【作者】 王慧；

【导师】 房明丽；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 宿主被病毒、细菌等外来病原微生物感染时,病原体相关分子模式可通过天然免疫细胞模式识别受体,激活机体固有免疫应答,保护宿主机体健康。然而,当免疫反应过激时,免疫细胞大量产生的细胞因子会对机体造成免疫性病理损伤。为了维持免疫的平衡,迫切需要一种抑制剂来调控过度激活的天然免疫反应与过度产生的炎症因子来维持机体的免疫平衡。免疫抑制性寡聚脱氧核苷酸(Inhibitory Oligodeoxynucleotide,ODN)是一种人工合成的具有负向免疫调节作用的单链DNA分子。在前期工作中,本科室模拟人类微卫星DNA的序列设计出了两种寡核苷酸(ODN),命名为MS ODN。分别为MS19(AAAGAAAGA AAGAAAGAAAGAAAG)及SAT05F(CCTCCTCCTCCTCCTCCTCCTCCT),该MS ODNs在败血症、系统性红斑狼疮、关节炎等免疫过激性疾病动物模型中起到了很好的疗效。但是MS ODNs通过哪种免疫细胞来调控免疫过激的机制还尚不清楚。因此,本论文通过从小鼠骨髓中诱导分化了各种天然免疫细胞,通过体外实验研究了MS ODNs对天然免疫细胞活化并产生细胞因子的抑制作用进行了分析;并对MS ODNs发挥生物学作用的序列特点进行了初步探讨;在体内实验中观察了MS ODNs对LPS诱导的小鼠急性肺损伤的影响,并对其作用机理进行了初步探讨。具体结果如下:1、MS ODNs(MS19、SAT05F)对DNA病毒、RNA病毒、细菌成分诱导小鼠骨髓来源的巨噬细胞、小鼠骨髓来源的树突状细胞及小鼠骨髓来源的浆细胞样树突状细胞活化分泌炎症因子的影响分离得到小鼠的骨髓单个核细胞,分别加入L929培养上清,集落刺激因子GM-CSF及FLT3L细胞因子,37℃培养7天后,获得小鼠原代骨髓来源的巨噬细胞(BMDM),小鼠骨髓来源的树突状细胞(BMDC,纯度大于80%)及小鼠骨髓来源的浆细胞样树突状细胞(命名为FLT3L~+细胞,包括10-15%BMpDC)。选用DNA病毒及DNA病毒模拟物(腺病毒(ADV),单纯疱疹病毒(HSV)及DNA病毒模拟物(VACV-70)),RNA病毒及RNA病毒代谢产物(流感病毒,呼肠孤病毒(Reov)及RNA病毒代谢产物(dsRNA long poly(I:C),LPIC)及细菌成分(脂多糖成分LPS、细菌的鞭毛蛋白Flagellin,及细菌成分凝胶多糖Curdlan)作为刺激剂,利用lipo 3000将MS19或者SAT05F转染入加入刺激剂的细胞中共同孵育,同时以只加Lipo 3000的细胞作为对照,培养16 h后,ELISA方法检测细胞培养上清中炎症因子TNF-a和IL-6的浓度。结果显示:(1)MS19、SAT05F能够抑制DNA病毒(VACV-70、HSV、ADV),RNA病毒(流感病毒、Reov、LPIC)及LPS诱导原代BMDM细胞分泌炎症因子IL-6和TNF-a的产生。对细菌成分Curdlan刺激BMDM分泌IL-6的下调作用无统计学意义。(2)MS19、SAT05F能够抑制流感病毒诱导BMDC细胞分泌IFN-a。(3)MS19、SAT05F能够抑制CpG A ODN诱导pDC分泌IFN-a的水平;抑制CpG B ODN诱导pDC分泌TNF-a的水平;抑制流感病毒刺激pDC分泌(40)型IFN-a和TNF-a的水平。上述结果说明:MS ODNs(MS19、SAT05F)对DNA病毒、RNA病毒及细菌成分活化小鼠原代骨髓来源的巨噬细胞、传统树突状细胞和浆细胞样树突状细胞分泌炎症因子及(40)型干扰素(IFN-a)有一定的抑制作用。2、MS19及其突变体对LPS刺激RAW264.7细胞分泌炎症因子的抑制作用我们将MS19序列中的基序AAAG突变为AAAC,该突变体命名为MS19-M1,在小鼠的巨噬细胞系中观察了MS19发挥生物学功能的序列特征。在小鼠RAW264.7巨噬细胞系中,检测了MS19及MS19-M1对LPS诱导RAW264.7细胞产生炎症因子IL-6和TNF-a的差异。结果显示,MS19能够抑制LPS诱导RAW264.7细胞分泌产生的的IL-6和TNF-a,而MS19-M1未起到抑制作用,说明AAAG基序是MS19发挥生物学功能的关键序列,其中G碱基可能是关键碱基。3、MS19及其突变体对不同刺激剂诱导RAW264.7细胞中NF-kB信号通路中p65蛋白活化的影响。为了检测MS19是否是通过抑制NF-kB信号通路中p65蛋白活化来发挥调控作用的。我们在小鼠巨噬细胞RAW264.7细胞中,以MS19-M1作为对照ODN,利用Western Blot方法检测了MS19对NF-kB信号通路中p65活化的影响。结果显示:MS19能够明显地抑制LPS、VACV-70、CpG ODN、流感病毒诱导RAW264.7细胞中p65的磷酸化,而MS19-M1对p65的磷酸化无明显抑制作用。以上结果说明:MS19可以抑制NF-kB信号通路中p65的活化,且MS19对p65活化的抑制作用具有序列依赖性。4、MS19对LPS诱导小鼠急性肺损伤的抑制作用小鼠的肺上皮中聚集着大量的巨噬细胞及树突状细胞,当机体发生过激免疫反应时,肺部的天然免疫细胞会分泌大量的炎症因子导致急性肺损伤。基于体外实验的结果,MS19对各种刺激剂诱导巨噬细胞和树突状细胞分泌炎症因子具有一定的抑制作用。因此,本论文中我们选择了LPS作为刺激剂,建立了LPS诱导小鼠的急性肺损伤模型。观察了MS19在小鼠体内对免疫性损伤的抑制作用。第0天,将LPS(20 mg/kg)通过滴鼻方式感染C57小鼠建立LPS诱导急性肺损伤小鼠模型。LPS感染前1天,我们将一种阳离子脂质体DOTAP与MS19体外孵育后滴入鼻腔,该组小鼠为LPS+MS19组。LPS感染前1天,我们将PBS滴入小鼠鼻腔,该组小鼠为LPS组。将未进行任何处理的小鼠作为健康对照组。每组8只小鼠。感染后第16 h,收集模型小鼠肺泡灌洗液,观察肺组织中炎症因子的表达情况;感染的第8天,收集小鼠的肺组织,进行H&E染色。与此同时,每天同一时间记录小鼠的身体状态,体重及生存期。结果显示:与LPS组小鼠相比,MS19给药组对模型小鼠的体重下降较缓;小鼠肺泡灌洗液细胞中炎症因子TNF-a、IL-6、IL-1b的mRNA表达水平较低;肺组织学结构少量炎细胞浸润,基本正常;MS19治疗组可以提高LPS模型组小鼠的生存率。该部分体内研究结果说明MS19对LPS诱导的小鼠急性肺损伤模型具有一定的保护作用。综上所述,本研究发现MS19和SAT05F可抑制小鼠骨髓来源的巨噬细胞和树突状细胞分泌IFN和炎症因子。其中,MS19可以抑制NF-kB信号通路中p65的活化,且MS19对p65活化的抑制作用具有序列依赖性。同时,在LPS诱导的小鼠急性肺损伤模型中,我们发现MS19可抑制模型小鼠的体重减轻;提高模型小鼠的存活率;降低小鼠肺泡灌洗液中细胞因子水平;减轻小鼠的肺部病理改变。提示MS19可能成为一种潜力的免疫负调节性药物,来治疗免疫过激性疾病所导致的病理性损伤。更多还原

【Abstract】 The innate immune system constitutes the first line of defense against invading microbial pathogens and relies on a large family of pattern recognition receptors(PRRs),which detect distinct evolutionarily conserved structures on pathogens,termed pathogen-associated molecular patterns(PAMPs).Upon PAMP engagement,PRRs trigger intracellular signaling cascades ultimately culminating in the expression of a variety of cytokines and prionflammatory molecules,which together orchestrate the host immune response against bacteria,viruses,fungi,and parasites.However,these damage-associated molecular patterns(DAMPs)released from damaged or necrotic cells are recognized by certain PRRs and subsequent over-activate the innate immune response and produce a large number of cytokines.The over-produced cytokines,as found in basic and clinical studies,contribute to immune pathological damage,even leading to a fatal outcome.Obviously,it is required to develop agents to curb the over-reaction and the over-produced cytokines for maintain immune balance.Inhibitory oligodeoxynucleotides(ODNs),an artificially synthesized single-stranded DNA molecule,could regulate innate immunity activation.In the previous study,SAT05f and MS19,the MS ODNs(microsatellite DNA mimicking oligonucleotides)designed with reference of the sequence of human microsatellite DNA,are capable of improve the survival rate in mouse model of immune over-activated disease,such as sepsis,systemic lupus erythematosus,and arthritis.However,which types of innate immune cell involved in the role of MS ODNs on controlling the immune over-activated disease are not entirely clear.In this study,we isolated and cultured various innate immune cells from mouse bone marrow.Then,we detected the inhibition role of MS ODNs and MS19 mutant on the production of pro-inflammatory cytokines form bone marrow-derived cells.stimu-lated by various PRRs.In vivo experiments,we observed the effect of MS ODNs on acute lung injury induced by LPS in mice and study the mechanism.1.The inhibitory effect of MS ODNs on secretion of inflammatory cytokines and interferon(IFN)in bone marrow-derived macrophages(BMDMs),bone marrow-derived dendritic cells(BMDCs)and bone marrow-derived plasmacytoid dendritic cells(BM-pDCs)stimulated by DNA virus,RNA virus and bacteria in vitro.BM mononucleocytes were isolated from mouse and added the L929-conditioned medium,recombinant murine granulocyte-macrophage colony stimulating factor(rmGM-CSF)and recombinant murine FMS-like tyrosine kinase 3ligand(rmFLT3L)to obtain BMDMs,BMDCs and FLT3L~+cells(including 10-15%BM-pDCs),respectively.We choose the DNA viruses(Adenovirus(ADV),Herpes simplex virus(HSV)and DNA virus mimics VACV-70),RNA virus(Influenza virus,Reovirus(Reov)and RNA virus metabolism products dsRNA long poly(I:C),(LPIC)and bacterial components(lipopolysaccharide(LPS),flagellin and Gram-positive bacteria cell wall component Curdlan)as stimulus.MS19(1mM)or SAT05F(1mM)transfected with Lipofectamine 3000(Lipo 3000)into cells and then incubated with different stimulus for 16 h.Cells with transfect Lipo 3000 were as medium control.The concentration of TNF-aand IL-6 in the supernatant was detected by ELISA.The results showed that MS19 and SAT05F could significantly inhibit the TNF-αand IL-6 production from BMDMs triggered by DNA virus(VACV-70,HSV,ADV),RNA virus(PR8,Reov,LPIC)and LPS(P<0.01).Notably,MS19 and SAT05F failed to inhibit the IL-6 production from BMDMs stimulated by curdlan,a bacterial cell wall component(P>0.05).Additionally,MS19 and SAT05f were capable of inhibiting IFN-asecretion of BMDCs induced by influenza virus.Like BMDMs and BMDCs,MS19 and SAT05F can reduce the production of IFN-aand TNF-ain FLT3L~+cells in response to CpG A ODN and CpG B ODN and influenza virus.Based on these results,we found that MS ODNs(MS19 and SAT05F)displayed a strong inhibition role on the secretion of cytokines from BMDMs,BMDCs and FLT3L~+cells stimulated by various microbial pathogens,except for curdlan.2.The inhibitory effect of MS ODN and MS ODN mutant on production of inflammatory cytokines.from RAW264.7 cells stimulated by LPS.Single site mutants(G to C)were employed to measure sequence-dependent mechanism properties of AAAG-rich ODN(MS19)on inhibiting the production of inflammatory cytokines from innate immune cells.In the mouse RAW264.7macrophage cell line,the mRNA expression level of IL-6 and TNF-aof RAW264.7stimulated by LPS with or with not MS19 or MS19 mutant(MS19-M1)by RT-PCR.The results showed that MS19 could inhibit the production of IL-6 and TNF-afrom RAW 264.7 cells induced by LPS,while MS19-M1 could not.It suggested that G base is a key site in AAAG motif,which is associated with biological functions of MS19.3.Regulatory effect of MS ODN and MS ODN mutant on activation of p65 in NF-kB signaling pathway in RAW264.7 cells.Activation of p65 in response to a variety of stimuli is most typical process of NF-kB activated in RAW264.7 cells.To find the initial mechanism of MS ODN on inhibiting the production of pro-inflammatory cytokines in innate immune cells,we detected the expression of p65 phosphorylation in RAW264.7 cells stimulated by LPS with or with not MS19 or MS19 mutant(MS19-M1)by western blot.In contrast to the RAW264.7 cells treated with various LPS or VACV70 or influenza virus stimulation alone,the phosphorylation of p65 intensity was dramatically decreased in RAW264.7 cells treated simultaneously with MS19 and various stimulus.We speculated that the function of MS19 on inhibition of IL-6 and TNF-aproduction in innate immune cells relies on inhibiting the phosphorylation of p65 in the NF-κB signaling pathway.4.Inhibitory effect of MS19 on LPS-induced acute lung injury in miceUpon these,MS 19 could block the production of IL-6 and TNF-aby blocking the phosphorylation of p65 in macrophages and dendritic cells.Based on the published articles,a large number of innate immune cells like macrophages and dendritic cells harbor into the lung epithelial cells.When the immune system was over-activated,the localized macrophages and dendritic cells in the lung firstly secreted a large number of inflammatory factors that constitute to the acute inflamma-tory response.Here,we want to observe the effect of MS19 on LPS-induced acute lung injury in mice.On day 0,mice were inoculated intranasally with LPS(20 mg/kg)to establish LPS-induced acute lung injury model mice.The mice(n=8)sham-inoculated with sterile PBS were used as health control.On days 1 before infection,the model mice(n=8)were translocate with PBS or MS19 by mixing the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP).We observed the effect of MS19 on the morbidity,mortality,and lung inflammatory injury of mice induced by LPS.The results showed that the model mice received PBS displayed 13%mean weight loss on day 4 post infection,while model mice treated with MS19 only displayed 10%mean weight loss,indicated that MS19 significantly inhibit the weight loss of mice treated with LPS.Compared with the model mice treated with PBS,the mortality rates of the mice treated with MS19 successively increased from 62.5%to 87.5%.The lung of the mice treated with MS19 manifested thinner alveolar walls,lacking intra-alveolar edema fluid and infiltrated leukocytes.Furthermore,the pathological score of the lung tissue of MS19-treated mice was lower than that in the mice treated with PBS.Besides,MS19 significantly inhibit the mRNA expression levels of TNF-a,IL-6,IL-1bin the Bronchoalveolar lavage fluid(BALF)of model mice.In present study,we found that MS19 and SAT05f could significantly inhibit the production of IFN and inflammatory cytokine by blocking the activation of p65 in NF-kB signaling pathway in macrophages and dendritic cells induced by various stimulis in vitro.Meanwhile,in mice model of LPS-induced acute lung injury,we observed that MS19 inhibited the weight loss of mice,increase the survival rate of the mice and displayed dramatic effect on lessening the acute lung injury by decreasing the cytokines level in BALF and reducing consolidation,intra-alveolar edema and leukocyte infiltration in lungs of the mice.The data suggested that MS19 might be developed a candidate ODN for the treatment of over activated immune disease.更多还原