本文選題：PKCλ/ι + Th17細(xì)胞��； 參考：《江蘇省血吸蟲病防治研究所》2017年碩士論文

【摘要】：免疫調(diào)節(jié)在免疫生理及免疫病理中起著非常重要的作用。當(dāng)今免疫學(xué)的熱點(diǎn),如自身免疫、腫瘤免疫和變態(tài)反應(yīng)等,均與免疫調(diào)節(jié)失衡有關(guān)。Th17細(xì)胞、調(diào)節(jié)性T細(xì)胞(regulatory T cell,Treg)、自然殺傷性T細(xì)胞(natural killer T cell,NKT)、濾泡輔助性T細(xì)胞(T follicular helper,Tfh)及其分泌的細(xì)胞因子(cytokine)、化學(xué)趨化因子(chemokine)等,均是參與免疫調(diào)節(jié)的重要細(xì)胞與介質(zhì)。我們在國際上首次證實(shí)了非典型蛋白激酶Cλ/ι(PKCλ/ι)是一個重要的信號轉(zhuǎn)導(dǎo)分子,參與調(diào)節(jié)Th2細(xì)胞的分化及功能,并在T細(xì)胞極化中起重要作用。蛋白激酶C(protein kinase C,PKC)廣泛存在于機(jī)體組織及細(xì)胞中,在跨膜信號傳遞過程中起著重要作用。PKC分為三組:傳統(tǒng)PKC(classical PKC,c PKC),包括α、βI、βⅡ和γ亞類;新型PKC(novel PKC,n PKC),包括δ、ε、η和θ亞類;非典型PKC(atypical PKC,a PKC),由PKCζ和PKCλ/ι亞類組成。PKC參與多種生理與病理反應(yīng),在腫瘤免疫、過敏性炎癥、糖尿病等的發(fā)病機(jī)理中起重要的作用,已成為新藥開發(fā)的靶標(biāo)(therapeutic target)。已經(jīng)證實(shí)非典型蛋白激酶Cλ/ι(protein kinase Cλ/ι,PKCλ/ι)參與調(diào)節(jié)Th2細(xì)胞的分化及功能,并在T細(xì)胞的極化中起重要作用。它們廣泛參與多種信號轉(zhuǎn)導(dǎo)通路,與寄生蟲感染、過敏性炎癥、腫瘤等的發(fā)生發(fā)展密切相關(guān)。Th17細(xì)胞是新近發(fā)現(xiàn)的CD4+T細(xì)胞亞群,區(qū)別于傳統(tǒng)的Th1/Th2細(xì)胞,并與之起相互拮抗作用。Th17細(xì)胞具有獨(dú)立的分化調(diào)節(jié)途徑,發(fā)揮著重要的免疫調(diào)節(jié)與免疫效應(yīng)功能。Th17細(xì)胞能分泌IL-17(也稱為IL-17A),IL-17F,IL-21和IL-22等效應(yīng)性細(xì)胞因子。IL-17和IL-22細(xì)胞因子的受體廣泛存在于多種組織,如上皮組織中。因此這兩種細(xì)胞因子在免疫系統(tǒng)和組織細(xì)胞的相互聯(lián)絡(luò)中起關(guān)鍵作用。越來越多的研究表明,Th17細(xì)胞在過敏性氣道炎癥中發(fā)揮重要作用。Th17細(xì)胞在過敏性哮喘、感染及自身免疫中起重要的免疫病理作用,已被視為開發(fā)治療以上疾病新藥的靶標(biāo)。有關(guān)PKCλ/ι對Th17細(xì)胞的作用,國內(nèi)外尚未有報(bào)道,屬空白領(lǐng)域。為此,本研究應(yīng)用PKCλ/ι條件性基因敲除鼠,開創(chuàng)性地研究PKCλ/ι對Th17細(xì)胞分化及功能的作用,闡明其分子機(jī)理。并探討PKCλ/ι-Th17軸(axis)對過敏性炎癥的調(diào)控作用。第一部分PKCλ/ι基因缺損對Th17細(xì)胞體外分化及細(xì)胞因子分泌的調(diào)節(jié)作用目的:探討PKCλ/ι對Th17細(xì)胞體外分化及細(xì)胞因子分泌的調(diào)節(jié)作用。擬運(yùn)用活化T細(xì)胞特異的PKCλ/ι條件性基因敲除鼠與對照鼠,分離純化其CD4+T細(xì)胞。采用非極化及Th17極化體外培養(yǎng)體系,探討PKCλ/ι缺損是否影響Th17細(xì)胞的分化及效應(yīng)細(xì)胞因子的分泌等。方法:選用PKCλ/ιflx/flxOX40Cre+(以下簡稱KO)及對照鼠PKCλ/ιflx/flxOX40Cre-(WT),采用全自動磁性細(xì)胞分離技術(shù)(Auto MACS Pro)從脾臟中純化初始(na?ve)CD4+T細(xì)胞。純化的CD4+T細(xì)胞用Anti-CD3/CD28刺激作體外非極化培養(yǎng)、Th17/Treg極化條件下培養(yǎng)。培養(yǎng)上清中細(xì)胞因子含量用酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immunosorbent assay,ELISA)、培養(yǎng)細(xì)胞用流式細(xì)胞技術(shù)(fluorescence-activated cell sorting,FACS)作細(xì)胞內(nèi)細(xì)胞因子染色(intracellular cytokine staining)。例如檢測Th2、Th17、Treg細(xì)胞的數(shù)量及細(xì)胞因子分泌,用流式細(xì)胞儀分別作細(xì)胞內(nèi)IL-4、IL-17、CD25/Foxp3染色,并測定培養(yǎng)上清中三種細(xì)胞所對應(yīng)的代表性效應(yīng)細(xì)胞因子IL-4/IL-13、IL-17A/IL-17F、TGF-β1/IL-10等。結(jié)果:PKCλ/ι缺損型CD4+T細(xì)胞在體外Th17極化條件(TGF-β+IL-6),培養(yǎng)4天,經(jīng)再刺激后IL-17分泌細(xì)胞(Th17細(xì)胞)明顯低于WT型CD4+T細(xì)胞!在體外Th17非極化條件下(anti-CD3/CD28刺激),培養(yǎng)上清中PKCλ/ι敲除鼠IL-17細(xì)胞因子水平明顯降低;在體外Th17極化條件下,PKCλ/ι敲除鼠IL-17細(xì)胞因子水平明顯降低。在脾細(xì)胞培養(yǎng)上清中(anti-CD3/CD28刺激),PKCλ/ι缺損組IL-17細(xì)胞因子水平明顯降低;在體外Th17極化條件下,PKCλ/ι缺損組的IL-17細(xì)胞因子水平也是明顯降低的,這與體內(nèi)的結(jié)果一致。結(jié)論:PKCλ/ι能調(diào)控Th17細(xì)胞體外的分化及細(xì)胞因子的分泌。第二部分PKCλ/ι基因缺損對Th17體內(nèi)功能的影響目的:研究PKCλ/ι缺損對Th17細(xì)胞體內(nèi)功能的影響;我們將研究活化T細(xì)胞特異的PKCλ/ι條件性基因敲除對小鼠哮喘模型的影響。方法:建立塵螨(house dust mite,HDM)誘導(dǎo)的小鼠哮喘模型,應(yīng)用PKCλ/ι條件性基因敲除鼠(PKCλ/ιflx/flxCreoxtuv,以下簡稱KO)和野生型對照鼠(PKCλ/ιflx/flxCreoxtuw,以下簡稱WT)。收集小鼠支氣管肺泡洗液(Bronchoalveolar lavage,BAL),取肺組織(按其生理分頁),分別做石蠟病理切片(HE),Kwik-Diff染色,以檢測氣道局部炎癥浸潤細(xì)胞;用ELISA及實(shí)時熒光定量PCR分別測定BAL液及肺組織中多種細(xì)胞因子/趨化因子,如IL-4,IL-17,IFN-γ,Gob-5,Eotaxin等。結(jié)果:本課題研究了PKCλ/ι敲除對HDM誘導(dǎo)的過敏性氣道局部炎癥的作用。氣道炎癥的一大特征就是CD4+T細(xì)胞、巨噬細(xì)胞、嗜中性粒細(xì)胞以及嗜酸性粒細(xì)胞的浸潤,繼而導(dǎo)致氣道高反應(yīng)性和結(jié)構(gòu)改變。在病理學(xué)切片結(jié)果中,PKCλ/ι敲除后,浸潤肺支氣管組織局部的嗜酸性粒細(xì)胞、中性粒細(xì)胞都明顯減少;與此相對應(yīng),實(shí)時熒光定量PCR檢測結(jié)果則表明,肺組織中的IL-4、IL-17、Eotaxin、Gob-5等炎癥相關(guān)的細(xì)胞因子m RNA水平的表達(dá)明顯下調(diào)。支氣管肺泡洗液(BAL)的ELISA結(jié)果顯示,PKCλ/ι缺損對HDM誘導(dǎo)的哮喘炎癥相關(guān)因子造成一定的影響,其中Th2細(xì)胞因子IL-4、IL-5與Th17細(xì)胞因子IL-17以及與炎癥相關(guān)的Eotaxin等都受到了抑制。結(jié)論:本課題運(yùn)用PKCλ/ι條件性基因敲除鼠,成功建立了HDM誘導(dǎo)的小鼠哮喘模型。PKCλ/ι條件性基因敲除能明顯抑制HDM誘導(dǎo)的過敏性氣道炎癥,表現(xiàn)為支氣管及肺組織中炎癥浸潤細(xì)胞,特別是中性粒細(xì)胞數(shù)目減少,Th2及Th17效應(yīng)細(xì)胞因子下調(diào)。PKCλ/ι能調(diào)控體內(nèi)Th17的功能。本研究是在國際上首次運(yùn)用PKCλ/ι條件性基因敲除鼠探討PKCλ/ι-Th17軸對過敏性氣道炎癥的作用。相關(guān)分子機(jī)理的深入研究正在進(jìn)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.

【學(xué)位授予單位】：江蘇省血吸蟲病防治研究所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R56

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