非典型蛋白激酶C对Th17细胞免疫调节作用的研究

发布时间：2018-04-25 20:31

本文选题：PKCλ/ι + Th17细胞　；参考：《江苏省血吸虫病防治研究所》2017年硕士论文

【摘要】：免疫调节在免疫生理及免疫病理中起着非常重要的作用。当今免疫学的热点,如自身免疫、肿瘤免疫和变态反应等,均与免疫调节失衡有关。Th17细胞、调节性T细胞(regulatory T cell,Treg)、自然杀伤性T细胞(natural killer T cell,NKT)、滤泡辅助性T细胞(T follicular helper,Tfh)及其分泌的细胞因子(cytokine)、化学趋化因子(chemokine)等,均是参与免疫调节的重要细胞与介质。我们在国际上首次证实了非典型蛋白激酶Cλ/ι(PKCλ/ι)是一个重要的信号转导分子,参与调节Th2细胞的分化及功能,并在T细胞极化中起重要作用。蛋白激酶C(protein kinase C,PKC)广泛存在于机体组织及细胞中,在跨膜信号传递过程中起着重要作用。PKC分为三组:传统PKC(classical PKC,c PKC),包括α、βI、βⅡ和γ亚类;新型PKC(novel PKC,n PKC),包括δ、ε、η和θ亚类;非典型PKC(atypical PKC,a PKC),由PKCζ和PKCλ/ι亚类组成。PKC参与多种生理与病理反应,在肿瘤免疫、过敏性炎症、糖尿病等的发病机理中起重要的作用,已成为新药开发的靶标(therapeutic target)。已经证实非典型蛋白激酶Cλ/ι(protein kinase Cλ/ι,PKCλ/ι)参与调节Th2细胞的分化及功能,并在T细胞的极化中起重要作用。它们广泛参与多种信号转导通路,与寄生虫感染、过敏性炎症、肿瘤等的发生发展密切相关。Th17细胞是新近发现的CD4+T细胞亚群,区别于传统的Th1/Th2细胞,并与之起相互拮抗作用。Th17细胞具有独立的分化调节途径,发挥着重要的免疫调节与免疫效应功能。Th17细胞能分泌IL-17(也称为IL-17A),IL-17F,IL-21和IL-22等效应性细胞因子。IL-17和IL-22细胞因子的受体广泛存在于多种组织,如上皮组织中。因此这两种细胞因子在免疫系统和组织细胞的相互联络中起关键作用。越来越多的研究表明,Th17细胞在过敏性气道炎症中发挥重要作用。Th17细胞在过敏性哮喘、感染及自身免疫中起重要的免疫病理作用,已被视为开发治疗以上疾病新药的靶标。有关PKCλ/ι对Th17细胞的作用,国内外尚未有报道,属空白领域。为此,本研究应用PKCλ/ι条件性基因敲除鼠,开创性地研究PKCλ/ι对Th17细胞分化及功能的作用,阐明其分子机理。并探讨PKCλ/ι-Th17轴(axis)对过敏性炎症的调控作用。第一部分PKCλ/ι基因缺损对Th17细胞体外分化及细胞因子分泌的调节作用目的:探讨PKCλ/ι对Th17细胞体外分化及细胞因子分泌的调节作用。拟运用活化T细胞特异的PKCλ/ι条件性基因敲除鼠与对照鼠,分离纯化其CD4+T细胞。采用非极化及Th17极化体外培养体系,探讨PKCλ/ι缺损是否影响Th17细胞的分化及效应细胞因子的分泌等。方法:选用PKCλ/ιflx/flxOX40Cre+(以下简称KO)及对照鼠PKCλ/ιflx/flxOX40Cre-(WT),采用全自动磁性细胞分离技术(Auto MACS Pro)从脾脏中纯化初始(na?ve)CD4+T细胞。纯化的CD4+T细胞用Anti-CD3/CD28刺激作体外非极化培养、Th17/Treg极化条件下培养。培养上清中细胞因子含量用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)、培养细胞用流式细胞技术(fluorescence-activated cell sorting,FACS)作细胞内细胞因子染色(intracellular cytokine staining)。例如检测Th2、Th17、Treg细胞的数量及细胞因子分泌,用流式细胞仪分别作细胞内IL-4、IL-17、CD25/Foxp3染色,并测定培养上清中三种细胞所对应的代表性效应细胞因子IL-4/IL-13、IL-17A/IL-17F、TGF-β1/IL-10等。结果:PKCλ/ι缺损型CD4+T细胞在体外Th17极化条件(TGF-β+IL-6),培养4天,经再刺激后IL-17分泌细胞(Th17细胞)明显低于WT型CD4+T细胞!在体外Th17非极化条件下(anti-CD3/CD28刺激),培养上清中PKCλ/ι敲除鼠IL-17细胞因子水平明显降低;在体外Th17极化条件下,PKCλ/ι敲除鼠IL-17细胞因子水平明显降低。在脾细胞培养上清中(anti-CD3/CD28刺激),PKCλ/ι缺损组IL-17细胞因子水平明显降低;在体外Th17极化条件下,PKCλ/ι缺损组的IL-17细胞因子水平也是明显降低的,这与体内的结果一致。结论:PKCλ/ι能调控Th17细胞体外的分化及细胞因子的分泌。第二部分PKCλ/ι基因缺损对Th17体内功能的影响目的:研究PKCλ/ι缺损对Th17细胞体内功能的影响;我们将研究活化T细胞特异的PKCλ/ι条件性基因敲除对小鼠哮喘模型的影响。方法:建立尘螨(house dust mite,HDM)诱导的小鼠哮喘模型,应用PKCλ/ι条件性基因敲除鼠(PKCλ/ιflx/flxCreoxtuv,以下简称KO)和野生型对照鼠(PKCλ/ιflx/flxCreoxtuw,以下简称WT)。收集小鼠支气管肺泡洗液(Bronchoalveolar lavage,BAL),取肺组织(按其生理分页),分别做石蜡病理切片(HE),Kwik-Diff染色,以检测气道局部炎症浸润细胞;用ELISA及实时荧光定量PCR分别测定BAL液及肺组织中多种细胞因子/趋化因子,如IL-4,IL-17,IFN-γ,Gob-5,Eotaxin等。结果:本课题研究了PKCλ/ι敲除对HDM诱导的过敏性气道局部炎症的作用。气道炎症的一大特征就是CD4+T细胞、巨噬细胞、嗜中性粒细胞以及嗜酸性粒细胞的浸润,继而导致气道高反应性和结构改变。在病理学切片结果中,PKCλ/ι敲除后,浸润肺支气管组织局部的嗜酸性粒细胞、中性粒细胞都明显减少;与此相对应,实时荧光定量PCR检测结果则表明,肺组织中的IL-4、IL-17、Eotaxin、Gob-5等炎症相关的细胞因子m RNA水平的表达明显下调。支气管肺泡洗液(BAL)的ELISA结果显示,PKCλ/ι缺损对HDM诱导的哮喘炎症相关因子造成一定的影响,其中Th2细胞因子IL-4、IL-5与Th17细胞因子IL-17以及与炎症相关的Eotaxin等都受到了抑制。结论:本课题运用PKCλ/ι条件性基因敲除鼠,成功建立了HDM诱导的小鼠哮喘模型。PKCλ/ι条件性基因敲除能明显抑制HDM诱导的过敏性气道炎症,表现为支气管及肺组织中炎症浸润细胞,特别是中性粒细胞数目减少,Th2及Th17效应细胞因子下调。PKCλ/ι能调控体内Th17的功能。本研究是在国际上首次运用PKCλ/ι条件性基因敲除鼠探讨PKCλ/ι-Th17轴对过敏性气道炎症的作用。相关分子机理的深入研究正在进行中。
[Abstract]:Immunoregulation plays a very important role in immunological and immunological pathology. Today's immunological hotspots, such as autoimmune, tumor immunity and allergy, are related to.Th17 cells, regulatory T cells (regulatory T cell, Treg), natural killer T cells (natural killer T cell, NKT), follicular auxiliary fine T follicular helper (Tfh) and its secreted cytokine (cytokine) and chemical chemokines (chemokine) are important cells and mediators involved in immunomodulation. We have first confirmed that the atypical protein kinase C lambda / PKC [PKC lambda] is an important signal transduction molecule, and is involved in regulating the differentiation and function of Th2 cells. Protein kinase C (PKC), which plays an important role in the tissue and cells of the body, plays an important role in the transmembrane signaling process, and.PKC is divided into three groups: the traditional PKC (classical PKC, C PKC), including the alpha, beta, beta, and gamma subclasses, including the Delta, epsilon, ETA and theta subclasses; the atypical protein KC (atypical PKC, a PKC), composed of PKC zeta and PKC lambda / subclass.PKC to participate in a variety of physiological and pathological reactions, plays an important role in the pathogenesis of tumor immunity, allergic inflammation, diabetes and so on. It has become a target for the development of new drugs (therapeutic target). And regulate the differentiation and function of Th2 cells and play an important role in the polarization of T cells. They are widely involved in a variety of signal transduction pathways, closely related to the occurrence and development of parasitic infection, allergic inflammation, and tumor..Th17 cells are newly discovered CD4+T cell subgroups, different from the traditional Th1/Th2 cells, and are antagonistic to each other. .Th17 cells have an independent pathway for differentiation and regulation, playing an important immunoregulation and immune effect function.Th17 cells can secrete IL-17 (also known as IL-17A), IL-17F, IL-21 and IL-22 and other effector cytokines.IL-17 and IL-22 cytokine receptors are widely distributed in a variety of groups, such as epithelial tissue. Therefore, these two cytokines are A growing number of studies have shown that Th17 cells play an important role in allergic airway inflammation, and.Th17 cells play an important immuno pathological role in allergic asthma, infection and autoimmunity, and have been considered as a target for the development of new drugs for the treatment of the above diseases. PKC [lambda] / T The role of H17 cells, which is not reported at home and abroad, is a blank field. Therefore, this study uses PKC lambda / conditional gene knockout mice to explore the role of PKC lambda / Th17 on the differentiation and function of Th17 cells, elucidate its molecular mechanism and explore the regulatory role of PKC lambda / -Th17 axis (axis) on allergic inflammation. The aim of the regulation of Th17 cells in vitro differentiation and cytokine secretion was to explore the regulation of PKC lambda / Th17 on the differentiation and cytokine secretion of Th17 cells. The CD4+T cells were isolated and purified by using the activated T cell specific PKC lambda / conditioned gene knockout mice and the control rats. To discuss whether the PKC lambda / flx/flxOX40Cre+ defect affects the differentiation of Th17 cells and the secretion of effector cytokine. Methods: PKC lambda / flx/flxOX40Cre+ (hereinafter referred to as KO) and the control rat PKC lambda / flx/flxOX40Cre- (WT) were used to purify the initial (Na?) cells from the spleen by the full automatic magnetic cell separation technique (Auto MACS Pro). CD3/CD28 stimulation was used in non polarizing culture in vitro and cultured in Th17/Treg polarization condition. The content of cytokine in the culture supernatant was detected by enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA). The cultured cells were stained by flow cytometry (fluorescence-activated cell sorting, FACS) for intracellular cytokine staining (intracellular cytokin). E staining). For example, detecting the number of Th2, Th17, Treg cells and cytokine secretion, using a flow cytometry to stain the intracellular IL-4, IL-17, CD25/Foxp3, and determine the representative effect of the three cells in the culture supernatant, IL-4/IL-13, IL-17A/IL-17F, TGF- beta 1/IL-10 and so on. The Th17 polarization condition (TGF- beta +IL-6) was cultured for 4 days, and the IL-17 secretory cells (Th17 cells) were significantly lower than WT CD4+T cells after the stimulation. Under the non polarization condition of Th17 in vitro (anti-CD3/CD28 stimulation), the level of IL-17 cytokine in the culture supernatant was significantly lower than that of the PKC lambda / knockout mouse. The level of IL-17 cytokine in the PKC lambda / Th17 defect group decreased obviously in the splenocytes culture supernatant (anti-CD3/CD28 stimulation), and the level of IL-17 cytokine in the PKC lambda / defect group was also significantly reduced under the condition of Th17 polarization in vitro, which was in accordance with the results in the body. Conclusion: PKC lambda / gland can regulate the differentiation and fine of Th17 cells in vitro. The effect of PKC lambda / PKC gene defect on the function of Th17 in vivo. Objective: To study the effect of PKC lambda / defect on the function of Th17 cells; we will study the effect of activated T cell specific PKC lambda / conditional knockout on the model of asthma in mice. Method: mouse asthma (house dust mite, HDM) induced asthma in mice. Model, PKC lambda / conditional gene knockout rat (PKC lambda / flx/flxCreoxtuv, hereinafter referred to as KO) and wild type control rats (PKC lambda / flx/flxCreoxtuw, hereinafter referred to as WT) were used to collect mouse bronchoalveolar lotion (Bronchoalveolar lavage, BAL), take the lung tissue (according to its pagination), to make paraffin pathological sections (HE), Kwik-Diff staining, for detection. Local inflammatory infiltration cells in the airway; ELISA and real-time fluorescence quantitative PCR were used to determine a variety of cytokines / chemotactic factors in BAL solution and lung tissue, such as IL-4, IL-17, IFN- gamma, Gob-5, Eotaxin, etc.. Results: this subject studied the effect of PKC lambda / Gob-5, Eotaxin on the allergic airway local inflammation induced by HDM. The infiltration of macrophages, macrophages, neutrophils, and eosinophils led to hyperresponsiveness and structural changes in the airway. In the pathological section, PKC lambda / inhibitor knockout, the local eosinophils infiltrated in the lung bronchus, and neutrophils were significantly reduced; corresponding to this, real-time fluorescence quantitative PCR detection results The expression of M RNA levels of IL-4, IL-17, Eotaxin, Gob-5, and other inflammatory cytokines in the lung tissue was obviously downregulated. The ELISA results of bronchial alveolar lotion (BAL) showed that PKC lambda / Gob-5 defects had a certain effect on HDM induced asthma related factors, including Th2 cytokine IL-4. The related Eotaxin and so on were suppressed. Conclusion: this subject uses PKC lambda / conditional gene knockout mice, and successfully established the HDM induced mouse asthma model.PKC lambda / conditional knockout can obviously inhibit the allergic airway inflammation induced by HDM, which is manifested in the inflammatory infiltrating cells in the bronchial and lung tissues, especially the number of neutrophils. Decrease, Th2 and Th17 effect cytokines down regulation of.PKC lambda / Th17 can regulate the function of Th17 in vivo. This study is the first time to use PKC lambda / conditional knockout rat to explore the effect of PKC lambda / -Th17 axis on allergic airway inflammation. The in-depth study of the molecular mechanism is under way.

【学位授予单位】：江苏省血吸虫病防治研究所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R56

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