基于蛋白组学的环境污染物毒性效应评价和作用机制研究
文献类型：学位论文
| 作者 | 梁雪芳 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 王子健,查金苗 |
| 关键词 | 双向电泳 DIGE Toxicity assessment Toxictiy pathways Protein biomarkers 差异凝胶电泳 毒性评价 毒性作用通路 蛋白标志物 2-DE |
| 其他题名 | Toxicity assessment and mechanisms investigation of environmental pollutants based on proteomic methods |
| 学位专业 | 环境科学 |
| 中文摘要 | 本研究旨在建立可用于水生态毒理学研究的基于胶的蛋白组学（双向电泳2-DE和差异凝胶电泳 DIGE）研究方法，并基于该方法筛选环境内分泌干扰物质的蛋白标志物，同时结合传统毒理学研究手段以及基因组学手段从多指标多终点对毒性效应尚不明确的化合物进行内分泌干扰效应和神经毒性的评价并阐明其作用机制，最后将所建立的方法应用于其他水生生物并从蛋白角度对污染物的毒性作用通路进行阐述和预测。 利用 DIGE技术，结合定量 PCR技术和 Western blot免疫印迹技术，对典型环境内分泌干扰物雌二醇 (E2)和甲基睾酮(MT)暴露的稀有鮈鲫肝脏蛋白组的进行了分析，并从差异蛋白图谱中筛选并验证了丙酮酸羧化酶在 E2和 MT暴露后mRNA水平和蛋白水平都发生显著下调，证明了该蛋白可作为环境内分泌干扰物的一个潜在蛋白标志物。同时证明了所建立的方法可适用于环境污染物的毒性评价以及生物标志物的筛选，为污染物早期预警提供有效的工具。 利用 DIGE技术，结合体征指数、组织病理学观察、血液类固醇指标和下丘脑-垂体-性腺作用轴相关基因表达图谱，对苯并三氮唑的毒性效应及其作用机制进行了多指标多终点的研究，证明了该化合物具有弱雌激素效应，且其在雌、雄鱼体中的作用机制不同。同时对苯并三氮唑暴露的稀有鮈鲫雌鱼的脑蛋白组的2-DE分析表明，苯并三氮唑可通过影响细胞呼吸、G蛋白信号通路、钙离子信号通路、细胞周期和凋亡等作用通路对水生生物产生神经毒性，且作用机制与剂量有关。同时，结合定量 PCR手段，筛选了 CKBB, GS, HPCA, VDAC1和 FLOT1A作为 BT神经毒性的潜在生物标志物。 将 2-DE技术应用于德克隆对中华鲟的毒性评价和毒性通路研究，得到 27个与细胞应激、小 G蛋白通路、钙离子信号通路以及糖代谢相关的差异蛋白(ratio≥ 2, p <0.05)，对德克隆作用机制的预测表明该化合物可能与多溴联苯醚类似，即通过影响钙离子平衡，激活 Ras信号通路，引起下游代谢过程和基因表达的变化。 |
| 英文摘要 | In this study, gel-based proteomic strategies, including two dimensional electrophoresis (2-DE) and differential in gel electrophoresis (DIGE), were developed for aquatic toxicology research and were employed to screen the potential protein biomarkers for endocrine disrupting chemicals (EDCs). Then, the toxicity and mechanisms of unclarified chemicals were assessed by these proteomic technologies and other toxic endpoints. In addition, we analyzed the proteomic response of other aquatic organisms in exposure to environmental contaminants using these methods and predicted the toxicity pathways of chemicals. DIGE was applied to analyze the hepatic proteome of Chinese rare minnow (Gobiocypris rarus) exposed to 17-estradiol (E2) and methyltestosterone (MT). Combined with qPCR and Western blot, PC was observed to be significantly decreased at both protein and mRNA levels after E2 and MT exposure. The results suggest that PC may be a sensitive potential protein biomarker for endocrine disruptors. Moreover, the gel-based proteomic strategies were demonstrated to be effective for toxicity assessment and biomarker screening. These methods may provide useful tools for early warning of aquatic pollution. To investigate the toxicity of benzotriazole (BT) on the rare minnow, many toxicological endpoints were examined. These endpoints included somatic indices, pathological changes, steroid hormone levels, hepatic proteomic responses, and transcriptional levels of the hypothalamic-pituitary-gonadal (HPG) axis-related genes. The results indicated the weak endocrine disruption effects of BT as well as its sex-dependent mechanisms. In addition, the response of the brain proteome of female rare minnow in exposure to BT was analyzed using 2-DE. The results revealed that BT may exert neurotoxicity to rare minnow through several pathways including cellular respiration, G-protein signal cascades, Ca2+-dependent signaling, cell cycle, and apoptosis. Moreover, the neurotoxicity of BT was related to doses. Furthermore, several sensitive proteins affected by BT identified in this study such as CKBB, GS, HPCA, VDAC1, and FLOT1A may have potential to be used as biomarker candidates of BT. Additionally, we used 2-DE to analyze the liver proteomes of juvenile Chinese sturgeon (Acipenser sinensis) exposed to dechlorane plus (DP). A total of 27 significantly altered proteins (ratio ≥ 2, p < 0.05) were successfully identified. Based upon protein responses, we suggest that DP has effects on the generalized stress response, small G-protein signal cascades, Ca2+ signaling pathway, and carbohydrate metabolism. The underlying mechanisms of DP appear comparable to polybrominated diphenyl ethers (PBDEs), impacting calcium homeostasis and activation of the Ras signal cascade. |
| 公开日期 | 2015-07-08 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/15638]  |
| 专题 | 生态环境研究中心_环境水质学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 梁雪芳. 基于蛋白组学的环境污染物毒性效应评价和作用机制研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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