【作者】 张涛；

【导师】 钱峰；

【作者基本信息】 上海交通大学 ， 药学， 2020， 硕士

【摘要】 炎症性肠病（Inflammatory bowel disease,IBD）包括克隆恩病（Crohn’s disease,CD）和溃疡性结肠炎（ulcerative colitis,UC）,是一类慢性自发性胃肠道炎症疾病,其主要临床表现为腹痛、腹泻、血便、体重减轻等一系列相关症状。炎症性肠病发病机制复杂且尚不明确,一般认为是由于肠道菌群激发免疫系统产生过度炎症引起的疾病,与基因的易感性和环境因素有关。近年来,炎症性肠病的发病率逐步上升,而临床缺乏有效治疗手段。因此,探究炎症性肠病的发病机制,鉴定潜在的药物靶标,开发有效的治疗药物对于炎症性肠病至关重要。蛋白激酶MK2是p38/MAPK信号的重要下游蛋白激酶,参与调控细胞因子产生以及相关转录因子活化,是机体调控炎症反应的关键分子。我们利用葡聚糖硫酸钠（dextran sulfate sodium,DSS）在MK2髓系基因敲除小鼠(MK2^Lyz2-KO)和同窝对照小鼠(MK2^Lyz2-WT)诱导炎症性肠病模型,探讨髓系细胞中MK2是否调控炎症性肠病。结果发现,经DSS诱导后,相较于MK2^Lyz2-WT小鼠,MK2^Lyz2-KO小鼠体重指数下降幅度较小,结肠长度更长,结肠组织中炎症因子释放更少,活性氧（reactive oxygen species,ROS）和髓过氧化物酶（myeloperoxidasedeficiency,MPO）水平更低。这些结果表明髓系细胞MK2基因缺失可以缓解DSS诱导的炎症性肠病。活性氧（reactive oxygen species,ROS）是一类广泛存在与生物体内的氧的单电子还原产物。ROS的过量产生以及氧化应激损伤一直被认为是促进炎症性肠病发病的重要因素之一。在本研究中,在DSS诱导的炎症性肠病模型中,髓系MK2基因敲除鼠结肠组织损伤较轻,隐窝结构更完整,杯状细胞数量更多,同时ROS的产生也较于对照组大量减少。提示髓系MK2基因敲除鼠炎症性肠病的损伤减轻可能与ROS产生减少有关。嗜中性粒细胞是ROS的重要来源。体外实验表明,嗜中性粒细胞MK2基因缺失下调f MLF诱导的小鼠骨髓来源的嗜中性粒细胞活性氧的产生。提示MK2可能通过调控嗜中性粒细胞产生ROS参与炎症性肠病。NOX-2一类主要存在于嗜中性粒细胞中的NADPH氧化酶,是影响嗜中性粒细胞活性氧产生的重要氧化酶之一。为进一步确认MK2对嗜中性粒细胞ROS产生的调控作用及机制,我们利用Western Blot检测蛋白激酶MK2对Akt/p38 MAPK/Mk2/NOX2(p47^phox)信号通路的影响,发现在f MLF刺激下,MK2缺失影响Akt、p38 MAPK、以及NOX2的关键组分p47^phox的磷酸化,最终影响NOX2的活性,降低ROS的产生,提示蛋白激酶MK2通过Akt/p38MAPK/Mk2/NOX2(p47^phox)信号通路调控嗜中性粒细胞ROS的产生。白细胞介素6（interleukin-6,IL-6）是一种与机体炎症与免疫反应密切相关的多效性细胞因子。在炎症性肠病模型中,我们发现细胞因子IL-6显著升高。因此,本研究通过制备IL-6抗体去探究其作为炎症性肠病治疗性抗体的可行性。本研究运用原核蛋白表达技术,表达纯化了人IL-6细胞因子。随后利用该重组蛋白免疫小鼠,采用杂交瘤技术获得抗人IL-6单克隆抗体并将其命名为9#01,并且利用蛋白免疫印迹法鉴定该抗体结合人IL-6的抗原表位,主要位于80-115位氨基酸之间。此外,验证该株单抗是否具有中和IL-6活性。将该抗体加入人源IL-6刺激的Hela细胞中发现,该抗体不能阻断IL-6引起的STAT1的磷酸化。综上所述,本课题制备了一种新型抗人IL-6单克隆抗体,不具有中和人IL-6作用,但可用来作为检测性抗体使用。综上所述,本研究一方面发现MK2可以调控嗜中性粒细胞ROS产生参与炎症性肠病的病理过程,Akt/p38 MAPK/MK2/p47^phox信号通路参与MK2调控嗜中性粒细胞在在炎症性肠病中的作用,干预这些潜在药物靶标分子可能治疗炎症性肠病。另一方面,本研究基于炎症性肠病IL-6升高这一发现,制备了一种新型抗人IL-6单克隆抗体,发现此抗体可以结合人IL-6的抗原表位但不具有中和人IL-6作用,因此可用来作为人IL-6检测性抗体使用。总而言之,本研究为探索炎症性肠病的治疗靶点及潜在药物提供了重要的理论依据。更多还原

【Abstract】 Inflammatory bowel diseases（IBD）,including Crohn’s disease（CD）and ulcerative colitis（UC）,are characterized by chronic uncontrolled intestinal inflammation.The patients with IBD display multiple symptoms such as weight loss,abdominal pain,recurrent diarrhoea and bleeding.IBD is a complex clinical disease which arises as a result of the interaction of genetic and environmental factors leading to immune responses and inflammation in the intestinal.Over the past half century,the incidence and prevalence of IBD showed a fast increasing tendency worldwide.In recent years,some progress has been made to treat IBD,but IBD remains an incurable and awful clinical problem.Thus,it is necessary to comprehensively understand under-lying mechanisms and potential treatment targets for IBD.Phosphorylated by p38 MAP kinase,MK2 is known to be involved in many cellular processes including stress and inflammatory responses,gene expression regulation and cell proliferation.In the present study,we used MK2^Lyz2-KO mice,a mouse strain with MK2 conditional deficiency in myeloid lineage cells,to identify whether the inhibition targeting MK2 in myeloid lineage cells could ameliorate IBD.We found that MK2conditional deficiency in myeloid lineage cells markedly alleviated colon damage and inflammation inflammatory reaction in a mouse IBD model.In response to DSS challenge,compared to MK2^Lyz2-WT mice,MK2^Lyz2-KOmice exhibited less damages of epithelium and goblet cells,lower concentration of IL-6,TNF-α,MPO and ROS and enhanced capability of cell proliferation.These data consistently suggested that MK2 in myeloid lineage cells contribute to the pathogenesis of IBD.Oxidative stress has been proved to involve in the pathogenesis of IBD,and ROS plays a vital role to damage intestinal tissues.In our study,we identified that MK2 in myeloid lineage cells contributed to the production of ROS in the pathogenies of IBD,causing persist intestinal tissue damage.In addition,we determined that MK2 in neutrophils was required for the production of neutrophil-derived ROS.Upon treatment with f MLF,the generation of ROS was attenuated in MK2 deficient neutrophils.Collectively,these findings indicated that MK2 was required for neutrophil-derived ROS production and inflammatory bowel disease.NOX-2 oxidase,one of the NADPH oxidase（NOX）isoforms,maily existed in neutrophils.As MK2^Lyz2-KO neutrophils produced less superoxide when stimulated by f MLF,we therefore detected the potential mechinams of ROS production regulated by MK2.Our results suggested that MK2 was required for f MLF-induced activation of Akt,p38 MAPK and NADPH oxidase in neutrophils,and MK2 ablation in the myeloid lineage cells had a negative impact on the activation of Akt,p38 MAPK and NADPH oxidase,resulting in the reduction of ROS production by neutrophils,therefore alleviating IBD.Interleukin-6（IL-6）,as a pleiotrpic cytokine,was involved in inflammation and immune response.In our DSS-induced IBD model,we found that the expression of IL-6 was at a high level.Therefore,we decided to development IL-6 antibody to explore the feasibility of it as a therapeutic antibody for IBD.We constructed the expression vector and expressed h-IL-6 protein.Subsequently,the recombinant protein and hybridoma technique were used to produce the anti-h IL-6 monoclonal antibody,which was named 9#01.We identified that the antigen epitopes of the antibody binding to h IL-6,mainly located between amino acids 80-115.In addition,we determined the neutralizing activity of 9#01 for h-IL-6.We found that9#01 couldn’t neutralize the h-IL-6 activity,which could induce the phosphorylation of STAT1 in Hela cell.In summary,we make a new anti-h IL-6 monoclonal antibody,which could not neutralize h IL-6.However,it can be used as a detection antibody.In summary,our studies identified critical roles of neutrophils’MK2 in accentuating intestinal mucosal inflammation through more production of pro-inflammatory cytokines and ROS.We also found that MK2 played a key role in ROS production through Akt and p38 MAPK signal pathways.In addition,we developed a new anti-h IL-6 antibody though without neutralizing activity.Collectively,our study provided an important theoretical basis for exploring therapeutic targets and potential drugs of inflammatory bowel disease.更多还原