BALB/c小鼠免疫复合物性肾损伤模型建立及肾损伤生物标志物初步探讨  

Establishment of a model of immune complex-induced renal injury in BALB/c mice and preliminary study of biomarkers of renal injury

作　　者：郑少秋 张亚群 缪成贤 李一昊 王娅 刘军 王先浩 王明 孙璐瑶 吕建军 汪溪洁 ZHENG Shaoqiu;ZHANG Yaqun;MIAO Chengxian;LI Yihao;WANG Ya;LIU Jun;WANG Xianhao;WANG Ming;SUN Luyao;LYU Jianjun;WANG Xijie(Jiangxi University of Traditional Chinese Medicine,Nanchang 330103,China;Innostar Biotechnology Nantong Co.,Ltd.,Nantong 226133,China;Yangtze Delta Advanced Research Institute,Yangtze Delta Pharmaceutical College Nantong,Nantong 226133,China;Shanghai Innostar Biotechnology Co.,Ltd.,Shanghai 201203,China)

机构地区：[1]江西中医药大学,江西南昌330103 [2]益诺思生物技术南通有限公司,江苏南通226133 [3]长三角药物高等研究院,长三角药物高等工程学院,江苏南通226133 [4]上海益诺思生物技术股份有限公司,上海201203

出　　处：《药物评价研究》2023年第5期934-943,共10页Drug Evaluation Research

基　　金：江苏省新药一站式高效非临床评价公共服务平台建设(BM2021002)。

摘　　要：目的构建BALB/c小鼠免疫复合物性肾损伤模型,探讨观察此类肾损伤的早期、简便、灵敏检测方法和候选生物标志物,为生物技术药物非临床安全性评价毒性试验中肾损伤检测提供参考。方法BALB/c小鼠随机分为溶媒对照组和阳离子化牛血清白蛋白(C-BSA)低、中、高剂量造模组,每组10只。诱导免疫时,造模组动物给予2 mg·mL^(−1) C-BSA和弗氏不完全佐剂1∶1等体积混合乳剂(1 mg·mL^(−1)、5 mL·kg^(−1)),而溶媒对照组动物给予磷酸盐(PBS)缓冲液与弗氏不完全佐剂1∶1等体积混合乳剂,sc诱导免疫,每周1次,共2次;加强免疫时,各组动物均尾iv给予C-BSA,溶媒对照组和C-BSA低、中、高剂量组剂量分别为0、2.5、5.0、10.0 mg·kg^(−1),每3天给予1次,共5次。造模期末,检测造模期末小鼠24 h尿蛋白含量;检测凝血指标,包括凝血酶原时间(PT)、活化部分凝血活酶时间(APPT)以及纤维蛋白原含量(Fbg);检测血清血尿素氮(BUN)、血肌酐(SCr)以及三酰甘油(TG)水平;摘取主要脏器称质量并计算脏体比、脏脑比;HE染色和特殊染色[过碘酸-希夫(PAS)染色、Masson三色染色、过碘酸六胺银(PASM)染色]后,光学显微镜下检查肾组织形态学改变;免疫组化观察肾脏补体成分3(C3)、免疫球蛋白G(IgG)、结蛋白水平;免疫荧光观察肾脏C3水平;透射电子显微镜观察肾脏肾小球基底膜、足细胞足突的变化及毛细血管内皮细胞下有无电子致密物沉积。结果C-BSA各剂量组动物在尾iv给予C-BSA后出现不同程度精神萎靡、活动减少、皮肤发红和呼吸急促等过敏反应症状,溶媒对照组动物给药后无明显异常;在加强免疫2周后,与溶媒对照组比较,C-BSA低、中和高剂量组动物体质量增长显著缓慢(P<0.05);C-BSA低、中、高剂量组动物尿蛋白呈阳性。与溶媒对照组比较,C-BSA各组动物BUN、SCr、TG均未见规律改变,仅高剂量组动物的BUN升高,TG降低;C-BSA中、Objective Construct an immune complex-induced renal injury model in BALB/c mice,explore early,simple,sensitive detection methods and candidate biomarkers for detecting such renal injury,and provide references for renal injury detection of biopharmaceuticals in toxicity studies during nonclinical safety evaluation.Methods BALB/c mice were randomly divided into solvent control group and C-BSA low,medium,and high dose modeling groups,with 10 mice in each group.During induction immunization,the animals of model groups were administrated with an emulsion made of a mixture of 2 mg·mL^(−1) C-BSA and equal volume of Freund's incomplete adjuvant via subcutaneous injection,while the animals of vehicle control group were given an emulsion made of a mixture of PBS buffer and equal volume of Freund's incomplete adjuvant.Once a week,twice in total.During booster immunization,all animals in each group were administered with 0,2.5,5.0,and 10.0 mg·kg^(−1) C-BSA via intravenous injection through tail vein for the vehicle control group and the low,medium,and high dose groups of C-BSA,respectively.Once every three days,five times in total.At the end of the modeling period,24-hour urine protein content of mice were detected.Coagulation indicators were also detected,including prothrombin time(PT),activated partial thromboplastin time(APPT),and fibrinogen(Fbg).Blood urea nitrogen(BUN),serum creatinine(SCr),and triglyceride(TG)levels were detected.The main organs were weighed and calculated the mass,organ weight to body weight ratio and organ weight to brain weight ratio.Slides of kidneys prepared with HE staining and special staining[periodic acid Schiff(PAS)staining,Masson trichrome staining,and periodic acid silver methenamine(PASM)staining],the morphological changes of renal tissue were examined under an optical microscope.Immunohistochemical examination of renal complement component 3(C3),immunoglobulin G(IgG),and desmin levels and immunofluorescence examination of renal C3 levels were carried out.Transmission electron micros

关 键 词：BALB/C小鼠 免疫复合物 肾损伤 阳离子化牛血清白蛋白 生物标志物 补体成分3 免疫球蛋白G 结蛋白 

分 类 号：R965.3[医药卫生—药理学]