土壤微塑料污染对高等动物免疫系统的毒性研究——以小鼠为例

As a new type of pollutant, microplastic has drawn more and more attentions recently. At present, there are many researches on marine microplastic pollution. However, few report have been reported on microplastic pollution and it’s ecotoxicology in the land, especially in the soil environment. And there is no relevant research on the immunotoxicity of microplastic to higher animals. In this study, Balb/c mice were used as model in laboratory simulation experiments about microplastic soil pollution. Fluorescence-labeled microplastic beads were added into quartz sand bedding and drinking water for mice feeding. And the toxic effects of different concentrations of microplastic on the murine immune system were observed. The distribution of microplastics with different particle sizes in organs was detected by the laser confocal microscopy. The organ injury and immune infiltration were detected by immunohistochemistry. The function of immune cells was analyzed by flow cytometry, ELISA and antibody binding assay. On this basis, the transcriptional levels of key functional genes and regulatory genes in immune cells were analyzed by high throughput sequencing technology in RNA transcriptional group. The research results will reveal the toxic effects of soil microplastic pollution to higher animal immune system, and clarify the toxic mechanism in gene, protein, cell, organ, and animal levels. This study will also supply a scientific basis of evaluating effects of soil microplastic pollution on human environmental health, predicting the environmental risk threshold of microplastic pollution and formulating the administrative regulation on this new type pollutant.

微塑料作为一种新型污染物越来越受到重视。当前关于海洋微塑料污染的研究较多，对陆地土壤环境中微塑料污染及生态毒理的研究则鲜有报道，而关于微塑料对高等动物免疫系统的毒性研究则未见报道。本研究采用土壤污染模拟试验，以Balb/c小鼠为模式生物，以荧光标记微塑料染毒石英砂垫料和饮水，考察不同浓度及粒径微塑料对小鼠免疫系统的毒性效应；以激光共聚焦显微镜检测不同粒径微塑料在脏器中的分布；以免疫组化检测脏器损伤和免疫浸润反应；以流式细胞术、ELISA、抗体结合试验分析免疫细胞功能的变化。在此基础上，利用RNA转录组高通量测序技术，解析免疫细胞中关键功能基因和调控基因转录水平的变化。研究结果可在基因、蛋白、细胞、器官、动物水平上揭示土壤微塑料污染对高等生物免疫系统的毒性效应，阐明其作用机制，从而为科学评估土壤微塑料污染对人类环境健康影响，预测微塑料污染环境风险阈值，制定新型污染物管理法规提供科学依据。

微塑料作为一种新型污染物越来越受到重视。当前关于海洋微塑料污染的研究较多，对陆地土壤环境中微塑料污染及生态毒理的研究则鲜有报道，而关于微塑料对高等动物免疫系统的毒性研究则未见报道。本研究采用污染模拟试验，以Balb/c小鼠脾淋巴细胞为模式免疫细胞，以聚苯乙烯微球PS-NP为微纳米塑料的代表，考察不同浓度及粒径微塑料对小鼠免疫系统的毒性效应特点及相关毒理机制。研究结果发现，PS-NP可被小鼠脾淋巴细胞内化进入细胞内部，其内化效率与粒径和表面电荷相关。不同粒径及不同电荷的PS-NP在不同浓度下均会对小鼠脾细胞造成影响。具体表现为影响细胞活力、诱导细胞凋亡、抑制细胞增殖、阻碍细胞活化及影响免疫反应功能等方面。在毒理机制研究方面，发现PS-NPs的毒性具有剂量相关性。不同粒径，不同电荷的PS-NP通过不同途径导致细胞活力降低，带负电和不带电的PS-NPs可导致ROS过量累积，带正电的PS-NP可能通过直接破坏线粒体膜造成线粒体膜电位降低。PS-NP均可抑制小鼠脾淋巴细胞表面标志物的表达，抑制CD8+T细胞因活化导致的分化，抑制淋巴细胞细胞因子分泌，下调免疫细胞激活与行使功能的重要通路PKCθ/NF-ĸB及IL-2R/STAT5相关蛋白的表达。本研究可在基因、蛋白、细胞、器官、动物水平上揭示微塑料污染对高等生物免疫系统的毒性效应，阐明其作用机制，从而为科学评估土壤微塑料污染对人类环境健康影响，预测微塑料污染环境风险阈值，制定新型污染物管理法规提供科学依据。