得克隆 602的免疫干扰效应及小鼠肝免疫毒理细胞模型的建立
文献类型：学位论文
| 作者 | 田纪景 |
| 学位类别 | 博士后 |
| 答辩日期 | 2015-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赵斌 |
| 关键词 | 得克隆 602，免疫干扰效应，胸腺，脾脏，KC，原代培养，Dechlorane 602，immuno-distuption，thymus，spleen，primary Kupffer cell，culture |
| 学位专业 | 环境科学 |
| 中文摘要 | 得克隆类化合物，作为阻燃剂被广泛应用于工业生产和人们生活中。得克隆类化合物包括得克隆（DP）、得克隆 602、得克隆 603、得克隆 604等，已在多种环境介质、野生动物，甚至人体检出，说明其可能存在健康风险。但是目前几乎没有关于此类物质健康效应的研究报道。得克隆 602是其中较为新型的阻燃剂，由于其在环境中比较稳定，并且较 DP相比具有更强的生物累积性和富集性，因此对其健康效应的研究具有更重要的意义。 免疫系统是环境污染物的潜在靶器官之一，且免疫系统功能的紊乱也可对神经、内分泌等系统产生影响，因此，本研究对得克隆 602的免疫干扰效应进行了初步的研究。鉴于得克隆 602健康效应的研究目前仍为空白，因此本研究结合目前一些新型的研究技术如悬浮芯片技术等，以中枢免疫器官胸腺及重要的外周免疫器官脾脏为主要对象，从体液免疫和细胞免疫的角度对得克隆 602的免疫干扰效应进行了初步的筛选和研究。发现得克隆 602对 T、B细胞的分化以及抗体的分泌有干扰作用，具体研究结果见下： （1）得克隆602暴露1周后小鼠胸腺指数有所下降，但差异不显著，流式细胞术检测显示胸腺 CD4+CD8-T淋巴细胞比例显著下降，同时 CD4+CD8+T淋巴细胞的比例显著升高，暗示得克隆 602对胸腺T细胞的阴性选择和阳性选择产生了影响，从而影响了胸腺 T细胞的分化。 （2）得克隆 602暴露小鼠脾脏中总的T细胞比例较对照组显著下降，其中CD4+CD8-和 CD4-CD8+T淋巴细胞比例显著下降，同时检测到 CD4+CD8-T淋巴细胞早期凋亡显著增加。其中 CD4+CD8-T淋巴细胞亚型分化转录因子的表达也出现异常，表现为 Th1核转录因子 T-bet的转录表达显著下降，Th2核转录因子GATA3的表达升高，提示上述辅助T细胞的分化也可能受到影响。在B细胞方面，得克隆 602虽然对脾脏总B细胞比例没有显著影响，但是却显著抑制了B细胞向成熟的浆细胞的转化。上述淋巴细胞分化的结果提示得克隆 602暴露扰乱了T和B细胞的分化，总体表现为对机体免疫功能的抑制作用。 （3）与上述淋巴细胞的分化相一致，得克隆 602暴露小鼠1周后，炎症相关细胞因子的检测结果显示抗炎活性增强，包括抗炎相关的细胞因子 IL10、IL13、IL4转录水平的显著上升，以及促炎相关细胞因子IL2、TNFα、IFNγ表达量的显著下降。另外，小鼠血清中免疫球蛋白 IgM和IgG的分泌量显著下降，其中免疫球蛋白亚型 IgG1在暴露组小鼠血清中显著升高，而IgG2a则显著下降，造成IgG1和IgG2a的比例显著升高。这些结果与前述的 CD4+CD8-T淋巴细胞、其亚型 Th1和 Th2细胞的分化紊乱及 B细胞的成熟障碍也是一致的。研究从细胞和分子层面提供了得克隆 602可能造成免疫功能抑制的最新实验数据。因此有必要进行更细致的效应研究及作用机制研究。 肝脏是机体重要的解毒器官，是外源性化合物生物转化的主要场所，因此肝脏常常是这些化合物健康效应的主要靶器官。免疫系统特别是肝脏自身免疫功能紊乱，是导致肝损伤如纤维化等的原因之一。肝脏原代细胞分离培养在病理和生物医学等领域已经有较普遍和成熟的应用，其在阐述肝脏疾病发病机制等方面发挥了重要的作用。但是，目前原代细胞分离培养技术在环境毒理学研究领域的应用尚少。枯否氏细胞（Kupffer cell，KC）是肝脏固有的巨噬细胞，它可以分泌多种细胞因子参与到肝脏炎症、纤维化等过程中。因此，本研究希望借鉴国外病理/医学实验室的先进技术手段和经验，建立小鼠原代 KC分离培养的方法，并将其应用于毒理学领域的研究，为揭示环境污染物的毒性作用提供新的技术手段。 目前已成功建立了两步酶消化法（collagenase、pronase消化）体外灌注提取小鼠原代 KC的方法。脱氧胆酸（deoxycholate，DCA）暴露细胞可以模拟胆汁淤积的环境，与肝脏的炎症损伤有关。实验表明所建立的原代 KC经DCA处理后表现出与预期相符的炎症效应，表现为炎症相关细胞因子IL10、IFNγ和IL-1β表达的显著升高，炎性小体信号通路相关蛋白 NLRP3和ASC的表达升高等。这种基于原代 KC培养的 DCA促发炎症模型不仅可以用于研究胆汁淤积性肝炎的肝损伤过程及机制，也可以作为外源物质对肝脏免疫系统相关毒性及机制研究的体外模型。 |
| 英文摘要 | Dechloranes are widely used in our daily life and industry. Dechlorane plus (DP), dechlorane 602 (Dec 602), dechlorane 603 (Dec 603) and dechlorane 604 (Dec 604)have been detected in environment, wildlife, and even in human body. However, little information has been reported about health effects of these chemicals. Dec 602 is a new substitute for mirex and is stable in environment. Compared to DP, Dec 602 is more bioavailable. Thus, it is important to address health effects of Dec 602. Immune system is one of the potential targets for environtal pollutants. Dysfunction of immune system might also have adverse effects on nervous and endocrine systems. In current study, we examined the immune-disrupting effects of Dec 602. Some of the new methods such as Luminex assay have been used in this study to test immune-disruption of Dec 602 both on thymocytes and splenocytes differentiation, as well as on humoral and cell-mediated immune function. The results are listed as follows. (1) Reduced index of thymus organ weight was observed after 7-day Dec 602 exposure. Percentage of CD4+ T cell decreased significantly, while ratio of CD4+CD8+ T cell increased significantly in Dec 602 exposed mice. It indicates that Dec 602 exposure has adverse effects on T cell differentiation via thymic negative and positive selection. (2) Percentage of splenic T cells, CD4+ and CD8+ T cells were found significant decrease in Dec 602 groups. Early apoptotic CD4+ T cells increased significantly after exposure. IL10, IL13 and IL4 expression levels were higher and IL2, TNFα, IFNγ were lower after exposure. Though no significant changes were found in percentage of B cells, inhibition of B cells to plasma cells was observed after Dec 602 exposure. The results suggest that mice immune function was suppressed after Dec 602 exposure, possibly via disturbance of differentiation of splenic T cell and B cell. (3) Levels of serum IgM and IgG were significantly decreased after 7-day Dec 602 exposure. One of the IgG subtype IgG1 increased significantly and another one IgG2a decreased significantly after exposure. The specific nuclear transcription factors T-bet for Th1 decreased significantly, and GATA3 for Th2 cells increased significantly, respectively. The results showed that disturbance of Th1 and Th2 cell differentiation might be one of the reasons for humoral immunity suppression. Liver is the detoxification organ and the mayor biotransformation site for xenobiotics. Thus, liver is an important tartget for these chemicals. Dysfunction of immune function, especially hepatic immune function is one of the reasons for hepatic fibrosis and liver injury. Primary hepatic cells have been used to address pathogenesis of liver diseases for years in pathological and biomedical studies but not toxicicolical studies. Kupffer cell is the liver-specific macrophage. Cytokines secreted by Kupffer cell participate in processes of liver inflammation and fibrosis. Thus, in the present study, we established a method for Kupffer cell isolation and we hope it will be helpful in the field of toxicology. We established a two-step protocol (collagenase/pronase digestion) for mice primary hepatic Kupffer cell isolation. Deoxycholate (DCA) exposure could mimic the extracellular environment of cholestasis, and is responsible for liver injury. We found that DCA exposure significantly induced the expression of IL10, IFNγ and IL-1β. Activation of NLRP3 inflammasome signaling, including induction of NLRP3 and ASC, was also abserved after expsosure. Activation of NLRP3 signaling is contributed to cell injury and is one of the reasons for the increase of pro-inflammatory cytokines. In summary, we examined biological effects of DCA via the in vitro system of primary hepatic Kupffer cell. It provides new insights into studies of hepatic immunotoxicity. |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/34375]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 田纪景. 得克隆 602的免疫干扰效应及小鼠肝免疫毒理细胞模型的建立[D]. 北京. 中国科学院研究生院. 2015. |
入库方式： OAI收割
来源：生态环境研究中心
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