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纳米银对紫贻贝的毒理效应研究
Alternative TitleStudies on the toxicological effects of silver nanoparticles on mytilus galloprovincialis
由丽萍
Subtype博士
Thesis Advisor吴惠丰 ; 赵建民
2014-05-21
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline环境科学
Keyword紫贻贝 Mytilus Galloprovincialis 纳米银 Silver Nanoparticles 毒理效应 Toxicological Effect 生物标记物 Biomarker
Abstract纳米科技是当今社会发展最快的学科之一。纳米材料具有多种不同于常规宏观材料的特殊性质,被广泛应用于生产、生活、医疗及环境治理等领域中,然而纳米材料无论是在生产还是产品应用的过程中都可以进入环境,最终会汇聚到水环境中,并可能对环境生物和人类健康造成不利的影响。
本研究选择纳米产品中应用最广泛的纳米银(AgNPs)为研究对象,以近岸海域广泛分布的海洋环境监测生物紫贻贝为受试生物,利用荧光定量PCR、酶活性测定、组织切片以及流式细胞术等手段,探讨AgNPs对紫贻贝的毒理效应机制,从而为纳米科技的健康和可持续发展,以及海洋环境生物监测的执行和发展提供科学依据。研究结果如下:
利用RACE技术从紫贻贝cDNA文库中的EST序列中克隆获得四种热休克蛋白基因(HSPA12ATCTPsHSP22sHSP24.1),其cDNA全长分别为2039、950、962和989 bp,编码区分别编码491、171、181和247个氨基酸。采用实时荧光定量PCR方法分析了四种HSPs的组织表达情况,发现HSPA12ATCTPsHSPs22sHSPs24.1分别在性腺、肌肉、肌肉和性腺中表达量最高,且四种HSPs基因在肝胰腺和鳃组织中均为组成性表达。
在AgNPs暴露实验中,分别从抗氧化酶活性、细胞凋亡、组织病理、免疫毒性及基因表达等方面,研究了30~50和~10 nm AgNPs在50和500 μg/L浓度条件下,对紫贻贝的综合毒理效应。结果表明:
(1)肝胰腺和鳃组织对Ag都有较强的富集作用,且Ag在肝胰腺中的富集量高于鳃组织,高浓度暴露组的Ag组织富集量高于低浓度组的。
(2)由四种HSPsHSPA12ATCTPsHSP22sHSP24.1)及六种凋亡相关基因(caspase8caspase2caspase3/7-1p63Bcl-2Ras)的表达变化情况可知,这些基因在AgNPs暴露96 h后,主要发挥抗凋亡作用,暴露30 d后,主要表现为促凋亡作用。且从中可推断出HSPs的两种可能的抗凋亡途径,即抑制凋亡诱导因子和结合Bcl-2抗凋亡家族的基因。
(3)由两种金属硫蛋白(MT10-IIIMT20-IV)的表达变化情况可知,重金属Ag的解毒过程主要发生在肝胰腺组织中。
(4)在AgNPs暴露过程中,鳃组织的氧化应激较肝胰腺强烈,且由抗氧化系统的反应可推测出SOD、CAT和GSH及sHSPs和GSH可能分别共同参与了紫贻贝肝胰腺抗氧化过程。
(5)暴露30 d后的肝胰腺组织病理现象较96 h严重,暴露96 h和30 d后的鳃组织都出现了相似程度的组织病理现象,且结合组织微结构的病理变化及细胞中荧光标记的DNA片段的出现,可推断AgNPs暴露使紫贻贝组织中出现了细胞早期凋亡现象。
(6)在AgNPs暴露过程中,血细胞免疫系统中未出现严重的毒性效应,但诱导产生了免疫应答反应。
Other AbstractNowadays, nanotechnology is one of the fastest developing subjects. Due to the particular characteristics different with the macro-materials, nano-materials are widely used in many areas, including production, life, health care, environmental management, etc. However, nano-materials in the products can be released into the environment and finally accumulated in the aquatic environment. And these nano-materials may have adverse effects on aquatic organisms and human health.
In the present study, the toxicological effects of the most widely used nanomaterials - silver nanoparticles (AgNPs) were studied in marine mussels Mytilus galloprovincialis, which are ubiquitous along the coastal and estuarine areas and frequently used in biomonitoring programs. Techniques of qRT-PCR, enzyme activity test, histological section, flow cytometry, etc. were applied to study the toxicological effects of AgNPs in the mussels. And the study was supposed to provide a scientific evidence for the healthy and sustainable development of nanotechnology and the biological monitoring for marine environment. The major results were summarized as follows.
The cDNAs of four heat shock proteins (HSPs) were indentified from M. galloprovincialis by RACE approaches. The full-length of HSPA12A, TCTP, sHSP22 and sHSP24.1 were of 2039, 950, 962 and 989 bp, and encoded a peptide of 491, 171, 181 and 247 amino acids, respectively. qRT-PCR was employed to analyze the gene tissue distribution, and it was found that the transcripts of HSPA12A, TCTP, sHSP22 and sHSP24.1 were dominantly expressed in gonad, muscle, muscle and gonad, respectively. And the four HSPs were all found to be constitutively expressed in the tissues of hepatopancreas and gills.
In the study of nanotoxicity, the mussels M. galloprovincialis were exposed to 500 and 50 µg · L-1 (nominal concentrations) of AgNPs with particle sizes of 30~50 and ~10 nm,respectively. Data of antioxidant enzymes activity, DNA fragmentation, histo- and ultrastrutural pathology, immunotoxicity, and gene expression were evaluated to study the comprehensive toxicology effects of AgNPs on the mussels. The summarized results were as following.
(1) After AgNPs exposures, silver was accumulated more highly in the hepatopancreas than that in the gills, and the accumulation of 500 µg · L-1 AgNPs was higher than that of 50 µg · L-1 AgNPs.
(2) Data of the expression levels of the four HSPs (HSPA12A, TCTP, sHSP22 and sHSP24.1) and six apoptosis-related genes (caspase8, caspase2, caspase3/7-1, p63, Bcl-2 and Ras) suggested the anti-apoptotic effect of the genes after 96 h exposures and the pro-apoptotic effect after 30 days exposures of AgNPs. And two possible anti-apoptotic pathways involving HSPs were speculated, that was the HSPs might antagonize the apoptosis inducing factor or bind to anti-apoptotic members of the Bcl-2 family.
(3) Data of the expression levels of the two MTs (MT10-III and MT20-IV) indicated that the detoxification of Ag mainly happened in the hepatopancreas.
(4) AgNPs induced higher oxidative stress in the gills than that in the hepatopancreas. And data of the reaction of antioxidant system might suggest two stress resistance groups of SOD-CAT-GSH and sHSPs-GSH in the hepatopancreas.
(5) Effects of AgNPs on histo- and ultrastructural pathology were more severe after 30 days exposure than that after 96 h in the hepatopancreas, and were both severe in the gills after 30 days and 96 h exposures. Data of the ultrastructural pathology and the appearance of DNA fragmentations in the cells of the two tissues suggested the occurance of early apoptosis.
(6) An immune response, rather than cytotoxicity was induced in the mussel immune system after AgNPs exposures.
Subject Area环境科学
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/7071
Collection中科院烟台海岸带研究所知识产出_学位论文
Recommended Citation
GB/T 7714
由丽萍. 纳米银对紫贻贝的毒理效应研究[D]. 北京. 中国科学院大学,2014.
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