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探讨阳离子牛血清蛋白制备纯化工艺及大鼠动物模型验证

Preparation of cationic bovine serum albumin and establishment of rats model of membranous nephropathy
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摘要 目的 探讨阳离子牛血清蛋白(C-BSA)制备提纯技术并构建其膜性肾病大鼠模型效应平台。方法 采用改良Border法制备C-BSA,采用十二烷基硫酸钠毛细管电泳法测定C-BSA纯度,毛细管电聚焦电泳法测定C-BSA的等电点;用C-BSA混合弗氏佐剂对SD大鼠预免疫1周后,尾静脉注射连续造模4周后进行生化指标检查和病理组织学检查。结果 C-BSA制备产率可达99.1%,纯度为84.56%,等电点为9~10;4周后模型大鼠尿蛋白明显高于对照组(P<0.05),血浆白蛋白含量明显低于对照组(P<0.05);三酰甘油与总胆固醇含量与对照组相比均有明显增加(P<0.05),PASM、HE组织染色显示出类似于早期膜性肾病的病理特征,IgG免疫荧光染色则显示出沿毛细血管壁有大量颗粒状物沉积,足细胞的特异性分子CD2AP、Podocin和Nephrin荧光表达均减弱,透射电镜下显示造模组有明显的足突广泛融合、基底膜增厚免疫复合物团块状沉积。结论 在改良Border法的基础上增加小剂量乙二胺、增加透析频率容易获取高产率纯度及等电点更高的C-BSA,大鼠动物实验造模成功率及稳定性更佳。 Objective To prepare cationic bovine serum albumin (C-BSA) and verify the success of C-BSA in constructing the rat model of membranous nephropathy.Methods C-BSA was prepared by modified Border method.Sodium dodecyl sulfate capillary electrophoresis was used to determine the purity of C-BSA.The isoelectric point of C-BSA was determined by capillary isoelectric focusing electrophoresis.SD rats were pre-immunized for one week,followed by tail vein injection for 4 weeks before clinical index examination and histopathological examination.Results The yield of C-BSA reached 99.1%,the purity of C-BSA was 84.56%,and the isoelectric point was 9~10.After four weeks,the urine protein of the model rats was significantly higher than that of the control group (P <0.05),and the plasma albumin content was significantly lower than that of the control group (P <0.05).Both the triglyceride and total cholesterol content were both significantly higher than those of the control group (P <0.05).PASM and HE tissue staining showed features similar to early membranous nephropathy.IgG immunofluorescence staining showed a large amount of granular deposit along the capillary wall.Particular matter was deposited.The mRNA expressions of podocin,CD2AP and nephrin in the model group were lower than those in the control group.The transmission electron microscope showed obvious fusion and effacement of podocyte foot processes and the deposits in the basement membrance.Conclusion The Border method can be used to prepare cationic bovine serum albumin with high yield,and could successfully construct a rat model of membranous nephropathy.
作者 高青青 纪越 国大亮 戴智强 贾丽娟 杨洪涛 刘玉璇 GAO Qing-qing;JI Yue;GUO Da-liang;DAI Zhi-qiang;JIA Li-juan;YANG Hong-tao;LIU Yuxuan(Tianjin University of Traditional Chinese Medicine,Tianjin 301617;The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 300193)
出处 《中南药学》 CAS 2022年第11期2495-2500,共6页 Central South Pharmacy
基金 国家自然科学基金面上项目(No.81873151) 国家科技重大专项课题(No.2019YFC1709400) 天津市卫健委中医中西医结合课题(No.2021012) 天津市研究生科研创新项目(No.YJSKC-20211003)。
关键词 阳离子牛血清蛋白 制备纯化 膜性肾病 大鼠模型 cationic bovine serum protein preparation and purification membranous nephropathy rat model
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