| 题名 | 纳米银的胚胎发育毒性机理研究 |
| 作者 | 王哲 |
| 学位类别 | 博士 |
| 答辩日期 | 2013-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 刘思金 |
| 关键词 | 纳米银 组织分布 胎盘屏障 胚胎发育迟缓 贫血 “木马”效应 转录 RNA 聚合酶 Nanosilver Biodistribution Placental barrier Embryonic developmental retardation Anemia Trojan horse Transcription RNA polymerase |
| 其他题名 | The effects on embryonic development from exposure to nanosilver and the related mechanisms |
| 学位专业 | 环境科学 |
| 中文摘要 | 由于其广谱的抗菌活性，纳米银被添加到许多商品中，在日常生活和医药卫生中广泛应用。纳米银的生物安全性和潜在毒性效应开始引起人们的关注。虽然纳米银的生物毒性效应已有报道，但是大部分研究是针对细胞水平的。目前有关纳米银在动物体内的组织分布、代谢和潜在毒性效应的研究十分有限，有关纳米银对哺乳动物胚胎发育毒性的研究比较缺乏。深入探索纳米银的胚胎毒性作用对于揭示纳米银的安全性和潜在健康风险具有重要意义。本论文研究了纳米银暴露对小鼠胚胎发育的毒性效应，并揭示了纳米银导致胚胎发育迟缓的分子机理。 胎盘组织是母体与胎儿之间进行物质交换的重要场所和生物屏障。对胎盘、胎儿（去除胎肝后的剩余部分）和胎肝中的银含量进行检测后发现，纳米银可以穿过小鼠的胎盘屏障进入胎内体内，并且累积于胎肝中。证实纳米银具有穿过胎盘屏障的能力，这是本论文的第一个创新点。对纳米银在小鼠体内的分布、代谢动力学过程和组织损伤的研究结果表明，纳米银进入小鼠体内后，主要分布于肝脏、脾脏和肾脏。纳米银在肝脏中的 累积造成肝脏的组织结构发生改变。纳米银在小鼠体内处于动态过程，随着时间的延长，靶器官中检测的银含量减少。纳米银可以在小鼠体内滞留至少4 个月。 纳米银进入小鼠胎儿体内后，导致胚胎发育迟缓。在胚胎发育第14.5（E14.5）天时，22 μg/kg 纳米银暴露组的胚胎重量与对照组相比减少40%。记录出生时的胎儿数目，发现22 μg/kg 纳米银暴露组的出生率与对照组相比减少一半。对胎儿的生长发育状况进行连续观察发现，由于生长稀释作用，纳米银对小鼠个体早期发育过程中的饮食、体重、 器官重量和血细胞参数没有影响。 E14.5 天时的胎肝是胎儿唯一的造血器官，纳米银抑制胚胎时期红细胞的基因表达水平，导致红细胞生成受到抑制，引发贫血，贫血可能是导致胚胎发育迟缓的原因之一。这是本论文的第二个创新点。对E14.5 天时的胎肝组织中的基因表达水平和血红蛋白含量进行检测后发现，22 μg/kg 纳米银暴露组中与造血和红细胞生成相关的基因的表达水平受到抑制，编码血红蛋白基因的表达水平与对照组相比减少了50%，血红蛋白含量与 对照组相比减少80%。 对胚胎外周血中红细胞的数量和血红蛋白含量的检测结果表明，22 μg/kg 纳米银暴露组的胚胎外周血中的无核红细胞数目减少，外周血中的血红蛋白含量与对照组相比减少25%。这些结果充分说明纳米银导致胎肝组织的造血功能减弱，血红蛋白减少，引发贫血。 纳米银对胎肝组织中基因表达水平的抑制作用，提示着纳米银可能干扰了RNA 的转录。证实纳米银与RNA 聚合酶结合，从而抑制细胞内整体基因的表达水平，这是本论文的第三个创新点。细胞内总RNA 的放射自显影结果表明，纳米银抑制红系细胞内总体基因的表达水平。当纳米银的暴露剂量对细胞的生存没有毒性时，纳米银对基因转录的抑制作用不依赖于银离子和ROS 的产生。此外，纳米银进入生物体和细胞后可以释放银离子。纳米银是否存在“颗粒效应”目前并没有定论。由于纳米银容易穿过质膜进入细胞，银离子则不易进入细胞，纳米银通过“木马”（载体）效应将银带入细胞，细胞内同时存在纳米颗粒和银离子，以颗粒为主。通过体外结合实验和免疫共沉淀实验，证实纳米银与RNA 聚合酶之间存在相互作用，从而干扰了RNA 聚合酶的转录效应，导致细胞内基因表达水平下降。低浓度条件下，在纳米银抑制基因转录的过程中，“颗粒效应”占主导。 |
| 英文摘要 | Among the popular engineered nanomaterials, nanosilver (nAg) is the most commonly used inorganic metal-based particles. Due to its novel properties, in particular its intrinsic antimicrobial activity, nAg has been applied to diverse fields with significant advantages, such as medicine and personal care. Previous studies have demonstrated that nano-silver exposure to various tissues and organs leads to adverse effects. The current understanding about the toxicity and health influence with exposure to nano-silver is limited, particularly about the influence on embryogenesis and early development. In the current study, we aim to closely investigate the effect on embryonic development from exposure to nAg and the related mechanism. The placenta is the important place and biological barrier for material exchange between fetus and its mother. In the current study, we exposed nAg to BALB/c mice at various concentrations via intraperitoneal (IP) and intravenous (IV) routes. For the first time, we demonstrated that nAg had the ability to cross the placental barrier and accumulate in fetuses, since a mount of silver were detected in the placentas, embryos and fetal liver cells from mice treated with 22 μg/kg nAg via IP. The results showed that nAg was predominantly localized in liver and spleen in mice for both administration methods. The accumulation of nAg in livers caused remarkable hepatic toxicity. Additionally, the pharmacokinetic process of nAg in mice was also assessed in this study. Our findings indicated that nAg retention in mouse body could last for more than 4 months, and the silver content in the major recipient organs decreased in a time-dependent manner. Nanosilver located in the embryos lead to embryonic developmental retardation. The weight of embryos from 22 μg/kg nAg-treated mice was nealy reduced 40%, compared to those from the control. To delineate the effect of nano-silver exposure on embryonic survival, fetal growth post birth and early development, we carried out in vivo nAg exposure in another group of mice that were allowed to give birth to babies. The average number of viable babies per litter was significantly reduced 50% in mice upon 22 μg/kg nAg exposure. No abnormal developmental parameters (such as activity, diet, and complete blood count indexes) were observed in those surviving mice from the parental mice with 22 μg/kg nAg administration. Fetal liver was the only hematopoietic organ around E14.5. For the first time, we demonstrated that a large reduction of erythropoiesis in fetal livers in embryos upon exposure to nAg, and the prominent repression of gene transcription in erythroid cells was likely the molecular basis for nAg-induced fetal anemia and developmental retardation. The microarray analysis revealed that a significant inhibition on transcription of a variety of vital genes in fetal liver cells upon exposure to nAg. The qRT-PCR analysis showed a significant decline (50%) of both α-globin and β-globin mRNAs in fetal liver cells from 22 μg/kg nAg-treated mice. Consistent with the qRT-PCR results, the globin protein concentration was reduced 80% in fetal liver cells from the mice treated with 22 μg/kg nAg compared to that from the other groups. The staining of embryonic peripheral blood smears indicated a great proportion of peripheral red blood cells were larger nucleated primitive yolk sac-derived erythrocytes in embryos from 22 μg/kg nAg-treated mice. Moreover, the hemoglobin content in embryonic peripheral blood was reduced 25% in embryos from 22 μg/kg nAg-treated mice. Significant inhibition on expression of a variety of vital genes in fetal liver cells upon exposure to nAg indicated that nanosilver could disturb RNA transcription. For the first time, we domenstrated that nAg exhibited a robust inhibition on RNA polymerase activity and overall RNA transcription through dirct Ag binding to RNA polymerase, which is separated from the cytotoxicity pathway induced by Ag ions and is independent on ROS. The result of autoradiography of radioactive labeled total RNA after agarose formaldehyde gel showed the accumulation of Ag in MEL cells substantially inhibited overall RNA transcription. To date, there is no conclusive answer to the sources of nAg-mediated toxicity: Ag ions or “particle-specific” effects, or both. We here demonstrated that both Ag ions and nAg particles simultaneously existed inside cells, demonstrating the “Trojan horse” effects of nAg particles in posing biological impacts on erythroid cells. Importantly, we demonstrated that intracellular nAg particles have a potential interaction between nAg and RNA polymerase in an acellular system and in cells through pull-down assay and immunoprecipitation. Moreover, our results suggested that “particle-specific” effects could be the predominant mediator in eliciting biological influences on erythroid cells under relatively low concentrations of nAg exposure |
| 公开日期 | 2014-10-28 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/7711]  |
| 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 | 王哲. 纳米银的胚胎发育毒性机理研究[D]. 北京. 中国科学院研究生院. 2013. |
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