鼠源性髓过氧化物酶特异性抗中性粒细胞胞质抗体的制备及鉴定  

Preparation and identification of mouse myeloperoxidase specific anti-neutrophil cytoplasmic antibody

作　　者：安晓宁 魏兆楠 倪莉燕[1] 沈艳 陈永熙[1] AN Xiao-ning;WEI Zhao-nan;NI Li-yan;SHEN Yan;CHEN Yong-xi(Department of Nephrology,Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Experimental Medical Research Center,Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China)

机构地区：[1]上海交通大学医学院附属瑞金医院肾脏科,上海200025 [2]上海交通大学医学院附属瑞金医院实验医学研究中心,上海200025

出　　处：《现代免疫学》2022年第3期218-223,共6页Current Immunology

基　　金：国家自然科学基金青年基金(81000285);国家自然科学基金面上项目(81470041)。

摘　　要：采用鼠源性髓过氧化物酶(myeloperoxidase,MPO)与弗氏佐剂混合注射的方式制备小鼠MPO特异性抗中性粒细胞胞质抗体(anti-neutrophil cytoplasmic antibody,ANCA),并予以定性及定量检测。使用B6.129X1-Mpo^(tm1Lus)/J小鼠(即MPO敲除小鼠),将mMPO与完全/不完全弗氏佐剂混合,通过腹腔注射方式诱导小鼠产生适应性免疫应答。采用定性ELISA以及间接免疫荧光法检测小鼠血清ANCA水平;采用肌酐检测试剂盒(肌氨酸氧化酶法)检测小鼠血清肌酐水平以评估其肾功能。ELISA检测结果显示,实验组小鼠血清MPO-ANCA的光密度D(450 nm)值较对照组显著升高(P<0.001),约为对照组的5.6倍;间接免疫荧光法结果提示,实验组中性粒细胞胞质有较强的荧光信号,与MPO-ANCA的核周型特点相一致;H-E染色示部分肾小球囊壁层上皮细胞过度增殖;实验组与对照组小鼠血清肌酐水平检测结果差异不显著(P>0.05)。由此,注射MPO与弗氏佐剂混合液诱导MPO-/-小鼠产生了抗MPO-ANCA,ELISA和细胞免疫荧光实验均验证了抗体的生成,可为进一步构建实验性血管炎小鼠模型提供重要基础。To prepare mouse myeloperoxidase(mMPO)-specific anti-neutrophil cytoplasmic antibody(ANCA),B6.129X1-Mpo^(tm1Lus)/J mice(MPO knockout mice)were immunized with mMPO along with Freund's complete/incomplete adjuvant by intraperitoneal injection.Qualitative ELISA and indirect immunofluorescence assay were used to detect serum ANCA level.The serum creatinine(Scr)level of mice was detected by a creatinine kit(sarcosine oxidase method)to evaluate their renal function.The results showed that the optical density D(450 nm)value of serum MPO-ANCA in the experimental group was 5.6 times higher than that in the control group(P<0.001).An indirect immunofluorescence assay revealed that the cytoplasm of neutrophils in the experimental group had a strong fluorescence signal,which was consistent with the perinuclear characteristics of MPO-ANCA.The H-E staining showed partial overproliferation of glomerular wall epithelium.The results indicated no significant difference in Scr level between the two groups(P>0.05).In summary,the anti-MPO-ANCA antibody was successfully generated in MPO-/-mice upon MPO/Freund's adjuvant injection verified by ELISA and immunofluorescence.Our work provides an important basis for further construction of the experimental vasculitis mouse model.

关 键 词：髓过氧化物酶 抗中性粒细胞胞质抗体 血管炎 

分 类 号：R392-3[医药卫生—免疫学]