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题名: 基于报告基因的二恶英类生物检测系统构建与应用
作者: 尹雪娇
学位类别: 硕士
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: 二恶英,芳香烃受体,二恶英生物检测法,报告基因,重组质粒, Aryl Hydrocarbon Receptor (AhR), Bioassays, Reporter Gene, Recombinant Plasmids
其他题名: Development and Application of Reporter Gene Based Bioassay for Dioxins
学位专业: 环境科学
中文摘要:     二恶英类污染物存在着较为广泛的毒性,包括肝毒性、免疫毒性、致癌性等等。目前,二恶英的检测方法有仪器分析法和生物检测法两大类。仪器分析法(HRGC-HRMS)是二恶英分析的“黄金标准”,其灵敏度和准确性都很高,但是检测周期较长、检测费用高昂,不适用于大规模的二恶英筛查。生物检测法具有检测通量高、成本低、周期短的优势,更适合对大量样品开展二恶英筛查。基于报告基因的二恶英生物检测法是其中应用比较广泛的一类,较其它生物检测法具有灵敏度高的优势,并可反映二恶英类的总体毒性风险,美国、欧盟以及日本等国家和地区已建立了相应的标准方法,在针对大量污染源样品、自然环境样品,以及生物样品的二恶英筛查方面都有重要的应用价值。但是我国相关的研究、技术开发和标准的建立尚未发展成熟。本研究主要针对该生物检测法的两个主要核心技术,即检测质粒的构建和检测细胞株的筛选开展研究,并在此基础上探索了该类检测方法在生物样品检测方面的新应用。
    在方法研发方面,本论文包含了多种人源化重组质粒的构建以及鼠源二恶英高响应单克隆细胞株的建立。基于报告基因的二恶英生物检测技术源于二恶英的经典毒性通路,芳香烃受体(AhR,二恶英受体)依赖的信号通路,该通路具有一定的种属差异性。目前国际上所应用的检测系统的核心检测质粒是基于鼠源DNA序列构建的,为了适应未来健康风险评估的需要,本研究进行了人源化的生物检测系统开展的研发。根据两个二恶英类效应基因CYP1A1CYP1A2分别构建了两类报告基因检测质粒,其中pCL-HCR2重组质粒在人源细胞中的最低检测限(MDL)达到0.3 pM,超过目前国际通用的鼠源系统的MDL;另外,质粒pCL-H2CR1仅需8小时暴露就可以达到最高响应,较鼠源质粒pGL6.1在人源细胞中响应更快。说明人源质粒较鼠源质粒在人源细胞中有明显的匹配优势,提示在健康风险评估中使用全人源系统的必要性。在开发人源检测质粒的同时,应用已开发的新型鼠源检测质粒,进行了鼠源二恶英高响应单克隆细胞株的筛选和鉴定,该新型检测质粒与国际通用的鼠源质粒pGL6.1有相同数量的二恶英响应元件(DRE),但是序列不同。所获得的检测细胞株CBG2.16D的EC50值达到2.551×10-11 M,与源于pGL6.1的国际通用细胞株Hepa 6.1相当。在响应倍数方面,将CBG2.16D细胞暴露于10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)24h后,细胞株的响应为DMSO处理组的99倍,较Hepa 6.1的响应倍数高。但是在灵敏度方面逊于实验室自行开发的另一个同源细胞株CBG2.8D,该检测质粒中DRE的数目是CBG2.16D检测质粒的一半,提示DRE的数目不是决定检测质粒对二恶英响应能力的唯一指标。
    在方法应用方面,本研究还初步探索了基于报告基因的二恶英生物检测法在实验动物的二恶英组织分布方面的应用。实验选择了更为灵敏的CBG2.8D检测细胞株,对长期低剂量TCDD暴露大鼠的部分组织进行了上述生物检测,并同时进行了HRGC-HRMS的比对实验,结果显示生物检测法的检测结果与仪器分析方法结果具有较好的一致性,提示应用生物检测法进行二恶英组织分布的研究具有一定的可行性,需要进行更完善的比对研究。
英文摘要:     Dioxins have a wide variety of health effects, such as liver, and immunotoxicity, and tumor promotion etc. There are two kinds of analytical methods for determining dioxins, including bioanalytical methods and chemical method. The commonly recognized chemical method (HRGC-HRMS) is the “golden standard” for dioxins with the advantage of high sensitivity and accuracy. While compared with chemical method, bioanalytical methods show the advantages of low cost, high throughout and short term, making it proper for screening large-scale samples. Among the bioanalytical methods, reporter-gene based bioanalytical methods are the most widely used because of their high sensitivity and the character of assessing total toxicity of the pollutants. The reporter-gene based bioassays have been standardized in USA, Europe and Japan, showing a great application value in the screening of dioxin in environmental and biological samples. In china, the related study, technology and standardization to reporter gene based bioassays have not been developed yet, thus we conduct our research on the two major process of the bioassay, namely the development of recombinant plasmids as well as monoclonal stable cell lines. Based on which we explored the application for biological samples.
    Several humanized recombinant plasmids and mouse-origin stably transfected cell lines for the detection of dioxins were developed in the respect of method development. Reporter gene based bioanalytical methods is based on the AhR dependent signaling pathway, which varies among different species. At present, the core detection plasmid of internationally used is constructed from mouse origin DNA sequences. To meet the demand of health assessment in the future, we focused on the development of humanized bioanalytical system. By analyzing the two responsive genes, CYP1A1 and CYP1A2, we developed two kinds of humanized plasmids,and the detection limit of the plasmid reached 0.3PM, which is equal to that of mouse origin detection system. In addition, the plasmid named pCL-H2CR1 can reach the highest response level within 8 hours’ exposure to TCDD after transfection into Hep G2 cells, and the exposure time is much shorter than that of the mouse origin plasmid. The results indicate the importance of humanized plasmid in response to dioxin in human origin cells. Except for the humanized system, we also screened a dioxin responsive monoclonal cell line named CBG2.16D with mouse origin plasmid developed earlier in the lab, which has the same number of DREs with the internationally used plasmid (pGL6.1). The EC50 value of CBG2.16D is 2.551E-11 M that is the same level with Hepa 6.1 stably express pGL6.1. The fold change of CBG2.16D cells exposed to TCDD is 99 compared to DMSO control, which is higher than that of Hepa 6.1, while the sensitivity of this cell line is not so high as another cell line called CBG2.8D developed by our team. The DRE number of CBG2.8D is half of that of CBG2.16D, showing that the number of DREs is not the only factor to determine the detection sensitivity.
    At last, we explored the application of reporter gene based bioanalytical method to revealing tissue distribution of TCDD in experimental animals. We choose the more sensitive cell line CBG2.8D as our detection tool and analyzed the tissue samples from SD rat exposed to low concentration TCDD for a long period, the result of which is compared to that from HRGC-HRMS. Comparable results were obtained by the the two methods, suggesting a new application potential of this bioanlytical method.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34463
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
尹雪娇. 基于报告基因的二恶英类生物检测系统构建与应用[D]. 北京. 中国科学院研究生院. 2015.
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