人硫氧还蛋白治疗小鼠溃疡性结肠炎的药效学作用  

The therapeutic effect of human thioredoxin on ulcerative colitis in mice

作　　者：周露露 刘兴 丁杨[1] 林琳[3] 华子春 ZHOU Lu-lu;LIU Xing;DING Yang;LIN Lin;HUA Zi-chun(School of Biopharmacy,China Pharmaceutical University,Nanjing 211198,China;The State Key Laboratory of Pharmaceutical Biotechnology,School of Life Sciences,Nanjing University,Nanjing 210023,China;School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China;High-tech Research Institute of Nanjing University at Changzhou and Jiangsu Target Pharma Laboratories Inc.,Changzhou 213164,China)

机构地区：[1]中国药科大学生物药物学院,江苏南京211198 [2]南京大学生命科学学院,医药生物技术国家重点实验室,江苏南京210023 [3]江苏大学食品与生物工程学院,江苏镇江212013 [4]常州南京大学高新技术研究院和江苏靶标生物医药研究所有限公司,江苏常州213164

出　　处：《药学学报》2022年第6期1816-1824,共9页Acta Pharmaceutica Sinica

基　　金：常州市科技局(CZ20210010,CJ20210024);江苏省前沿引领技术基础研究专项资助(BK20192005)。

摘　　要：本研究旨在利用基因工程技术表达重组人硫氧还蛋白(recombinant human thioredoxin,rhTXN),并在葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的小鼠溃疡性结肠炎模型上验证其治疗效果。从Jurkat细胞的cDNA中克隆出人硫氧还蛋白基因TXN,采用限制性酶切法构建pCold TF-rhTXN重组表达质粒,在大肠杆菌中表达后,利用镍柱纯化、再通过酶切去除融合表达标签、获得完整的rhTXN重组蛋白,并用胰岛素还原法检测其二硫键还原酶活性。本研究中的动物实验是按照南京大学实验动物福利伦理审查委员会的伦理指导方针进行的。3%DSS自由饮用诱导小鼠溃疡性结肠炎(ulcerative colitis,UC)模型,腹腔注射rhTXN,通过体重变化、结肠长度和苏木精-伊红(hematoxylin and eosin,HE)染色切片研究其治疗效果。利用活体成像研究rhTXN对DSS小鼠的靶向性。利用GEO数据库的GSE107499数据集筛选UC炎症部位的枢纽基因并研究与TXN的相关性。实验结果显示,具有活性的rhTXN成功表达和纯化,rhTXN(100μg·kg^(-1))在维持DSS小鼠体重变化(P=0.0005)和减少肠道损伤上(P<0.0001)有显著治疗效果,并对DSS小鼠有结肠靶向性。GSE107499数据集中UC患者炎症部位TXN有极显著的下调(P<0.01),并与枢纽基因CD40有极显著的负相关(P<0.01),与纤连蛋白1(fibronectin 1,FN1)有极显著的正相关(P<0.01)。本研究成功制备出具有生物活性的rhTXN,并证明对DSS小鼠模型有较好的治疗效果,而TXN基因与UC枢纽基因CD40和FN1有显著相关性。This study was designed to obtain recombinant human thioredoxin(rhTXN)by gene cloning and prokaryotic expression,and evaluated its therapeutic effect in the mouse ulcerative colitis(UC)model induced by dextran sulfate sodium(DSS).The human thioredoxin gene TXN was cloned from the cDNA of Jurkat cells.The recombinant expression plasmid pCold TF-rhTXN was constructed by restriction enzyme digestion.After expression in E.coli BL21(DE3),recombinant human thioredoxin was purified by a nickel column.Intact rhTXN recombinant protein was obtained after removal of the fusion partner-tag by enzyme digestion and the activity of disulfide reductase was detected by the insulin reduction method.The animal experiments in this study were performed in accordance with the ethical guidelines of the Laboratory Animal Welfare Ethical Review Committee of Nanjing University.Experiment ulcerative colitis was induced by providing mice with sterilized drinking water which contained 3%DSS.rhTXN was injected intraperitoneally.The therapeutic effect was studied by weight change,colon length and HE(hematoxylin and eosin)stained sections.In vivo imaging was used to study the targeting of rhTXN to DSS mice.The GSE107499 data set of GEO database was used to screen the hub genes at the lesional sites of UC and study the correlation with TXN.The experimental results showed that rhTXN was successfully expressed and purified with disulfide reductase activity.rhTXN(100μg·kg^(-1))had a significant therapeutic effect on maintaining the weight change of mice(P=0.0005)and reducing intestinal injury(P<0.0001),and had a colon targeting effect on DSS mice.In GSE107499 data set,TXN in inflammatory sites of UC patients was significantly down regulated(P<0.01)and negatively correlated with hub gene CD40(P<0.01)and positively correlated with hub gene fibronectin 1(FN1)(P<0.01).In this study,biologically active rhTXN was successfully prepared and proved to have a promising therapeutic effect on the DSS mouse model,and TXN gene was significantly correlated with

关 键 词：重组人硫氧还蛋白 蛋白纯化 溃疡性结肠炎 葡聚糖硫酸钠 枢纽基因 

分 类 号：R967[医药卫生—药理学]