一种循环高表达IL-6慢性系统性炎症小鼠模型的建立

Establishment of a mouse model of chronic systemic inflammation with high circulating IL-6

目的通过外源性引入IL-6载体,实现在循环中长期高表达IL-6,构建一种慢性系统性炎症的动物模型。方法构建重组鼠IL-6编码腺相关病毒(IL-6-encoding adeno-associated virus,AAV-IL-6)。24周龄雄性C57BL/6J小鼠随机分为AAV-IL-6组、空白载体对照组、空白对照组,每组各7只,分别在干预开始0、8、16周尾静脉注射100μl 0.5×10^(10) vp/ml的AAV-IL-6、空白载体AAV或相同体积的生理盐水。ELISA检测小鼠血清IL-6水平。观察小鼠一般情况、血常规、血生化、C反应蛋白(C-reactive protein,CRP)。在24周干预结束后处死小鼠,取心肌、肝脏、脾脏、股四头肌、膝关节、股骨中段做HE染色。结果在干预后4、8、16及24周,AAV-IL-6组小鼠血清IL-6水平为(75.41~169.28)pg/ml,而两对照组均低于检测下限(7.8 pg/ml)。干预后24周,AAV-IL-6组小鼠体重明显低于两对照组;AAV-IL-6组小鼠中性粒细胞计数和CRP水平均高于两对照组,而白蛋白、肌酐、甘油三酯和胆固醇水平低于两对照组,上述指标在对照组之间差异无统计学意义;AAV-IL-6组及空白载体对照组小鼠淋巴细胞均高于空白对照组;3组小鼠肝、肾功能在干预结束后均正常。组织病理显示,AAV-IL-6组小鼠肝脏中央静脉区及心肌肌纤维间隙、骨骼肌肌纤维间隙灶性淋巴细胞浸润,脾脏弥漫性多核巨细胞浸润;骨骼肌肌纤维萎缩;骺板结构紊乱和软骨增生区变小,骨小梁变薄、稀疏;骨皮质变薄、类骨质减少,两对照组小鼠无相应的组织病理改变表现。结论通过重复尾静脉注射AAV-IL-6能够实现小鼠长期循环高表达IL-6,初步检测该小鼠具有慢性系统性炎症表型,为相关研究提供了一种安全、有效且相对简单可及的动物模型。

Objective To establish an animal model of chronic systemic inflammation with long-term high expression of circulating IL-6 by introducing exogenous IL-6 gene transfer vector.Methods Recombinant murine IL-6-encoding adeno-associated virus(AAV-IL-6)was constructed.Twenty-one 24-week-old male C57BL/6J mice were randomly divided into three groups with seven in each group:AAV-IL-6 group,vector control(AAV-ctrl)group and blank control group.At 0,8 and 16 weeks of intervention,the mice in the three groups were injected with AAV-IL-6(100μl 0.5×1010 vp/ml),unloaded AAV(100μl 0.5×10^(10) vp/ml)and the same volume of saline in the tail vein,respectively.IL-6 levels in mouse serum were measured by ELISA.The general condition of mice was observed and blood routine tests were performed.Changes in blood biochemical parameters and C-reactive protein(CRP)levels were detected.At the end of 24-week intervention,the mice were sacrificed and the myocardium,liver,spleen,quadriceps femoris,knee joint and middle femur were taken for HE staining.Results At 4,8,16 and 24 weeks after intervention,serum IL-6 levels were(75.41-169.28)pg/ml in the AAV-IL-6 group,while in the two control groups,the levels were below the lower limit of detection(7.8 pg/ml).At 24 weeks after intervention,the body weight of mice in the AAV-IL-6 group was significantly lower than that of mice in the two control groups;the neutrophil counts and CRP level in the AAV-IL-6 group were higher than those in the two control groups,while the levels of albumin,creatinine,triglyceride and cholesterol were lower than those in the two control groups.There were no differences in the aforementioned parameters between the two control groups.Compared with the blank control group,both AAV-IL-6 and AAV-ctrl groups showed increased lymphocyte counts.All mice had normal liver and kidney functions at the end of intervention.Histopathological findings indicated that the mice in the AAV-IL-6 group had focal infiltration of lymphocytes in the central venous area of the liver and around the myocardial and the skeletal muscle fibers,diffuse infiltration of multinucleated giant cells in the spleen,atrophic skeletal muscle,disorganized growth plate,reduced chondrocyte hypertrophic zone,thinner bone cortex and trabecular,and reduced osteoid.There were no histopathological changes in mice of the two control groups.Conclusions Repeated tail vein injection of AAV-IL-6 could achieve long-term high expression of circulating IL-6 in mice,which manifested the phenotype of chronic systemic inflammation in preliminary detection and provided a safe,effective and simply accessible animal model for related studies.