慢性肾脏病血管钙化大鼠血清炎症因子抗体芯片检测及分析

Antibody microarray analysis of serum inflammatory cytokines in rats with chronic renal disease vascular calcification

目的应用抗体芯片技术检测慢性肾脏病(chronic renal disease,CKD)血管钙化大鼠模型血清中的炎症细胞因子水平,并探讨炎症与血管钙化之间的关系。方法 30只SD雄性大鼠分成对照组和模型组,每组15只。模型组大鼠联合腺嘌呤灌胃和阿霉素尾静脉注射建立大鼠慢性肾脏病血管钙化模型;对照组大鼠给予相应的生理盐水处理。给药28 d后取材检测肾功能、电解质等生化指标,天狼猩红染色法检测肾纤维化病变,HE染色检测血管病理改变,von Kossa染色法检测血管钙化程度,免疫组织化学染色法检测平滑肌肌动蛋白α(α-SMA)及Runt相关转录因子2(Runx2)表达。细胞因子抗体芯片检测两组大鼠血清中27种细胞因子水平的变化。结果模型组大鼠血肌酐、尿素氮及钙磷水平升高;天狼猩红染色显示模型组大鼠肾有明显的纤维化病变; HE染色显示主动脉中膜有弹性纤维断裂; von Kossa染色显示模型组大鼠主动脉中膜层有大量黑色颗粒沉积;免疫组织化学染色显示模型组较对照组α-SMA表达减少而Runx2表达增多。抗体芯片结果显示模型组CINC、GM-CSF、IFN-γ、IL-1α、IL-6、MCP-1、ICAM-1、TIMP-1、L-selectin、IL-2及IL-10较对照组均有不同程度的升高,而IL-4、IL-13、fractaikine、IL-1β、TNF-α较对照组降低。结论慢性肾脏病血管钙化大鼠发生了明显的炎症反应失衡,其中致炎因子占主导作用。

Objective To detect and analyze the expressions of serum inflammatory factors in chronic renal disease(CKD)rats with vascular calcification by antibody microarrays.Methods Thirty SD male rats were randomly divided into control and model groups.The model group was treated with adenine gavage and adriamycin caudal intravenous injection to establish a model of CKD with vascular calcification,while the control group was given corresponding saline treatment.Twenty-eight days after administration,the levels of serum creatinine,urea nitrogen,blood calcium and blood phosphorus were measured.Sirius red staining,HE and von Kossa staining were used to detect morphological changes in renal and aortic tissues.Immunohistochemical staining was used to detect the expressions and distributions ofα-smooth muscle actin(α-SMA)and Runt related transcription factor 2(Runx2)in the aorta.Antibody microarray was used to detect 27 inflammatory cytokines in serum samples from the two groups.Results Compared with the control group,serum creatinine,urea nitrogen,calcium,and phosphorus levels were increased.Sirius red staining showed marked renal fibrosis,and HE and von Kossa staining showed elastic fiber rupture and calcium deposition in aortic media.Immunohistochemical staining showed that the expression ofα-SMA was decreased and that of Runx2 was increased in the model group compared with the control group.Antibody microarray results showed that compared with the control group,the levels of CINC,GM-CSF,IFN-γ,IL-1α,IL-6,MCP-1,ICAM-1,TIMP-1,L-selectin,IL-2 and IL-10 were increased by different degrees from those in the control group,while IL-4,IL-13,fractalkine,IL-1βand TNF-αwere lower than in the control group.Conclusions There is a significant imbalance in the levels of inflammatory factors in CKD rats with vascular calcification,in which proinflammatory factors play a leading role.