意义；致敏组血清中白细胞介素4（IL-4）水平为（18.9±3.1）pg/ml，对照组为（8.3±1.4）pg/ml，致敏组显著高于对照组，差异有统计学意义。 结论： 体外行TSLP受体阻断可抑制T淋巴细胞向TH2方向转化，这可能为临床变应性鼻炎及哮喘等TH2优势的疾病提供新的治疗策略。
[Abstract]:Background: The treatment effect of allergic rhinitis is not ideal. In recent years, the non-specific immunotherapy around the enhancement of the immune regulation of the body and inducing the immune tolerance gradually causes people The TSLP receptor can stimulate the activation and maturation of the DC by stimulating the TSLP receptor on the surface of the dendritic cell (DC), and further promote the immune response of the TH2. It happens that blocking the TSLP action can block or inhibit the process of the TH2 reaction, thereby inducing immune tolerance to change the rotation of the allergic disease. What's the matter? We're simulating the process in vitro. Research. Objective: To establish an immature dendritic cell (imDC) culture model in vitro, to isolate the total T-lymphocytes and to detect the changes of the cytokines in coculture after the TSLP receptors on the imDC surface. In the TH2 immune equilibrium, it is possible to block or inhibit the process of the TH2 reaction and then to change The model of allergic rhinitis in mice was established, and the morphological observation and IL-4 detection were carried out to the next. experimental base The bone marrow of the 6-8-week-old female BALB/ c mice was used as the DC source, the granulocyte-macrophage colony-stimulating factor (GM-CSF) and the interleukin-4 (IL-4) were used for the in vitro proliferation. Immature dendritic cells (imDC), mature dendritic cells (mDC), transmission electron microscopy (TEM) and micro-electron microscope (TEM) were obtained by flow cytometry. The instrument detects the maturity of the surface marker. The imDC cultured to the sixth day is divided into two groups, one group is added to the TSLPR and the antibody is closed, and the FITC-MHC-II and the PE-CD8 are respectively added after two days of culture. 0. The fluorescent double-label and flow cytometry were used to detect the phenotype. The spleen of the 6-8-week-old female BALB/ c mice was used as the source of T-lymphocytes and the immunomagnetic beads were used. The samples were co-cultured with T-lymphocytes and divided into 4 groups, TSLP-stimulated group, TSLP-stimulated and TSLP-receptor blocking group, ovalbumin (OVA) stimulation group, ovalbumin-stimulated and TSLPR blocking group. 2 days after co-culture The levels of IL-4 and IL-8 in the supernatant of each group were not detected. 20 BALB/ c mice were randomly divided into the group-sensitive group and the control group. A model of mouse allergic rhinitis was established by using ovalbumin (OVA). The general remodeling of the nasal mucosa of mice was observed by HE staining. A) Act The level of interleukin-4 (IL-4) in the serum of the mice was detected. The results showed that the morphology of the cells was irregular under the microscope and the microscopic electron microscope, and the surface had more short protrusions, which met the morphological characteristics of the immature dendritic cells. The fluorescence intensity of FITC-CD11c was detected by flow cytometry. The fluorescence intensity of MHC-II, CD80 and MHC-II and CD86 was 2.61% and 0.11%, respectively. The fluorescence intensity of FITC-MHC-II and PE-CD80 in both groups was detected by flow cytometry. To study the significance of the study, it is concluded that the TSLP receptor on the surface of the closed imDC can effectively maintain its immature state. The results of ELISA showed that the level of IL-4 in TSLP receptor blocking group was lower than that of TSLP. The effect of TSLP receptor blockade on the level of IL-8 expression in the supernatant of cell culture was not significantly affected by the stimulation group and the OVA-stimulated group. The level of IL-4 after TSLP receptor is significantly reduced, indicating that the blocking of TSLP receptor can be a certain extent. In response to the inhibition of TH2, the biological behavior of the model of allergic rhinitis in mice was 6. 5-1.3, suggesting that the model was successful. In contrast to the control group, the epithelial cells of the nasal mucosa of the sensitive group were shed, necrosis, the proliferation of the cup-like cells, and the squamous epithelium. The number of eosinophils in the nasal mucosa of the sensitive group and the count of the cup-like cells were (26. 4, 5.72) and (24.14, 3.12), respectively. The levels of interleukin4 (IL-4) in the serum of the sensitive group were (18.9% 3.1). pg/ Results: TSLP receptor in vitro was found to be higher in the control group than in the control group.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R765.21
本文编号：2339832
[Abstract]:Background: The treatment effect of allergic rhinitis is not ideal. In recent years, the non-specific immunotherapy around the enhancement of the immune regulation of the body and inducing the immune tolerance gradually causes people The TSLP receptor can stimulate the activation and maturation of the DC by stimulating the TSLP receptor on the surface of the dendritic cell (DC), and further promote the immune response of the TH2. It happens that blocking the TSLP action can block or inhibit the process of the TH2 reaction, thereby inducing immune tolerance to change the rotation of the allergic disease. What's the matter? We're simulating the process in vitro. Research. Objective: To establish an immature dendritic cell (imDC) culture model in vitro, to isolate the total T-lymphocytes and to detect the changes of the cytokines in coculture after the TSLP receptors on the imDC surface. In the TH2 immune equilibrium, it is possible to block or inhibit the process of the TH2 reaction and then to change The model of allergic rhinitis in mice was established, and the morphological observation and IL-4 detection were carried out to the next. experimental base The bone marrow of the 6-8-week-old female BALB/ c mice was used as the DC source, the granulocyte-macrophage colony-stimulating factor (GM-CSF) and the interleukin-4 (IL-4) were used for the in vitro proliferation. Immature dendritic cells (imDC), mature dendritic cells (mDC), transmission electron microscopy (TEM) and micro-electron microscope (TEM) were obtained by flow cytometry. The instrument detects the maturity of the surface marker. The imDC cultured to the sixth day is divided into two groups, one group is added to the TSLPR and the antibody is closed, and the FITC-MHC-II and the PE-CD8 are respectively added after two days of culture. 0. The fluorescent double-label and flow cytometry were used to detect the phenotype. The spleen of the 6-8-week-old female BALB/ c mice was used as the source of T-lymphocytes and the immunomagnetic beads were used. The samples were co-cultured with T-lymphocytes and divided into 4 groups, TSLP-stimulated group, TSLP-stimulated and TSLP-receptor blocking group, ovalbumin (OVA) stimulation group, ovalbumin-stimulated and TSLPR blocking group. 2 days after co-culture The levels of IL-4 and IL-8 in the supernatant of each group were not detected. 20 BALB/ c mice were randomly divided into the group-sensitive group and the control group. A model of mouse allergic rhinitis was established by using ovalbumin (OVA). The general remodeling of the nasal mucosa of mice was observed by HE staining. A) Act The level of interleukin-4 (IL-4) in the serum of the mice was detected. The results showed that the morphology of the cells was irregular under the microscope and the microscopic electron microscope, and the surface had more short protrusions, which met the morphological characteristics of the immature dendritic cells. The fluorescence intensity of FITC-CD11c was detected by flow cytometry. The fluorescence intensity of MHC-II, CD80 and MHC-II and CD86 was 2.61% and 0.11%, respectively. The fluorescence intensity of FITC-MHC-II and PE-CD80 in both groups was detected by flow cytometry. To study the significance of the study, it is concluded that the TSLP receptor on the surface of the closed imDC can effectively maintain its immature state. The results of ELISA showed that the level of IL-4 in TSLP receptor blocking group was lower than that of TSLP. The effect of TSLP receptor blockade on the level of IL-8 expression in the supernatant of cell culture was not significantly affected by the stimulation group and the OVA-stimulated group. The level of IL-4 after TSLP receptor is significantly reduced, indicating that the blocking of TSLP receptor can be a certain extent. In response to the inhibition of TH2, the biological behavior of the model of allergic rhinitis in mice was 6. 5-1.3, suggesting that the model was successful. In contrast to the control group, the epithelial cells of the nasal mucosa of the sensitive group were shed, necrosis, the proliferation of the cup-like cells, and the squamous epithelium. The number of eosinophils in the nasal mucosa of the sensitive group and the count of the cup-like cells were (26. 4, 5.72) and (24.14, 3.12), respectively. The levels of interleukin4 (IL-4) in the serum of the sensitive group were (18.9% 3.1). pg/ Results: TSLP receptor in vitro was found to be higher in the control group than in the control group.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R765.21
【引证文献】
相关期刊论文 前2条
1 田阳;饶欢;薛文通;;花生过敏原检测、降敏加工及脱敏治疗研究进展[J];食品工业科技;2017年18期
2 姚娟;郑晓汾;李冰;;胸腺基质淋巴细胞生成素在过敏性结膜炎发病中的作用[J];国际眼科纵览;2015年03期
相关硕士学位论文 前1条
1 姚娟;TSLP、IL-13在不同类型过敏性结膜炎患者眼表的表达[D];山西医科大学;2015年
本文编号：2339832
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