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
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Q522

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国家重点研发计划(2020YFA0112604)


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

    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(1 250 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。

    Abstract:

    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(1 250 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.

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周慧,王雁,李华,汪溪洁,陈华英,马璟.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[J].南京医科大学学报(自然科学版),2024,(2):154-161

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  • 收稿日期:2023-03-30
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  • 在线发布日期: 2024-02-05
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