【作者】 黄维琳；

【导师】 刘荣玉；

【作者基本信息】 安徽医科大学 ， 老年医学， 2008， 硕士

【摘要】 背景支气管哮喘是由淋巴细胞、嗜酸性粒细胞和肥大细胞等多种炎性细胞、炎症介质和细胞因子共同参与的一种气道慢性炎症。免疫反应在哮喘的发病机制中占有重要地位。目前有人研究发现哮喘的发病的重要基础是辅助性T细胞两种亚型Th1和Th2细胞组成的比例和功能失衡。肺泡巨噬细胞可通过吞噬凋亡的嗜酸性粒细胞、中性粒细胞等而有利于哮喘气道炎症的消退,同时也可通过释放多种细胞因子和蛋白酶,参与哮喘免疫炎症反应及气道的修复和重构,为支气管哮喘的重要效应细胞之一。因此,研究巨噬细胞在支气管哮喘发病机制中的作用,调控巨噬细胞功能可能为支气管哮喘治疗提供一个新思路。CpG-ODN是含未甲基化的CpG二核苷酸的寡核苷酸序列,和细菌DNA一样具有强烈的免疫刺激作用,能激活哺乳动物巨噬细胞、树突状细胞等多种免疫细胞。CpG-ODN还能诱导Th1细胞因子产生,抑制Th2细胞因子的释放,诱导Th1反应,下调Th2反应,并使Th2反应向Th1反应转变,使Th1/Th2免疫反应达到平衡的状态。而调节Th1/Th2平衡对哮喘的治疗具有重要意义。其激活效应细胞途径是通过Toll样受体9(Toll like-receptor,TLR-9)介导的。TLR-9在识别CpG-ODN后,通过结构域募集髓样分化因子(Myeloid differentiation factor 88,MyD88),核因子κB等多条信号途径,启动细胞活化进程,上调主要组织相容性复合物和共刺激分子表达等,进而诱导各种炎性细胞因子的表达。因此研究CpG-ODN与TLR-9相互识别后对巨噬细胞细胞因子的分泌,TLR-9自身表达的影响,对于揭开CpG-ODN调控巨噬细胞功能的分子机制具有重要意义。目的(1)从mRNA和蛋白水平分析CpG-ODN刺激鼠巨噬细胞后对鼠巨噬细胞TLR-9的表达。探讨CpG-ODN调控巨噬细胞TLR-9表达的特征。(2)检测CpG-ODN刺激前后巨噬细胞上清液中Th1类细胞因子TNF-α、IL-12及Th2类细胞因子IL-6的表达水平,了解CpG-ODN刺激鼠巨噬细胞后对Th1/Th2类细胞因子分泌的影响。探讨CpG-ODN调控巨噬细胞功能及TLR-9表达的分子机制。方法(1)本研究采用体外培养小鼠单核-巨噬细胞系RAW264.7细胞,分别用CpG-ODN,Non-CpG-ODN,CpG-ODN+氯喹和培养基刺激巨噬细胞24小时,采用逆转录聚合酶链反应(RT-PCR)检测并比较各组细胞TLR-9mRNA的表达情况,同时用免疫印迹法(Western blotting)进一步验证TLR-9蛋白的表达。(2)放免法检测各组巨噬细胞上清液中TNF-α、IL-6的表达水平。酶联免疫吸附法(ELISA)检测各组巨噬细胞上清液中IL-12的表达水平。结果(1)刺激24小时后,CpG-ODN与CpG-ODN+氯喹组巨噬细胞上清中TLR-9mRNA的表达均显著高于Non-CpG-ODN及对照组(P＜0.05),用Western blotting法进一步证实了CpG-ODN与CpG-ODN+氯喹组巨噬细胞上清中TLR-9蛋白的表达较Non-CpG-ODN及对照组明显增加(P＜0.05)。(2)巨噬细胞上清液中TNF-α水平CpG-ODN组明显高于其他组,差异有显著性(P＜0.05)。而各组巨噬细胞上清液中IL-6,IL-12水平无显著性差异(p＞0.05)。结论CpG-ODN刺激鼠巨噬细胞24小时后可引起鼠巨噬细胞TLR-9无论在蛋白水平还是在核酸水平表达均增高,并且能增加巨噬细胞上清液中Th1类细胞因子TNF-α水平,但不增加Th2类细胞因子IL-6及Th1类细胞因子IL-12水平,从而调节Th1/Th2类细胞因子的分泌。CpG-ODN能很好的调控巨噬细胞功能,将为支气管哮喘治疗提供一个新思路。更多还原

【Abstract】 BacgroundBronchial asthma（asthma） is a inflammatory lung disease including lymphocyte、eosinophil、mast cell、inflammation matter and many cytokines.Immune response has an important position in asthma pathogenesy. Rccently,someone preferred nosogenic foundation of asthma is the constitutive ratio and function in balance of Th1,Th2 which are the subgroups of T helper cells.The removal of these apoptotic neutrophils and eosinophils through macrophages is one prerequisite of resolution of acute inflammation.Macrophages can also release many cytokines and protein enzyme,participate in asthma inflammation and airway repair and restructure.So it is necessary to study the function of macrophages in asthma and it is a good idea for asthma therapy through adjusting macrophages.CpG-ODN are oligodeoxynucleotides including CpG.It has intensity immunity stimulation like bacteria DNA.It can arose many immune cells,like macrophage and dendritic cell of mammal.DNA.CpG-ODN can induce Th1 cytokines and reduce Th2 cytokines,down-regulate Th2 but induce Th1 response,which can also shift the Th2 responses substantially toward Th1 and modulate the Th1 / Th2 immune response balance.The balance has significant meaning in cure of asthma.The approach of stimulate functionary cell through Toll like receptor-9（TLR-9）.After TLR-9 recognises CpG-ODN,it initiate cellactivation through myeloid differentiation factor 88 and nuclear factor,then upregulate major histocompatibility complex（MHC） and costimulatoty molecules expression,secrete cytokine and so on.So it is important to study cytokines secretion in macrophages after CpG-ODN meets TLR-9,and TLR-9 expression,It has signification to know how CpG-ODN adjusts macrophage.Objective（1） The study was designed to investigate the protein and mRNA expression of TLR-9 after CpG-ODN stimulated macrophages.To explore the character in CpG-ODN adjusting macrophages.（2） Investigate secretion on Th1 cytokines like TNF-α、IL-12 and Th2 cytokines like IL-6 before and after CpG-ODN stimulated the murine-macrophages-like Raw264.7 Cells.To know how CpG-ODN adjusts macrophage.Method（1） In this study,we cultivated RAW264.7 cells.They were seeded in 6-well culture plates and grew overnight in DMEM supplemented with 10%fetal calf serum in a Rumidified 5%CO₂ in cubator at 37℃.Then they were treated with DMEM,CpG-ODN,Non-CpG-ODN or CpG-ODN+chloroquine for 24 hours.The expression of TLR-9 mRNA and protein of each group were analyzed by RT-PCR and Western blot respectively.（2） The concentration of TNF-αand IL-6 in the supernatants were e xamined by Radioimmunoasssy（RIA） and the level of IL-12 was analyzed by ELISA.Result（1） After treated for 24 hours,the expression of TLR-9 mRNA in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.（P＜0.05） Using western blotting also prived that the protein expression of TLR-9 in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.（P＜0.05）（2） The level of TNF-αin the supernatant in the CpG-ODN group was higher than that in other groups.（P＜0.05）.The levels of IL-6 and IL-12 in the supernatants had no significant difference in these groups （p＞0.05）.ConclusionsThe protein and mRNA expression of TLR-9 both incarase after CpG-ODN stimulated macrophages.CpG-ODN can induce Th1 cytokines like TNF-α,but Th2 cytokines like IL-6 and Th1 cytokines like IL-12 not.These results demonstrate that CpG-ODN can up-regulate the expression of TLR-9 and adjust the expression of Th1 /Th2 cytokines secretion from macrophages. It is a good idea for asthma therapy through adjusting macrophages.更多还原