RCEES OpenIR  > 环境化学与生态毒理学国家重点实验室
干细胞毒理学在超细碳颗粒物潜在发育毒性评价中的应用
Alternative TitleThe application of a stem cell toxicology model to assess the potential developmental toxicity of ultrafine carbon particles
程战文
Subtype硕士
Thesis AdvisorFrancesco Faiola
2019-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学硕士
Degree Discipline环境科学
Keyword超细碳颗粒物,干细胞毒理学,胚胎干细胞,角质形成细胞 Ultrafine Carbon Particles, Stem Cell Toxicology, Embryonic Stem Cells, Keratinocytes
Abstract

      由于纳米材料具有良好且稳定的理化性质,目前已被广泛 应用 于工业生产 、生物医疗 以及生活中的方方面面。流行病学研究表明,纳米材料 的使用 会 危害人体健康 ,特别是对婴幼儿健康造成影响 。一般认为,纳米材料能通过呼吸道、消化道以及皮肤暴露的形式进入人体 组织,在细胞内造成 自由基 升高以及炎症因子的 异常 表达。同时,许多纳米材料还被证实能 透 过胎盘屏障进入胚胎,对胚胎发育造成影响。因此,从发育角度衡量纳米材料的毒性,将有助于更好 地 对纳米材料生物安全性 进行 评估。 干细胞毒理学的出现, 可在体外水平 帮助我们探究纳米材料的发育毒性 。此研究中我们的研究对象为超细碳纳米颗粒,目前 被 广泛应用于橡胶、印染等各个行业。为了全面而详细 地 评价其毒性,我们采用了 胚胎干细胞毒理学模型对其进行毒性探究。
      首先, 我们利用 多种手段如 电镜 等对超细碳颗粒物进行 了 表征。实验结果表明,超细碳颗粒 粒径 大小约为 50 nm,能在 培养基 中均匀 分散 。 急性毒性实验数据表明, 超细碳颗粒 72 h给药处理 不会造成 胚胎干细胞 细胞 活力降低 ,但却能特异性下调其多能性基因 SOX2的表达。此外,通过高光谱技术对暴露的碳颗粒进行定位, 我们还 发现碳颗粒倾向于分布 在 细胞与细胞之间。
      其次, 通过诱导 胚胎干细胞分化为拟胚体,我们 探究 了超细碳颗粒物暴露 对早期胚胎发育三胚层的影响 。 通过冰冻切片 图片 发现 ,在 拟胚体 形成过程中超细碳颗粒物能主动蓄积在 拟胚 体内;虽然此蓄积效应对神经外胚层、中胚层以及内胚层标志基因表达无显著影响,但是却能促进非神经外胚层基因的 过量 表达。此结果提示我们,超细碳颗粒物可能会影响非神经外胚层 相关 细胞,特别是表皮中角质形成细胞的 发育 过程。
      为了进一步探究超细碳颗粒暴露对非神经外胚层的潜在危害,我们定向将胚
胎干细胞分化为角质形成细胞并暴露在超细碳颗粒物中,重点关注了超细碳颗粒
物对表皮发育过程的影响 。 研究结果表明 1 μg/mL至 10 μg/mL超细碳颗粒物
给药处理 会 干扰角质形成细胞分化过程,具体表现在促进角质形成细胞早期标志
基因 KRT8、 KRT18和 ΔNP63的 异常 表达 同时 抑制角质形成细胞成熟基因 KRT14、KRT5、 KRT16和 COL7A1的表达。同时,此过程还伴随着牛皮癣相关基因 S100A7和 S100A9上调,以及炎症相关因子的高表达。因 此,我们认为,超细颗粒物会影响表皮发育过程,增加表皮炎症疾病的发病率。
      综上所述,本研究通过胚胎干细胞毒理学模型,证明了环境相关浓度的超细
碳颗粒物暴露对人体健康 存在 潜在危害,具体表现在影响胚胎干细胞的自我更新,干扰角质形成细胞分化并伴随炎症反应的产生。 该结果对于评价超细碳颗粒物毒性效应,理解纳米材料潜在发育毒性具有十分重要的意义。

Other Abstract

      Due to their stable and excellent properties, nanomaterials have been widely used in industry, medical equipments and daily life products. However, epidemiological studies have already shown that the use of nanomaterials could affect human health, especially in infants and young children. It was generally believed that nanomaterials could enter the human body through the respiratory and digestive tracts, and the skin, thus causing elevated ROS and disrupting the expression of inflammatory factors in tissues. Also, many nanomaterials have been proved to penetrate the placental barrier and potentially affect embryonic development. Therefore, measuring the toxicity of nanomaterials from the perspective of development, which can be achieved with stem cell toxicology, will contribute to better assess the safety of nanomaterials. In this study, we used embryonic stem cell-based toxicity models to evaluate the health effects of ultrafine carbon particles, that have been widely used in rubber, printing and dyeing industries.

      First of all, we characterized the ultrafine carbon particles with various methods. The results showed that ultrafine particles could distribute in the medium evenly, and the particle size was about 50 nm. 72-hour exposure did not significantly affect embryonic stem cell viability, but specifically down-regulated the expression of the pluripotent gene SOX2. In addition, we have analyzed the cells exposed carbon particles by hyperspectral techniques and found that carbon particles tended to be distributed between cells.
      Secondly, we explored the effects of ultrafine carbon particles on the three germ layer development by inducing the differentiation of human embryonic stem cells into embryoid bodies. With cryosection techniques, we found ultrafine carbon particles could be accumulated inside the embryoid bodies. However, they had no significant effects on neuroectodermal,  mesodermal and endodermal marker gene expression,while promoting the expression of non-neural ectodermal genes. This gave us a hint that ultrafine carbon particles may affect the differentiation of non-neuroectoderm, especially the differentiation into the keratinocytes of the epidermis.
      In order to explore the effects of ultrafine carbon particle on non-neuroectoderm, we differentiated human embryonic stem cells into keratinocytes. We found that exposure to ultrafine carbon particles, especially from 1 μg/mL to 10 μg/mL, could interfere with the differentiation into keratinocytes, by promoting the expression of the early marker genes KRT8, KRT18 and ΔNP63, and inhibiting the mature genes KRT14, KRT5, KRT16 and COL7A1. This process was accompanied by up-regulation of the psoriasis-related genes S100A7 and S100A9, as well as inflammation-related factors. Therefore, we believed that the exposure of ultrafine particles could disrupt epidermal development and increase the incidence of epidermal inflammatory diseases.
      In conclusion, this thesis demonstrated that the exposure of environmental relevant ultrafine carbon particles could damage human health by affecting the pluripotency of human embryonic stem cell, disrupting the differentiation of keratinocyte and causing the inflammation during development. This result is of great significance for evaluating the toxic effects of ultrafine carbon particles and understanding the potential developmental toxicity of nanomaterials.

Pages92
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42188
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
程战文. 干细胞毒理学在超细碳颗粒物潜在发育毒性评价中的应用[D]. 北京. 中国科学院生态环境研究中心,2019.
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