目的 構(gòu)建BALB/c小鼠免疫復(fù)合物性腎損傷模型，探討觀察此類腎損傷的早期、簡便、靈敏檢測方法和候選生物標(biāo)志物，為生物技術(shù)藥物非臨床安全性評價毒性試驗中腎損傷檢測提供參考。方法 BALB/c小鼠隨機(jī)分為溶媒對照組和陽離子化牛血清白蛋白（C-BSA）低、中、高劑量造模組，每組10只。誘導(dǎo)免疫時，造模組動物給予2 mg·mL^-1 C-BSA和弗氏不完全佐劑 1∶1等體積混合乳劑（1 mg·mL^-1、5 mL·kg^-1），而溶媒對照組動物給予磷酸鹽（PBS）緩沖液與弗氏不完全佐劑 1∶1等體積混合乳劑，sc誘導(dǎo)免疫，每周1次，共2次；加強(qiáng)免疫時，各組動物均尾iv給予C-BSA，溶媒對照組和C-BSA低、中、高劑量組劑量分別為 0、2.5、5.0、10.0 mg·kg^-1，每 3天給予 1次，共 5次。造模期末，檢測造模期末小鼠 24 h尿蛋白含量；檢測凝血指標(biāo)，包括凝血酶原時間（PT）、活化部分凝血活酶時間（APPT）以及纖維蛋白原含量（Fbg）；檢測血清血尿素氮（BUN）、血肌酐（SCr）以及三酰甘油（TG）水平；摘取主要臟器稱質(zhì)量并計算臟體比、臟腦比；HE染色和特殊染色［過碘酸-希夫（PAS）染色、Masson三色染色、過碘酸六胺銀（PASM）染色］后，光學(xué)顯微鏡下檢查腎組織形態(tài)學(xué)改變；免疫組化觀察腎臟補(bǔ)體成分3（C3）、免疫球蛋白G（IgG）、結(jié)蛋白水平；免疫熒光觀察腎臟C3水平；透射電子顯微鏡觀察腎臟腎小球基底膜、足細(xì)胞足突的變化及毛細(xì)血管內(nèi)皮細(xì)胞下有無電子致密物沉積。結(jié)果 C-BSA各劑量組動物在尾iv給予C-BSA后出現(xiàn)不同程度精神萎靡、活動減少、皮膚發(fā)紅和呼吸急促等過敏反應(yīng)癥狀，溶媒對照組動物給藥后無明顯異常；在加強(qiáng)免疫2周后，與溶媒對照組比較，C-BSA低、中和高劑量組動物體質(zhì)量增長顯著緩慢（P＜0.05）；C-BSA低、中、高劑量組動物尿蛋白呈陽性。與溶媒對照組比較，C-BSA各組動物 BUN、SCr、TG 均未見規(guī)律改變，僅高劑量組動物的 BUN 升高，TG 降低；C-BSA 中 、高劑量組動物 PT 顯著降低（P＜0.05），低、中劑量組動物 APPT 顯著降低（P＜0.05），低、中、高劑量組動物Fbg含量顯著降低（P＜0.001）；C-BSA低、中、高劑量組動物腎臟的臟體比顯著升高（P＜0.01），且低、中和高劑量組的脾臟絕對質(zhì)量和臟體比亦顯著升高（P＜0.05、0.01）。腎臟組織病理學(xué)檢查及特殊染色檢查發(fā)現(xiàn)C-BSA中、高劑量組動物腎小球系膜基質(zhì)增加、基底膜增厚；免疫組化結(jié)果顯示，與溶媒對照組比較，C-BSA低、中、高劑量組動物的C3、IgG、結(jié)蛋白表達(dá)水平均顯著升高（P＜0.001），高劑量組 IgG水平顯著高于低、中劑量組（P＜0.05）；免疫熒光結(jié)果顯示，與溶媒對照組比較，C-BSA高劑量組C3水平顯著升高（P＜0.001）；透射電鏡結(jié)果顯示C-BSA低、中、高劑量組動物腎臟腎小球基底膜增厚、足細(xì)胞足突部分融合，C-BSA中、高劑量組動物腎臟腎小球基底膜可見少量電子致密物沉積。結(jié)論 成功建立了免疫復(fù)合物性腎損傷模型和早期、簡便、靈敏的檢測方法，C3、IgG和結(jié)蛋白可作為生物技術(shù)藥物非臨床安全性評價毒性試驗中腎損傷檢測的候選生物標(biāo)志物。

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 microscopy (TEM) was used to observe the changes in the glomerular basement membrane and foot process of podocytes of the kidneys, as well as the presence of electron dense deposits under capillary endothelial cells.Results The animals in each modeling group showed varying degrees of allergic reaction symptoms such as lethargy, decreased activity, skin redness, and short breath after administration of C-BSA via intravenous injection, whereas the animals in the solvent control group showed no significant abnormalities after the administration. After two weeks of booster immunization, compared with the vehicle control group, the animals in the low, medium, and high dose modeling group showed significant slow body mass growth (P < 0.05). The low, medium, and high dose modeling groups of C-BSA showed positive urine protein. Compared with the solvent control group, there were no regular changes in BUN, SCr, and TG in the animals of modeling groups. Only the animals of high dose modeling group showed an increase in BUN and a decrease in TG. PT of animals in the medium and high dose modeling groups was significantly reduced (P < 0.05), APPT of animals in the low and medium dose modeling groups was significantly reduced (P < 0.05), and the Fbg of animals in the low, medium, and high dose modeling groups was significantly reduced (P < 0.001). The organ weight to body weight ratio of the kidneys in the low, medium, and high dose modeling groups was significantly increased (P < 0.01), and the absolute mass and organ weight to body weight ratio of the spleen in the low, medium, and high dose modeling groups were also significantly increased (P < 0.05, 0.01). Histopathological examination of kidneys showed that the increased mesangial matrix and thickened basement membrane of animals in the middle and high dose modeling groups. The immunohistochemical examination results showed that compared with the vehicle control group, the expression levels of C3, IgG, and desmin in the low, medium, and high dose modeling groups were significantly increased (P < 0.001), while the IgG levels in the high dose modeling group were significantly higher than those in the low and medium dose modeling groups (P < 0.05). The immunofluorescence examination results showed that compared with the vehicle control group, the high dose modeling group had a significant increase in C3 levels (P < 0.001). The TEM results showed that animals in the low, medium, and high dose modeling groups had thickened glomerular basement membrane and partially fused foot processes of podocytes. A small amount of electron dense material was observed in the glomerular basement membrane of animals in the medium and high dose modeling groups.Conclusion An immune complex-induced renal injury model, as well as early, simple and sensitive detection methods were successfully established. C3, IgG and desmin might be candidate biomarkers for detecting immune complexinduced renal injury in the toxicity studies during nonclinical safety evaluation of biopharmaceuticals.

R965.3

江蘇省新藥一站式高效非臨床評價公共服務(wù)平臺建設(shè)（BM2021002）