IL-33促進(jìn)Th2應(yīng)答和抑制Th17應(yīng)答的效應(yīng)能夠緩解小鼠缺血性腦損傷
發(fā)布時(shí)間：2018-08-07 13:53
【摘要】：白細(xì)胞介素(Interleukin, IL)-33是近年來發(fā)現(xiàn)的IL-1家族新成員,它作為細(xì)胞因子,可通過IL-33/ST2信號(hào)通路促進(jìn)Th2型免疫反應(yīng),同時(shí)也是一種核蛋白,可參與調(diào)控基因轉(zhuǎn)錄。在炎性刺激下,IL-33還可以作為一種“警報(bào)素”警示組織損傷或應(yīng)激。IL-33是ST2受體的唯一特異性配體,其生物學(xué)活性的發(fā)揮主要依賴與跨膜型ST2受體(ST2L)結(jié)合。IL-33調(diào)節(jié)免疫反應(yīng)的作用具有多樣性,取決于不同的疾病和模型。IL-33可促進(jìn)Th2型炎癥性疾病的發(fā)病,如哮喘、特應(yīng)性皮炎和過敏反應(yīng)等,然而在一些與Th2型免疫反應(yīng)相關(guān)的疾病或模型,如動(dòng)脈粥樣硬化、多發(fā)性硬化和實(shí)驗(yàn)性腸炎等,IL-33可通過促進(jìn)Th1/Th2平衡向Th2偏移而起到保護(hù)性作用。此外,新近研究還發(fā)現(xiàn)了IL-33可調(diào)節(jié)Th17型免疫應(yīng)答。IL-33在多組織器官高表達(dá),而腦和脊髓是IL-33表達(dá)量最高的器官之一,提示IL-33在調(diào)節(jié)中樞神經(jīng)系統(tǒng)的病理生理學(xué)過程中可能起著重要作用。近年來,有關(guān)IL-33或IL-33/ST2信號(hào)通路在中樞神經(jīng)系統(tǒng)疾病中的作用是神經(jīng)免疫學(xué)的研究熱點(diǎn)之一。 缺血性腦卒中是中老年人常見的腦血管疾病,可以導(dǎo)致腦組織損傷并伴隨炎癥反應(yīng),從而引發(fā)一系列癥候群。研究表明,Th1/Th2介導(dǎo)的免疫應(yīng)答以及Th17型免疫應(yīng)答在缺血性腦損傷的炎性病理機(jī)制中發(fā)揮重要作用。IL-33作為Th2型免疫反應(yīng)的關(guān)鍵活化分子,在缺血性腦卒中發(fā)病機(jī)制中的作用仍有待闡明。為了探討IL-33在缺血性腦卒中發(fā)病過程中的作用及其機(jī)制,本研究利用缺血性腦卒中動(dòng)物模型,觀察缺血性腦損傷時(shí)IL-33及其受體ST2L表達(dá)的變化規(guī)律,并探討IL-33對(duì)缺血性腦損傷程度的影響。論文共分為三個(gè)部分,主要內(nèi)容和結(jié)果如下： 一、短暫性腦缺血后IL-33及其受體ST2L的表達(dá) 目的：檢測IL-33及其受體ST2L在短暫性腦缺血后不同時(shí)程和不同腦區(qū)的表達(dá)情況,探討缺血性腦損傷時(shí)IL-33及其受體ST2L表達(dá)的規(guī)律性。 方法：利用線栓法閉塞大腦中動(dòng)脈(MCAO)30min誘導(dǎo)建立小鼠可逆性局灶性腦缺血再灌注損傷模型,通過半定量RT-PCR和Western blot檢測缺血再灌注后超急性期(6h)、急性期(24h)和延遲期(3d)缺血側(cè)腦組織IL-33mRNA和蛋白表達(dá)水平,并通過免疫組織化學(xué)染色觀察了IL-33在不同缺血腦區(qū)(運(yùn)動(dòng)皮層、感覺皮層、海馬和紋狀體)各時(shí)間點(diǎn)的表達(dá)情況。此外,對(duì)IL-33的受體ST2L mRNA表達(dá)水平也進(jìn)行了檢測。 結(jié)果：與假手術(shù)組相比,IL-33mRNA和蛋白水平在MCAO6h和3d表達(dá)減少,ST2L mRNA表達(dá)水平在MCAO24h和3d表達(dá)減少,其它時(shí)間點(diǎn)IL-33和ST2L表達(dá)無顯著改變。除了MCAO24h組運(yùn)動(dòng)皮層和紋狀體陽性染色增加外,IL-33陽性細(xì)胞數(shù)在缺血后不同時(shí)程各腦區(qū)均有不同程度減少。 結(jié)論：MCAO模型小鼠缺血再灌注后IL-33和ST2L表達(dá)的總體趨勢是下降的,提示短暫性腦缺血后IL-33/ST2L介導(dǎo)的Th2型免疫應(yīng)答是下調(diào)的。 二、側(cè)腦室注射IL-33對(duì)MCAO模型腦損傷的影響 目的：觀察外源性IL-33作用于MCAO模型后,對(duì)缺血性腦損傷嚴(yán)重程度、腦組織炎癥及脾臟免疫功能的影響。 方法：利用GST表達(dá)純化系統(tǒng)構(gòu)建小鼠IL-33成熟肽的重組質(zhì)粒,并純化制備具有生物學(xué)活性的IL-33蛋白。在小鼠MCAO模型前30min側(cè)腦室注射IL-33(2μg,5μl)或PBS5μl,于缺血再灌注后不同時(shí)間點(diǎn)(6h,24h和3d)通過Zea Longa評(píng)分法評(píng)估神經(jīng)行為學(xué)癥狀,TTC染色法評(píng)估腦梗死體積,HE染色觀察缺血側(cè)海馬及皮層炎性細(xì)胞浸潤情況,并觀察脾臟大小的改變。 結(jié)果：與側(cè)腦室注射PBS的MCAO對(duì)照組相比,側(cè)腦室注射IL-33可減輕MCAO24h和MCAO3d的神經(jīng)行為學(xué)癥狀(24h:2.0±0.302vs2.875±0.125;3d:1.833±0.167vs2.917±0.193；p0.05)和腦梗死體積(24h:21.89±4.8%vs36.27±1.6%;3d:8.52±4.175vs37.08±3.35%；p0.05).組織學(xué)染色顯示側(cè)腦室注射IL-33的MCAO24h和MCAO3d缺血側(cè)海馬和皮層炎性細(xì)胞浸潤數(shù)量較少。此外,側(cè)腦室注射IL-33也增加了MCAO24h和MCAO3d的脾臟指數(shù)(24h:0.36±0.02vs0.31±0.04;3d:0.30±0.05vs0.22±0.01;p0.05),脾臟萎縮不明顯。與對(duì)照組相比,側(cè)腦室注射IL-33對(duì)MCAO6h組的神經(jīng)行為學(xué)癥狀、腦梗死體積、腦組織炎性浸潤、脾臟指數(shù)及大小均無明顯差異。 結(jié)論：側(cè)腦室注射IL-33可減緩短暫性腦缺血再灌注后急性期和延遲期的腦損傷及炎癥程度,并緩解了脾臟免疫功能的抑制。 三、IL-33減緩短暫性腦缺血后腦損傷的免疫學(xué)機(jī)制 目的：探討側(cè)腦室注射IL-33緩解MCAO模型缺血再灌注后急性期和延遲期腦損傷及炎癥程度的免疫學(xué)機(jī)制。 方法：分離側(cè)腦室注射IL-33的MCAO24h和MCAO3d組缺血腦和脾臟中的單個(gè)核細(xì)胞,通過流式細(xì)胞術(shù)檢測Thl、Th2和Th17細(xì)胞亞群的改變,以及用ELISA檢測血清中細(xì)胞因子IFN-γ、IL-4和IL-17的表達(dá)。 結(jié)果：與側(cè)腦室注射PBS的MCAO對(duì)照組相比,側(cè)腦室注射IL-33可減少M(fèi)CAO24h和MCAO3d腦組織中IL-17+T細(xì)胞和IFN-γ+T細(xì)胞的百分比,并增加了IL-4+T細(xì)胞的陽性率,Th1/Th2比值下降。與此同時(shí),側(cè)腦室注射IL-33也減少了脾臟中IL-17+T細(xì)胞和IFN-y+T細(xì)胞的百分率,但對(duì)IL-4+T細(xì)胞的陽性率無明顯影響。相應(yīng)地,側(cè)腦室注射IL-33也減少了血清中IL-17和IFN-γ的表達(dá)量,增加了IL-4的表達(dá)量。 結(jié)論：IL-33減緩短暫性腦缺血后急性期和延遲期損傷嚴(yán)重程度的作用可能與促進(jìn)Th2型免疫反應(yīng)和抑制Thl7型免疫反應(yīng)有關(guān)。 本研究首次在小鼠MCAO模型的基礎(chǔ)上檢測了IL-33及其受體ST2L在缺血再灌注后超急性期到延遲期的表達(dá)情況,發(fā)現(xiàn)IL-33和ST2L在短暫性腦缺血后的總體表達(dá)趨勢是下降的,通過側(cè)腦室注射IL-33可顯著改善MCAO模型小鼠缺血再灌注后急性期和延遲期的損傷嚴(yán)重程度,其可能機(jī)制是外源性IL-33促進(jìn)Th1/Th2平衡向Th2偏移和抑制Th17型免疫應(yīng)答有關(guān)。本研究為改善腦卒中預(yù)后和治療提供了新思路。
[Abstract]:Interleukin (IL) -33 is a new member of the IL-1 family found in recent years. As a cytokine, it can promote Th2 type immune response by IL-33/ST2 signaling pathway. It is also a nuclear protein that can regulate gene transcription. Under inflammatory stimulation, IL-33 can also be a warning tissue damage or stress.IL as a "warning hormone" warning. -33 is the only specific ligand for the ST2 receptor. Its biological activity is mainly dependent on the diversity of the role of the transmembrane ST2 receptor (ST2L) combined with.IL-33 to regulate the immune response, depending on the different diseases and models.IL-33 can promote the pathogenesis of Th2 type inflammatory diseases, such as asthma, atopic dermatitis and allergic reactions, however, in some cases Diseases or models associated with type Th2 immunoreaction, such as atherosclerosis, multiple sclerosis, and experimental enteritis, IL-33 can play a protective role by promoting the migration of Th1/Th2 to Th2. In addition, the recent study also found that the IL-33 adjustable Th17 immune response.IL-33 is highly expressed in multiple tissues, while the brain and spinal cord are the IL-33 tables. One of the highest amounts of organs suggests that IL-33 may play an important role in the regulation of the pathophysiology of the central nervous system. In recent years, the role of IL-33 or IL-33/ST2 signaling pathways in central nervous system diseases is one of the hot topics in neuroimmunology.
Ischemic stroke is a common cerebrovascular disease in middle-aged and elderly people, which can lead to brain tissue damage and associated with inflammatory response, which leads to a series of syndrome. The study shows that Th1/Th2 mediated immune response and Th17 type immune response play an important role in the inflammatory pathological mechanism of ischemic brain damage as Th2 type immune response. The role of key activating molecules in the pathogenesis of ischemic stroke remains to be elucidated. In order to explore the role and mechanism of IL-33 in the pathogenesis of ischemic stroke, this study uses ischemic stroke animal model to observe the changes in the expression of IL-33 and its receptor ST2L in ischemic brain injury, and to explore the effect of IL-33 on ischemia. The paper is divided into three parts. The main contents and results are as follows:
1. Expression of IL-33 and its receptor ST2L after transient ischemic attack
Objective: to detect the expression of IL-33 and its receptor ST2L in different time history and different brain regions after transient ischemic stroke, and to explore the regularity of the expression of IL-33 and its receptor ST2L in ischemic brain injury.
Methods: the rat model of reversible focal cerebral ischemia reperfusion injury was induced by occlusion of the middle cerebral artery (MCAO) 30min by the method of thread thrombus, and the semi quantitative RT-PCR and Western blot were used to detect the hyperacute phase (6h) after ischemia-reperfusion, and the level of IL-33mRNA and protein expression in the cerebral tissue of the acute phase (24h) and delay period (3D), and through the immune group. The expression of IL-33 in different ischemic brain regions (motor cortex, sensory cortex, hippocampus and striatum) was observed at various time points in different ischemic brain regions. In addition, the expression level of IL-33 receptor ST2L mRNA was also detected.
Results: compared with the sham operation group, the expression of IL-33mRNA and protein decreased in MCAO6h and 3D, the expression level of ST2L mRNA decreased in MCAO24h and 3D, and there was no significant change in the expression of IL-33 and ST2L at other time points. Except for the positive staining of the motor cortex and striatum in the MCAO24h group, the number of IL-33 positive cells was all in the different brain regions after the ischemia. There is a decrease in different degrees.
Conclusion: the overall trend of IL-33 and ST2L expression in the MCAO model mice after ischemia-reperfusion was decreased, suggesting that the Th2 type immune response mediated by IL-33/ST2L was downregulated after transient ischemic stroke.
Two, the effect of intracerebroventricular injection of IL-33 on MCAO brain injury.
AIM: To observe the effects of exogenous IL-33 on the severity of ischemic brain injury, brain inflammation and spleen immune function in MCAO model.
Methods: the recombinant plasmid of mouse IL-33 mature peptide was constructed by GST expression and purification system, and the biological activity of IL-33 protein was purified. IL-33 (2 mu g, 5 U L) or PBS5 Mu l were injected into the ventricle of 30min side of the mouse MCAO model before the mice, and the neurobehavioral symptoms were evaluated at different time points after ischemia reperfusion. The volume of cerebral infarction was assessed by TTC staining. Inflammatory cell infiltration in hippocampus and cortex was observed by HE staining, and the size of spleen was also observed.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the neurobehavioral symptoms of MCAO24h and MCAO3d (24h:2.0 + 0.302vs2.875 + 0.125; 3d:1.833 + 0.167vs2.917 + 0.193; P0.05) and the volume of cerebral infarction (24h:21.89 + 4.8%vs36.27 + 1.6%; 8.52 + 3.35%); histological staining showed In the lateral ventricle, the number of MCAO24h and MCAO3d in the hippocampal and cortical inflammatory cells in the ischemic side of the IL-33 was less. In addition, the injection of IL-33 in the lateral ventricle also increased the spleen index of MCAO24h and MCAO3d (24h:0.36 + 0.02vs0.31 + 0.04; 3d:0.30 + 0.05vs0.22 + 0.01; P0.05), and the spleen atrophy was unknown. There were no significant differences in neurobehavioral symptoms, infarct volume, inflammatory infiltration in the brain, spleen index and size.
Conclusion: the injection of IL-33 in the lateral ventricle can slow down the brain damage and inflammation in the acute and delayed period of transient cerebral ischemia and reperfusion, and alleviate the suppression of spleen immune function.
Three, IL-33 slowed down the immunological mechanism of brain injury after transient ischemic attack.
AIM: To investigate the immunological mechanism of intraventricular injection of IL-33 to alleviate acute and delayed brain injury and inflammation in MCAO model after ischemia-reperfusion.
Methods: the ischemic brain and spleen cells in group MCAO24h and MCAO3d of IL-33 were injected into the lateral ventricle, and the changes of Thl, Th2 and Th17 cell subgroups were detected by flow cytometry, and the expression of serum cytokines IFN- y, IL-4 and IL-17 in serum was detected by ELISA.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the percentage of IL-17+T cells and IFN- gamma +T cells in the MCAO24h and MCAO3d brain tissues, and increased the positive rate of IL-4+T cells and the Th1/Th2 ratio decreased. Meanwhile, the lateral ventricle injection IL-33 also reduced the percentage of the spleen and the cells. There was no significant effect on the positive rate of IL-4+T cells. Accordingly, the injection of IL-33 in the lateral ventricle also reduced the expression of IL-17 and IFN- gamma in the serum, and increased the expression of IL-4.
Conclusion: the effect of IL-33 on reducing the severity of acute and delayed injury after transient ischemic stroke may be related to the promotion of Th2 type immune response and the inhibition of Thl7 type immune response.
This study was the first to detect the expression of IL-33 and its receptor ST2L in the hyperacute to delay period after ischemia reperfusion on the basis of the mouse MCAO model. It was found that the overall expression trend of IL-33 and ST2L was decreased after transient ischemic stroke. The acute phase after ischemia reperfusion in MCAO model mice could be improved significantly through the injection of IL-33 in the lateral ventricle. The possible mechanism of injury severity in the delay period is that exogenous IL-33 promotes the migration of Th1/Th2 balance to Th2 and inhibits the Th17 type immune response. This study provides a new idea to improve the prognosis and treatment of stroke.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3
本文編號(hào)：2170225
[Abstract]:Interleukin (IL) -33 is a new member of the IL-1 family found in recent years. As a cytokine, it can promote Th2 type immune response by IL-33/ST2 signaling pathway. It is also a nuclear protein that can regulate gene transcription. Under inflammatory stimulation, IL-33 can also be a warning tissue damage or stress.IL as a "warning hormone" warning. -33 is the only specific ligand for the ST2 receptor. Its biological activity is mainly dependent on the diversity of the role of the transmembrane ST2 receptor (ST2L) combined with.IL-33 to regulate the immune response, depending on the different diseases and models.IL-33 can promote the pathogenesis of Th2 type inflammatory diseases, such as asthma, atopic dermatitis and allergic reactions, however, in some cases Diseases or models associated with type Th2 immunoreaction, such as atherosclerosis, multiple sclerosis, and experimental enteritis, IL-33 can play a protective role by promoting the migration of Th1/Th2 to Th2. In addition, the recent study also found that the IL-33 adjustable Th17 immune response.IL-33 is highly expressed in multiple tissues, while the brain and spinal cord are the IL-33 tables. One of the highest amounts of organs suggests that IL-33 may play an important role in the regulation of the pathophysiology of the central nervous system. In recent years, the role of IL-33 or IL-33/ST2 signaling pathways in central nervous system diseases is one of the hot topics in neuroimmunology.
Ischemic stroke is a common cerebrovascular disease in middle-aged and elderly people, which can lead to brain tissue damage and associated with inflammatory response, which leads to a series of syndrome. The study shows that Th1/Th2 mediated immune response and Th17 type immune response play an important role in the inflammatory pathological mechanism of ischemic brain damage as Th2 type immune response. The role of key activating molecules in the pathogenesis of ischemic stroke remains to be elucidated. In order to explore the role and mechanism of IL-33 in the pathogenesis of ischemic stroke, this study uses ischemic stroke animal model to observe the changes in the expression of IL-33 and its receptor ST2L in ischemic brain injury, and to explore the effect of IL-33 on ischemia. The paper is divided into three parts. The main contents and results are as follows:
1. Expression of IL-33 and its receptor ST2L after transient ischemic attack
Objective: to detect the expression of IL-33 and its receptor ST2L in different time history and different brain regions after transient ischemic stroke, and to explore the regularity of the expression of IL-33 and its receptor ST2L in ischemic brain injury.
Methods: the rat model of reversible focal cerebral ischemia reperfusion injury was induced by occlusion of the middle cerebral artery (MCAO) 30min by the method of thread thrombus, and the semi quantitative RT-PCR and Western blot were used to detect the hyperacute phase (6h) after ischemia-reperfusion, and the level of IL-33mRNA and protein expression in the cerebral tissue of the acute phase (24h) and delay period (3D), and through the immune group. The expression of IL-33 in different ischemic brain regions (motor cortex, sensory cortex, hippocampus and striatum) was observed at various time points in different ischemic brain regions. In addition, the expression level of IL-33 receptor ST2L mRNA was also detected.
Results: compared with the sham operation group, the expression of IL-33mRNA and protein decreased in MCAO6h and 3D, the expression level of ST2L mRNA decreased in MCAO24h and 3D, and there was no significant change in the expression of IL-33 and ST2L at other time points. Except for the positive staining of the motor cortex and striatum in the MCAO24h group, the number of IL-33 positive cells was all in the different brain regions after the ischemia. There is a decrease in different degrees.
Conclusion: the overall trend of IL-33 and ST2L expression in the MCAO model mice after ischemia-reperfusion was decreased, suggesting that the Th2 type immune response mediated by IL-33/ST2L was downregulated after transient ischemic stroke.
Two, the effect of intracerebroventricular injection of IL-33 on MCAO brain injury.
AIM: To observe the effects of exogenous IL-33 on the severity of ischemic brain injury, brain inflammation and spleen immune function in MCAO model.
Methods: the recombinant plasmid of mouse IL-33 mature peptide was constructed by GST expression and purification system, and the biological activity of IL-33 protein was purified. IL-33 (2 mu g, 5 U L) or PBS5 Mu l were injected into the ventricle of 30min side of the mouse MCAO model before the mice, and the neurobehavioral symptoms were evaluated at different time points after ischemia reperfusion. The volume of cerebral infarction was assessed by TTC staining. Inflammatory cell infiltration in hippocampus and cortex was observed by HE staining, and the size of spleen was also observed.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the neurobehavioral symptoms of MCAO24h and MCAO3d (24h:2.0 + 0.302vs2.875 + 0.125; 3d:1.833 + 0.167vs2.917 + 0.193; P0.05) and the volume of cerebral infarction (24h:21.89 + 4.8%vs36.27 + 1.6%; 8.52 + 3.35%); histological staining showed In the lateral ventricle, the number of MCAO24h and MCAO3d in the hippocampal and cortical inflammatory cells in the ischemic side of the IL-33 was less. In addition, the injection of IL-33 in the lateral ventricle also increased the spleen index of MCAO24h and MCAO3d (24h:0.36 + 0.02vs0.31 + 0.04; 3d:0.30 + 0.05vs0.22 + 0.01; P0.05), and the spleen atrophy was unknown. There were no significant differences in neurobehavioral symptoms, infarct volume, inflammatory infiltration in the brain, spleen index and size.
Conclusion: the injection of IL-33 in the lateral ventricle can slow down the brain damage and inflammation in the acute and delayed period of transient cerebral ischemia and reperfusion, and alleviate the suppression of spleen immune function.
Three, IL-33 slowed down the immunological mechanism of brain injury after transient ischemic attack.
AIM: To investigate the immunological mechanism of intraventricular injection of IL-33 to alleviate acute and delayed brain injury and inflammation in MCAO model after ischemia-reperfusion.
Methods: the ischemic brain and spleen cells in group MCAO24h and MCAO3d of IL-33 were injected into the lateral ventricle, and the changes of Thl, Th2 and Th17 cell subgroups were detected by flow cytometry, and the expression of serum cytokines IFN- y, IL-4 and IL-17 in serum was detected by ELISA.
Results: compared with the MCAO control group injected with PBS in the lateral ventricle, the lateral ventricle injection of IL-33 could reduce the percentage of IL-17+T cells and IFN- gamma +T cells in the MCAO24h and MCAO3d brain tissues, and increased the positive rate of IL-4+T cells and the Th1/Th2 ratio decreased. Meanwhile, the lateral ventricle injection IL-33 also reduced the percentage of the spleen and the cells. There was no significant effect on the positive rate of IL-4+T cells. Accordingly, the injection of IL-33 in the lateral ventricle also reduced the expression of IL-17 and IFN- gamma in the serum, and increased the expression of IL-4.
Conclusion: the effect of IL-33 on reducing the severity of acute and delayed injury after transient ischemic stroke may be related to the promotion of Th2 type immune response and the inhibition of Thl7 type immune response.
This study was the first to detect the expression of IL-33 and its receptor ST2L in the hyperacute to delay period after ischemia reperfusion on the basis of the mouse MCAO model. It was found that the overall expression trend of IL-33 and ST2L was decreased after transient ischemic stroke. The acute phase after ischemia reperfusion in MCAO model mice could be improved significantly through the injection of IL-33 in the lateral ventricle. The possible mechanism of injury severity in the delay period is that exogenous IL-33 promotes the migration of Th1/Th2 balance to Th2 and inhibits the Th17 type immune response. This study provides a new idea to improve the prognosis and treatment of stroke.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 蘇革,黃榮芳,聞立時(shí),成會(huì)明;采用熱絲CVD法在多種基材上沉積金剛石薄膜[J];表面技術(shù);1998年01期
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