适合免疫治疗的大鼠抗肾小球基底膜肾炎模型的建立

Establishment of Anti-glomerular Basement Membrane Nephritis Model Suitable for Immunotherapy in Rats

以线性表位肽P14(a3_(127-148))作为抗原建立适合免疫治疗的抗肾小球基底膜(anti-glomerular basement membrane,GBM)病大鼠模型。采用大鼠后脚垫三点注射P14(a3_(127-148))与弗氏佐剂乳化物的方法进行单次免疫,免疫前后每周采集24 h尿样和血样,所有大鼠在免疫后7 w处死。大鼠免疫后,肾炎模型组在各时间点的24 h尿蛋白、尿蛋白肌酐比值(albumin/urine creatinine ratio,ACR)、血肌酐及血尿素氮均明显高于对照组(P<0.05);循环抗P14(a3_(127-148))IgG抗体水平明显高于对照组(P<0.001);PAS染色可见节段性纤维素样坏死,富于细胞型新月体;免疫组化染色可见肾小球有明显的中性粒细胞和巨噬细胞浸润;免疫荧光检测可见IgG沿GBM呈强线性沉积;电镜观察到GBM的断裂和收缩;而对照组均未见改变。HE染色在所有大鼠中均未观察到肺部病变。使用P14(a3_(127-148))线性肽免疫WKY大鼠成功建立了大鼠抗GBM病模型,有助于开发更为特异的免疫疗法。

Linear epitope peptide P14(a3_(127-148))was used as antigen to establish the rat model of anti-glomerular basement membrane(GBM)disease suitable for immunotherapy.Rats were immunized with P14(a3_(127-148))and Freund's adjuvant emulsion at three points on the rear foot pad.24 hours urine and blood samples were collected per week before and after immunization.All rats were killed 7 weeks after immunization.After immunization,24 h urinary protein,albumin/urine creatinine ratio(ACR),serum creatinine and blood urea nitrogen in nephritis model group were significantly higher than those in control group(P<0.05).The level of circulating anti-P14(a3_(127-148))IgG antibody was significantly higher than that of control group(P<0.001).PAS staining showed segmental-cellular-like necrosis,rich in cellular crescents.Immunohistochemical staining showed obvious infiltration of neutrophils and macrophages in the glomerulus.Immunofluorescence detection showed strong linear deposition of IgG along GBM.The fracture and contraction of GBM were observed by electron microscope.No change was observed in the control group.No lung lesion was observed in all rats by HE staining.WKY rats were successfully immunized with P14(a3_(127-148))linear peptide to establish a rat model of anti-GBM disease,which would be helpful for the development of more specific immunotherapy.