循环miR⁃124作为铅神经毒性标志物的灵敏性、稳定性和组织特异性研究

Study on the sensitivity,stability and tissue specificity of circulating miR⁃124 as a biomarker of lead neurotoxicity

目的:验证循环微小核糖核酸(microRNA,miRNA)⁃124的稳定性、组织特异性及其检测铅神经毒性的灵敏性,为循环miR⁃124用于铅神经毒性评价提供依据。方法:采用Zea⁃Longa线栓法建立大鼠大脑中动脉闭塞模型,采集血液样本离心、分装后立即检测或在室温、2~8℃和-70~-90℃条件下储存不同时间后,采用实时荧光定量聚合酶链式反应(quantitative real⁃time polymerase chain reaction,RT⁃qPCR)检测miR⁃124的表达;分别使用对乙酰氨基酚(1250 mg/kg)、异丙肾上腺素(2.5 mg/kg)和庆大霉素(80 mg/kg)建立大鼠肝毒性、心脏毒性和肾毒性模型,采集血液样本检测并比较造模前后循环miR⁃124的变化;使用醋酸铅(300、600 mg/kg)建立大鼠神经毒性模型,采用酶联免疫吸附测定方法(enzyme linked immunosorbent assay,ELISA)检测白细胞介素(interleukin,IL)⁃10、IL⁃1β和肿瘤坏死因子⁃α(tumor necrosis factorα,TNF⁃α)的变化,比较检测到细胞因子和循环miR⁃124含量变化的时间评估其灵敏性。结果:以新鲜血液样本作为对照,血液样本室温保存6 h,2~8℃保存24 h,-70~-90℃保存36 d以及3次冻融后循环miR⁃124仍保持稳定,稳定性可以支持实验室需求;循环miR⁃124在大鼠肝毒性、心脏毒性和肾毒性模型中均无明显改变,具有良好的组织特异性;与细胞因子相比,循环miR⁃124可以更灵敏地检测铅暴露引起的神经炎症。结论:循环miR⁃124具有良好的稳定性、组织特异性和灵敏性,可作为评价铅神经毒性的潜在的生物标志物。

Objective:To validate the stability,tissue specificity,and sensitivity of circulating miR⁃124 for detecting lead neurotoxicity,so as to provide a basis for the evaluation of lead neurotoxicity using circulating miR⁃124.Methods:A rat middle cerebral artery occlusion model was established using Zea⁃Longa line plug method.Blood samples were collected,centrifuged,and aliquoted,then immediately tested or stored at different times under conditions of room temperature,2℃to 8℃,and-70℃to-90℃the expression of miR⁃124 was detected by RT⁃qPCR method.Hepatotoxicity,cardiotoxicity and nephrotoxicity models were established with acetaminophen(1250 mg/kg),isoprenaline(2.5 mg/kg)and gentamycin(80 mg/kg),respectively.The expression of circulating miR⁃124 was detected and compared in the collected pre⁃modeling and post⁃modeling blood samples.Rat neurotoxicity models were established with lead acetate(300 and 600 mg/kg).ELISA method was used to detect the changes of IL⁃10,IL⁃1β,and TNF⁃α.The sensitivity was evaluated by comparing the time of changes detected of cytokines and circulating miR⁃124.Results:Compared with fresh blood samples,circulating miR⁃124 remained stable when the blood samples were stored at room temperature for 6 h,at 2℃to 8℃for 24 h,at-70℃to-90℃for 36 d,and for three freeze⁃thaw cycles.The stability could support the requirement so flaboratory.Circulating miR⁃124 has good tissue specificity since no significant changes were noted in rat hepatotoxicity,cardiotoxicity and nephrotoxicity models.Compared with the cytokines,circulating miR⁃124 could evaluate neuroinflammation caused by lead exposure with a more sensitive manner.Conclusion:Circulating miR⁃124 has good stability,tissue specificity and sensitivity,and can be used as a potential biomarker of evaluating lead neurotoxicity.