基于TLR4-Ig融合蛋白探究TLR4信号在溃疡性结肠炎中的关键作用  

To study the key role of TLR4 signal in ulcerative colitis using TLR4-Ig fusion protein

作　　者：钱珂文 王楚棋 张淑怡 李光耀 邹宜覃 郑欣亚 艾泓如 傅文燕 雷长海 胡适 QIAN Kewen;WANG Chuqi;ZHANG Shuyi;LI Guangyao;ZOU Yitan;ZHENG Xinya;AI Hongru;FU Wenyan;LEI Changhai;HU Shi(Department of Biomedical Engineering,College of Basic Medical Sciences,Naval Medical University(Second Military Medical University),Shanghai 200433,China;Department of Pharmacy,National University of Singapore,Singapore 119077,Singapore;Department of Biophysics,College of Basic Medical Sciences,Naval Medical University(Second Military Medical University),Shanghai 200433,China;Department of Assisted Reproduction,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China)

机构地区：[1]海军军医大学(第二军医大学)基础医学院生物医学工程教研室,上海200433 [2]新加坡国立大学药学系,新加坡119077 [3]海军军医大学(第二军医大学)基础医学院生物物理学教研室,上海200433 [4]上海交通大学医学院附属第九人民医院辅助生殖科,上海200011

出　　处：《海军军医大学学报》2024年第5期535-543,共9页Academic Journal of Naval Medical University

基　　金：国家自然科学基金(82322055,82272792,81903140,92169115).

摘　　要：目的探究Toll样受体4(TLR4)信号与溃疡性结肠炎(UC)发生、发展的关系。方法收集UC患者结直肠活检组织样本(n=63),根据Matts组织病理学分级标准分为1~5级,利用免疫组织化学染色检测TLR4表达情况。利用FreeStyle 293表达系统制备TLR4-Ig融合蛋白。体外培养健康者来源的外周血单个核细胞(PBMC),在培养基中添加100μg/mL脂多糖诱导炎症模型,并加入100μg/mL TLR4-Ig阻断TLR4,检测粒细胞-巨噬细胞集落刺激因子、干扰素γ、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10和TNF-α的分泌水平。以葡聚糖硫酸钠(DSS)诱导小鼠急性结肠炎模型,分别采用20 mg/kg TLR4-Ig、40 mg/kg TLR4-Ig、抗生素+40 mg/kg TLR4-Ig进行干预,取小鼠结肠组织测量结肠长度并进行组织病理学评估。结果UC患者结肠组织Matts分级越高TLR4高表达的样本比例越高(P<0.001)。TLR4-Ig融合蛋白能够以高亲和力结合TLR4配体,阻断TLR4信号介导的PBMC活化和炎症因子分泌。TLR4-Ig融合蛋白对TLR4信号的阻断加重了DSS诱导的小鼠急性结肠炎,而抗生素处理则对TLR4信号阻断造成的小鼠急性结肠炎加重有缓解作用。结论TLR4信号与肠道菌群的信号交流是UC发生早期的重要保护机制,TLR4信号的缺失不利于缓解急性炎症和修复肠黏膜。Objective To investigate the correlation between Toll-like receptor 4(TLR4)signal and the development and progression of ulcerative colitis(UC).Methods The colon biopsy samples(n=63)were collected from UC patients and were divided into Matts’histological grade 1-5.The TLR4 expression level was detected by immunohistochemistry.TLR4-immunoglobulin(Ig)fusion protein was prepared using FreeStyle 293 expression system.Peripheral blood mononuclear cells(PBMCs)from healthy individuals were cultured in vitro,100μg/mL lipopolysaccharide was added to induce inflammation,and 100μg/mL TLR4-Ig was added to block TLR4.Then the secretion levels of 10 different inflammatory cytokines,including granulocyte-macrophage colony-stimulating factor,interferonγ,interleukin(IL)-1β,IL-2,IL-4,IL-5,IL-6,IL-8,IL-10,and tumor necrosis factorα,were detected.The acute colitis mouse model was induced by dextran sulfate sodium(DSS),and was intervened with 20 mg/kg TLR4-Ig,40 mg/kg TLR4-Ig or antibiotics+40 mg/kg TLR4-Ig,respectively.Then the colon length of mice was measured and histopathological evaluation was performed.Results The expression level of TLR4 in colon tissues of UC patients was positively correlated with Matts’histological grades(P<0.001).TLR4-Ig fusion protein could bind TLR4 ligands with high affinity,blocking the activation of PBMCs and the secretion of inflammatory cytokines mediated by TLR4 signal.The blocking of TLR4 signal by TLR4-Ig fusion protein aggravated DSS-induced acute colitis in mice,and treatment with antibiotics alleviated the aggravation of the colitis caused by TLR4 signal blocking.Conclusion The communication between TLR4 signal and intestinal flora is an important protective mechanism at the early stage of UC.The absence of TLR4 signal is not conducive to relieving acute inflammation and repairing intestinal mucosa.

关 键 词：TOLL样受体4 溃疡性结肠炎 抗体融合蛋白 免疫组织化学 葡聚糖硫酸钠 

分 类 号：R574.62[医药卫生—消化系统]