摘要
支气管哮喘是一种全球性疾病,无地域和种族的局限性,也无年龄和性别的明显差异。支气管哮喘病因复杂,是以具有间歇性、可逆性的气道阻塞、支气管高反应性和淋巴细胞、嗜酸性粒细胞浸润气道粘膜下层为特征性的慢性气道炎症。关于气道炎症的发生,以往的研究普遍认为主要是免疫源性炎症,但其具体机制尚不十分清楚还有待进一步的研究。多数学者认为辅助性T淋巴细胞两种亚型(Thl和Th2)的失衡是哮喘发生的重要机制,具体则表现为Th2细胞的活化亢进,这是哮喘发病机制中的关键环节。Th1、Th2是由共同的前体细胞,即ThO分化而来,转录因子GATA-3是特异性调控ThO分化,起着Thl/Th2转换开关作用的关键因子之一,IFN-γ和IL-4则分别是Thl和Th2类细胞因子的主要代表。
近年来,气道炎症的Thl7细胞因素日益受到重视。Thl7细胞是机体中一类能产生IL-17的T细胞,RORγt是其特异性转录因子。IL-17A是近期发现的一种细胞因子,是属于一个独特类型的具有多种功能的细胞因子,可以通过诱导释放的促炎症因子和促中性粒细胞动员的细胞因子协调局部组织的炎症,在炎症性疾病和自身免疫性疾病,特别是在哮喘过敏性气道炎症中发挥重要作用。
RNA干扰(RNAi)是高度保守的基因沉默技术,它在功能上与基因敲除或基因沉默一致,但比基因敲除技术更为方便快捷。在本研究中,我们利用RNA干扰技术在急性哮喘小鼠模型中探讨GATA-3对RORγt表达以及对Thl7细胞分化及其致炎机能的影响。研究的主要目的之一是探讨RORγt对急性哮喘小鼠脾源性CD4+T细胞亚群Th17细胞分化和致炎机能的影响；研究的主要目的之二是通过RNA干扰GATA-3基因表达,观察急性哮喘小鼠脾源性CD4+T细胞中RORγt的表达变化及其亚群Thl7细胞分化和致炎机能是否受到影响。通过此研究,我们希望发现GATA-3在哮喘发生过程中Th2/Th17淋巴细胞分化调控失衡现象中的作用及可能机制,以期为哮喘防治提供新思路和理论依据。
目的通过补充完善急性哮喘动物模型评价标准,鉴定改良建立的急性哮喘小鼠模型是否成功。
方法将35只SPF级BALB/c小鼠随机分为两组：急性哮喘组(20只)以OVA致敏、激发；正常对照组(15只)以等体积的PBS代替OVA致敏、激发；两组均在末次激发24 h后处理小鼠。处理包括：1.查看小鼠的一般行为活动；2.乙酰胆碱激发小鼠后,有创肺阻抗法测定小鼠的气道反应性；3. BALF行细胞学分类、计数；4.观察肺组织的病理变化；5.计算脾源性CD4+T细胞总数和Th17细胞的阳性率；6.测定BALF和脾源性CD4+T细胞培养上清中IL-4和IL-17的浓度。
结果
(1)急性哮喘组小鼠出现类似于人的哮喘发作症状,而正常对照组小鼠表现基本正常。
(2)与正常对照组相比,急性哮喘组小鼠对乙酰胆碱的刺激反应明显,浓度反应曲线明显上移(P<0.01)。
(3)急性哮喘组小鼠BALF白细胞总数及中性粒细胞、嗜酸性粒细胞和淋巴细胞的百分比明显高于正常对照组(P均<0.01)。
(4)与正常对照组相比,急性哮喘组小鼠肺组织支气管及血管周围大量炎性细胞浸润(P<0.01)。
(5)急性哮喘组小鼠脾源性CD4+T细胞总数和Thl7细胞阳性率明显高于正常对照组(P均<0.01)。
(6)与正常对照组相比,急性哮喘组小鼠BALF和脾源性CD4+T细胞培养上清中IL-4和IL-17的浓度明显增高(P均<0.01)。
结论
(1)改良并建立了急性哮喘小鼠动物模型,并且模型鉴定成功。
(2)成功完善了急性哮喘小鼠动物模型的评价标准。
目的探讨急性哮喘小鼠脾源性CD4+T细胞中RORγt表达的变化以及RORγt对急性哮喘小鼠脾源性CD4+T细胞亚群Th17细胞分化和致炎机能的影响。
方法首先研磨小鼠脾脏制成单细胞悬液后裂解红细胞,再予免疫磁珠分离小鼠脾源性CD4+T细胞,悬于加有胎牛血清的RPMI 1640培养液,加入ConA和PMA刺激培养24小时后行real-time PCR、Western blotting测定小鼠脾源性CD4+T细胞亚群Th17细胞中RORγt的表达,流式细胞仪测定Thl7细胞阳性率,ELISA法检测脾源性CD4+T细胞培养上清中IL-17的浓度,并分析急性哮喘组小鼠RORγt的表达与Th17细胞阳性率及IL-17浓度的相关性。
结果
(1)急性哮喘组小鼠脾源性CD4-T细胞RORγt的mRNA及蛋白表达明显高于正常对照组(P<0.01)。
(2)急性哮喘组小鼠脾源性CD4+T细胞中Thl7细胞阳性率明显高于正常对照组(P<0.01)。
(3)急性哮喘组小鼠脾源性CD4+T细胞培养上清中IL-17的浓度明显高于正常对照组(P<0.01)。
(4)急性哮喘组小鼠脾源性CD4+T细胞中RORγt的mRNA表达与Thl7细胞阳性率、IL-17的浓度均呈正相关(r=0.854,0.872；P均<0.01)。
(5)急性哮喘组小鼠脾源性CD4+T细胞中RORγt的蛋白表达与Thl7细胞阳性率、IL-17的浓度均呈正相关(r=0.902,0.891；P均<0.01)。
结论
(1)急性哮喘小鼠脾源性CD4+T细胞中RORγt的表达显著增高。
(2)急性哮喘小鼠脾源性Thl7细胞的分化和致炎机能显著增高。
(3)急性哮喘小鼠脾源性CD4+T细胞中RORγt的表达与Th17细胞的分化和致炎机能呈正相关。
目的：通过RNA干扰GATA-3基因表达,观察急性哮喘小鼠脾源性CD4+T细胞中RORyt的表达及其亚群Th17细胞分化和致炎机能是否受到影响。
方法：通过基因瞬时转染的方法,将三对siRNA-GATA-3片段和阴性对照片段分别转染入急性哮喘组小鼠脾源性CD4+T细胞72小时后,进行RT-PCR和Western blotting检测GATA-3的表达,筛选出其中具有最佳沉默效果的片段。再将此片段转染入急性哮喘组小鼠脾源性CD4+T细胞,ConA和PMA刺激培养24小时后,行real-time PCR和Western blotting分别检测正常对照组小鼠、未转染siRNA组哮喘小鼠和转染最佳沉默片段siRNA组哮喘小鼠脾源性CD4+T细胞中GATA-3和RORyt的表达,以及行流式细胞术和ELISA法分别检测Th17细胞阳性率和细胞培养上清中IL-17浓度,并分析转染最佳沉默片段siRNA组急性哮喘小鼠脾源性CD4+T细胞中GATA-3的表达与RORyt的表达、Th17阳性率及IL-17浓度的相关性。
结果：
(1)在三对siRNA片段中以siRNA-GATA-3-201沉默效果最为明显(P<0.01)。
(2)转染siRNA-GATA-3-201组急性哮喘小鼠的脾源性CD4+T细胞中GATA-3 mRNA的表达明显低于正常对照组小鼠和未转染siRNA组哮喘小鼠(P均<0.01)；转染siRNA-GATA-3-201组哮喘小鼠脾源性CD4+T细胞中GATA-3蛋白的表达明显低于正常对照组小鼠和未转染siRNA组哮喘小鼠(P均<0.01)。
(3)转染siRNA-GATA-3-201组急性哮喘小鼠脾源性CD4+T细胞中RORγtmRNA的表达明显高于正常对照组小鼠和未转染siRNA组哮喘小鼠(P均<0.01)；转染siRNA-GATA-3-201组哮喘小鼠脾源性CD4+T细胞中RORyt蛋白的表达明显高于正常对照组小鼠和未转染siRNA组哮喘小鼠(P均<0.01)。
(4)转染siRNA-GATA-3-201组急性哮喘小鼠脾源性CD4+T细胞中Th17的阳性率明显高于正常对照组小鼠和未转染siRNA组急性哮喘小鼠(P均<0.01)。
(5)转染siRNA-GATA-3-201组急性哮喘小鼠脾源性CD4+T细胞培养上清中IL-17的浓度明显高于正常对照组小鼠和未转染siRNA组哮喘小鼠(P均<0.01)
(6)转染siRNA-GATA-3-201组急性哮喘小鼠脾源性CD4+T细胞中GATA-3的mRNA表达与RORyt的mRNA表达、Th17阳性率及IL-17的浓度呈负相关(r=-0.622,-0.692,-0.877；P均<0.01)。
(7)转染siRNA-GATA-3-201组急性哮喘小鼠脾源性CD4+T细胞中GATA-3的蛋白表达与RORyt的蛋白表达、Th17阳性率及IL-17的浓度呈负相关(r=-0.894,-0.728,-0.886；P均<0.01)。
结论：
(1)RNA干扰技术可应用于支气管哮喘小鼠动物模型,实现对靶基因的沉默。
(2)RNA干扰GATA-3基因表达可能促进急性哮喘小鼠脾源性CD4+T细胞中RORγt的表达。
(3)RNA干扰GATA-3基因表达可能促进急性哮喘小鼠脾源性Th17细胞的分化和致炎机能。
(4)RNA干扰GATA-3基因表达可能通过促进急性哮喘小鼠脾源性CD4+T细胞RORγt的表达进而促进Thl7细胞的分化和致炎机能。
Preface
Bronchial asthma is one of global diseases, and there is little difference both in area and ethnicity, or in age and sex. The Etiopathogenisis of bronchial asthma is complex. Asthma is chronic airway inflammation with the characteristics of intermittent and reversible airway obstruction, bronchial hyperresponsiveness, lymphocytes, eosinophil infiltration of airway submucosal. Regarding the induction of airway inflammation, it was widely recognized that it is a result of immune response in the bronchial airway. However, the underlying mechanism is not clear and requires further investigation. Many believe that imbalance of two types of T-helper cells, Th1 and Th2, is the underlying cause for asthma. Specifically, the increased activity of Th2 cells is a key event in asthma induction. Thl and Th2 are differentiated from a common precursor cells, ThO. Transcription factor GATA-3 regulates ThO differentiation in a specific manner. Such regulation is critical in controlling Thl and Th2. IFN-γand IL-4 are typical cytokines from Thl and Th2.
In recent years, the roles of Th17 cells in airway inflammation are receiving increasing attention. Th17 cells,which can produce IL-17 cytokines, are a new lineage of T-cells that are controlled by the transcription factor RORyt. IL-17 is a cytokine that discovered recently, belongs to a unique type of multi-functional cytokines, which coordinates the local inflammation of tissues by inducing the release of proinflammatory factors and mobilizing neutrophils, so it plays an important role in inflammatory diseases and autoimmune diseases, especially in allergic airway inflammation in asthma.
RNA interference is a kind of highly conserved gene silencing technology. RNAi technology is the same as gene knockout and gene silencing in functions, and more convenient than Gene knockout technology. In this study, we used acute mouse model of asthma to study the function of GATA-3 and RORyt in differentiation and inflammation of Th17 cells. One goal of this study is to elucidate the effect of RORyt on differentiation and inflammation of Th17 cells which is a subset of spleen CD4+T cells from acute mouse model of asthma. Furthermore, the other goal of our study is to detect the effect of downregulating GATA-3 expression by transfecting GATA-3-siRNA on expression of RORyt, and on differentiation and inflammation of Th17 cells from acute mouse model of asthma. Through this study, we hope to open new avenues and provide further experimental evidence for therapeutic treatment of asthma.
Objective:To establish and improve acute mouse model of asthma and confirm reliability of the model with modified evaluation criterion.
Methods:35 SPF female BALB/c mice were randomly divided into normal control group and acute asthma group, with 20 mice in acute asthma group and 15 mice in normal control group. The acute asthma model was established by sensitization with intraperitoneal injection of OVA and aerosol challenge with repeated inhalation of OVA in the acute asthma group. The control group received PBS as the substitution of OVA. After 24 hours of the last inhalation, asthmatic symptoms were observed; The changes in airway response were determined by lung resistance(RL) stimulated by acetylcholine(Ach); The white cell count, neutrophils(%), eosinophils(%), lymphocytes(%) of bronchoalveolar lavage fluid (BALF) were measured; Lung tissue sections were stained for general pathology; The total cellular count of spleen CD4+T cells and ratio of positive Th17 cells were detected; The levels of IL-4 and IL-17 concentration of BALF and splenocyte culture supernatant were examined.
Results:
(1) Asthma symptoms were more severe in acute asthma group, compared with normal control group.
(2) Total RL was obviously increased in acute asthma group, compared with normal control group(p<0.01).
(3) Total white cell count, Neu(%), Eos(%) and Lym(%) in the BALF of acute asthma group were more than those of normal control group (respectively p<0.01).
(4) There were more extensive inflammatory cells infiltration around the bronchi in acute asthma group, compared with normal control group(p<0.01).
(5) The total cellular count of spleen CD4+T cells and the ratio of positive Th17 cells in acute asthma group were obviously higher than those of normal control group(respectively p<0.01).
(6) In either splenocyte culture supernatant or BALF, the levels of IL-4 and IL-17 concentration were significantly elevated in acute asthma group, compared with normal control group(respectively p<0.01).
Conclusions:
(1) The reliability of the improved and established acute mouse model of asthma was successfully confirmed.
(2) The modified evaluation criterion of acute asthmatic mouse model was successful.
Objective:To investigate the change of RORyt expression in acute mouse model of asthma, and to elucidate the effect of RORyt on differentiation and inflammation of Th17 cells which is a subset of spleen CD4+T cells from acute mouse model of asthma.
Methods:Spleen mononuclear cells from acute mouse model of asthma were removed red blood cells, and, followed by isolation of CD4+T cells with immuno-magnetic beads, they were cultivated in RPMI-1640 with 10% Fetal Bovine Serum, stimulated by ConA and PMA. After 24 hours, all cells were detected the expression of RORyt mRNA by real-time PCR, the level of RORyt protein by Western blotting, the ratio of positive Th17 cells by flow cytometer (FCM), and the production of IL-17 by ELISA. The correlation between RORyt expression and the ratio of positive Th17 cells, and IL-17 production were analyzed.
Results:
(1) The expression of RORyt mRNA and protein in spleen CD4+T cells from acute asthma group were significantly elevated, compared with normal control group(respectively p<0.01).
(2) The ratio of positive Th17 cells from acute asthma group was obviously higher than that of normal control group(p<0.01).
(3) The more extensive production of IL-17 in splenocyte culture supernatant was found in acute asthma group, compared with normal control group(p<0.01)
(4) The RORyt mRNA expression of spleen CD4+T cells from acute asthma group was positively correlated with the ratio of positive Th17 cells and the production of IL-17(respectively r=0.854,0.872;p<0.01).
(5) The RORyt protein expression of spleen CD4+T cells from acute asthma group was positively correlated with the ratio of positive Th17 cells and the production of IL-17(respectively r=0.902,0.891;p<0.01).
Conclusions:
(1) RORyt expression is significantly increased in spleen CD4+T cells from acute mouse model of asthma.
(2) The differentiation and inflammation of Th17 cells are significantly elevated in acute mouse model of asthma.
(3)RORyt expression is associated with differentiation and inflammation of Th17 cells in acute mouse model of asthma.
Objective:To detect the effect of downregulating GATA-3 expression by transfecting GATA-3-siRNA on expression of RORγt, and on differentiation and inflammation of Th17 cells from acute mouse model of asthma.
Methods:Spleen CD4+T cells from acute mouse model of asthma were transfected with three chemosynthesis siRNA sequences by transient electroporation. After cultivating and stimulation with cytokine, the best efficient siRNA-GATA-3 was choosed by detection GATA-3 expression. This siRNA-GATA-3 was trasnsfected into Spleen CD4+T cells from asthma group. Cells, divided into three groups:control group, untransfected asthmatic group and transfected asthmatic group, were measured the expression of RORγt and GATA-3 mRNA by real-time PCR, the level of RORγt and GATA-3 protein by Western blotting, the ratio of positive Th17 cells by FCM and the production of IL-17 by ELISA. The correlation between GATA-3 expression and RORγt expression, the ratio of positive Th17 cells, and IL-17 production were analyzed.
Results:
(1) siRNA-GATA-3-201 had better silencing effect than other siRNA sequences(p<0.01).
(2) The expression of GATA-3 mRNA and protein in spleen CD4+T cells from transfected asthmatic group were lower than those of controls (respectively p<0.01).
(3) The expression of RORyt mRNA and protein in spleen CD4+T cells from transfected asthmatic group were higher than those of controls (respectively p<0.01).
(4) The ratio of positive Th17 cells from transfected asthmatic group was significantly increased, compared with controls (respectively p<0.01).
(5) The IL-17 production of splenocyte culture supernatant from transfected asthmatic group was significantly elevated, compared with controls (respectively p<0.01).
(6) The GATA-3 mRNA expression on spleen CD4+T cells from transfected asthmatic group was negatively correlated with RORyt mRNA expression, the ratio of positive Th17 cells and the production of IL-17 (respectively r=-0.622,-0.692,-0.877;p<0.01).
(7) The GATA-3 protein expression on spleen CD4+T cells from transfected asthmatic group was negatively correlated with RORyt protein expression, the ratio of positive Th17 cells and the production of IL-17 (respectively r=-0.894,-0.728,-0.886; p<0.01).
Conclusions:
(1) Application of RNA interference technique in asthmatic mouse model may implement target gene silencing.
(2) RNAi-mediated GATA-3 gene silencing could increase RORyt expression in spleen CD4+T cells from acute mouse model of asthma.
(3) RNAi-mediated GATA-3 gene silencing could promote the differentiation and inflammation of Th17 cells in acute mouse model of asthma.
(4) RNAi-mediated GATA-3 gene silencing could increase RORyt expression, which could promote the differentiation and inflammation of Th17 cells in spleen CD4+T cells from acute mouse model of asthma.
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