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检索条件"任意字段=2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会"

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环境超细粒子呼吸暴露致心血管系统损伤效应的发生机制研究

环境超细粒子呼吸暴露致心血管系统损伤效应的发生机制研究

引用

2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者：林治卿程文文刘丽华黄洁华王书美袭革军事医学科学院卫生学环境医学研究所

目的:大气颗粒物和人工纳米材料对环境和健康的影响是公众社会关注的焦点,关注纳米材料安全性,是源于空气颗粒物对人群健康危害研究。作为空气污染中独特而重要的组成成分,UFPs对心血管系统有复杂而重要的影响,是空气污染引起心血管事... 详细信息

目的:大气颗粒物和人工纳米材料对环境和健康的影响是公众社会关注的焦点,关注纳米材料安全性,是源于空气颗粒物对人群健康危害研究。作为空气污染中独特而重要的组成成分,UFPs对心血管系统有复杂而重要的影响,是空气污染引起心血管事件的重要启动因子。本项目探讨环境超细粒子呼吸暴露致心血管系统损伤的作用及发生机制,为建立呼吸暴露诱发心血管健康危害评估技术提供科学依据。方法:采集自然环境中空气动力学直径在0.1nm以下的超细颗粒物(PM)以及人工合成的典型纳米材料进行实验室模拟暴露研究。采用Iodogen方法对超细粒子进行放射性I标记,经气管滴注暴露健康成年雄性Wistar大鼠观察其在体内的生物分布并观察心血管系统的损伤效应。结合体外细胞培养,从细胞和分子水平探讨损伤作用的发生机制。结果:整体水平研究结果表明,环境超细粒子呼吸暴露能够透过肺血屏障进入血液循环。与对照组比较,血管外膜胶原蛋白Ⅰ和胶原蛋白Ⅲ表达量显著增加;HE染色显示,与对照组比较,低剂量超细粒子长期暴露组大鼠胸主动脉血管外膜组织发生血管重塑,而中、高剂量长期暴露组则出现不同程度的血管内弹力板断裂。超细粒子在穿透肺血屏障进入血流首先受损的是血管外膜。作为血管外膜主要细胞成分的成纤维细胞（AFs）能够通过感受各种刺激、改变自身功能以及表型转化在血管损伤中发挥关键作用。分子水平研究结果提示,超细粒子暴露早期可通过激活非Smads依赖的JNK途径,后期主要通过激活TGF-β1/Smads信号传导途径,二者共同调控SMα蛋白的表达,促进AFs转化为MFs;RAECs出现明显的氧化应激,活性氧激活血管内皮细胞内的核转录因子κB,启动血管内皮细胞内核转录因子κB信号途径而增加血管内皮细胞表面细胞间粘附分子-1的表达,介导中性粒细胞与血管内皮细胞的牢固粘附。结论:环境超细粒子呼吸暴露可致血管外膜损伤与内膜病变的形成。其发生机制可能为暴露致血管外膜发生氧化应激损伤致外膜成纤维细胞发生表型转化为肌成纤维细胞,后者发生增殖、迁移至内膜诱导血管内皮细胞分泌黏附因子,致血管内皮功能紊乱,促进血管内膜损伤的形成及发展。

关键词：环境超细粒子呼吸暴露心血管系统损伤效应发生机制

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利用CRISPR/Cas9系统构建ALDH2基因敲除的HepG2细胞株

利用CRISPR/Cas9系统构建ALDH2基因敲除的HepG2细胞株

引用

2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者：郭涛王婷王飞曾妮李若碧江红梅王庆中山大学公共卫生学院预防医学系

目的:采用不同基因编辑方法构建ALDH2基因沉默和基因敲除的细胞株,比较不同基因编辑方法的编辑效率,并探究ALDH2基因缺失对酒精毒效应的影响,为治疗ALDH2基因相关的疾病提供体外研究模型。方法:分别设计RNA干扰序列(shRNA)和短导向RNA序... 详细信息

目的:采用不同基因编辑方法构建ALDH2基因沉默和基因敲除的细胞株,比较不同基因编辑方法的编辑效率,并探究ALDH2基因缺失对酒精毒效应的影响,为治疗ALDH2基因相关的疾病提供体外研究模型。方法:分别设计RNA干扰序列(shRNA)和短导向RNA序列(sgRNA),并与载体pLKO.1、Lenticrispr v2和PX458进行连接,对构建的重组质粒进行测序鉴定。利用293FT细胞生产病毒液,收集病毒液并感染HepG2细胞,用终浓度为2μg/mL的嘌呤霉素筛选ALDH2基因敲除或缺失的HepG2细胞;瞬时转染PX458-sgRNA真核质粒,采用单克隆细胞有限稀释法筛选ALDH2基因敲除的HepG2细胞株。分别提取HepG2沉默和敲除细胞株的细胞总RNA,设计并合成ALDH2 QRT-PCR引物,通过QRT-PCR法检测HepG2细胞株ALDH2基因的mRNA表达量;蛋白印迹法检测HepG2细胞株ALDH2的蛋白表达量,通过MTS法检测酒精染毒后的细胞存活率。结果:NCBI比对结果显示,ALDH2-shRNA,Lencrispr v2 ALDH2-sgRNA和PX458 ALDH2-sgRNA重组质粒构建成功,没有出现碱基缺失或错配情况。经嘌呤霉素筛选获得ALDH2基因沉默和敲除的HepG2稳定细胞株。此外,通过单克隆细胞有限稀释法成功筛选出一株ALDH2敲除的HepG2细胞株。QRT-PCR结果显示,与对照组比较,shRNA介导的ALDH2沉默HepG2细胞株和CRISPR-sgRNA介导的HepG2敲除细胞株中,ALDH2基因mRNA的表达量均显著降低,但shRNA的抑制效率低于sgRNA抑制效率;westernblot结果显示,RNA干扰技术(shRNA)能有效抑制ALDH2蛋白的表达,但CRISPR/Cas9技术则能完全敲除ALDH2的蛋白表达;MTS结果显示,与HepG2野生型相比,shRNA介导的ALDH2沉默HepG2细胞株和CRISPR-sgRNA介导的ALDH2敲除HepG2细胞株对酒精染毒的敏感性均显著上升,但沉默细胞株的存活率略高于敲除细胞株的存活率。结论:CRISPR/Cas9技术能完全抑制ALDH2基因的蛋白表达;与RNA干扰技术相比,CRISPR/Cas9技术介导的ALDH2敲除HepG2细胞株对酒精毒效应的敏感性更高;慢病毒转染方式构建的基因敲除模型所需时间短,操作简便,但遗传背景较复杂,有回复成野生型细胞群的可能,适用于短期分子机制研究;利用单克隆有限稀释法构建的基因缺陷细胞株所需的时间较长,但遗传背景单一,更适用于长期毒性检测实验,如致癌物筛选实验。

关键词： ALDH2 慢病毒系统 CRISPR/Cas9 有限稀释法 RNA干扰

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AFB1协同HBx诱导肝细胞脂质蓄积的坏死性凋亡调控机制研究

AFB1协同HBx诱导肝细胞脂质蓄积的坏死性凋亡调控机制研究

引用

2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者：韩佩宇陈圆圆张力引蒋盛威何承勇林育纯林忠宁分子疫苗学和分子诊断学国家重点实验室厦门大学公共卫生学院

目的:黄曲霉毒素B1(AFB1)靶向肝脏发挥Ⅰ类致癌物生物学效应;乙肝病毒(HBV)的x蛋白(HBx)作为"病毒癌蛋白"之一促进HBV相关肝细胞癌(HCC)的发展。目前公认的人群中引起HCC的两大因素为HBV慢性感染和饮食摄入AFB1,已有文献证明... 详细信息

目的:黄曲霉毒素B1(AFB1)靶向肝脏发挥Ⅰ类致癌物生物学效应;乙肝病毒(HBV)的x蛋白(HBx)作为"病毒癌蛋白"之一促进HBV相关肝细胞癌(HCC)的发展。目前公认的人群中引起HCC的两大因素为HBV慢性感染和饮食摄入AFB1,已有文献证明HBx与AFB1具有协同致肝癌作用,但其早期毒性损伤效应仍不明确。坏死性凋亡(necroptosis,又称程序性坏死)是一种受体相互作用蛋白(RIPs)介导的细胞死亡形式,在肝脏损伤和疾病过程中起重要作用。本研究拟针对肝致癌作用早期脂质代谢过程,探讨AFB1联合HBx作用经由坏死性凋亡介导肝细胞脂质蓄积的调控机制,为脂质代谢异常在肝损伤和致癌过程的靶向干预提供线索。方法:人正常肝HepaRG细胞诱导分化28天,pcDNA3.1-HBx质粒(1μg/ml)转染,给予AFB1(0.5μM)处理,建立联合暴露模型。分为4组,对照组(Ctrl)、AFB1组、HBx组、和AFB1+HBx组(pcDNA3.1-HBx瞬转12 h后给予AFB1共同处理)。结果:(1)各组HepaRG细胞处理96 h,结果显示,油红染色AFB1组和HBx组细胞内脂滴轻微增多,AFB1+HBx组脂滴显著性增多(P)<0.05);总胆固醇(TC)含量在HBx组细胞中升高、且AFB1+HBx组显著性增高(P<0.05)。(2)处理组HepaRG细胞中脂质合成相关蛋白PPARγ的rmRNA和蛋白表达水平上调,线粒体β氧化限速酶CPT1A的mRNA水平上升,脂质代谢相关基因PPARα的mRNA和蛋白水平降低,提示线粒体β氧化以及PPARα和PPARγ在AFB1协同HBx诱导肝细胞脂质蓄积中起调控作用。(3)进一步检测发现AFB1+HBx组细胞中坏死性凋亡关键蛋白RIP1、p-MLKL水平较AFB1组或HBx组显著性增高,提示AFB1和HBx协同诱导肝细胞坏死性凋亡的发生;并通过RIP1特异性抑制剂Nec-1处理后细胞存活率拯救试验得到验证。(4)采用坏死性凋亡抑制剂Nec-1或NSA预处理进行干预,结果发现可抑制AFB1+HBx组坏死性凋亡,且油红染色的脂滴减少、TC水平下降。结论:AFB1协同HBx可经由坏死性凋亡通路介导肝细胞脂质蓄积的发生,与线粒体脂质代谢的调控机制有关。

关键词：黄曲霉毒素B1 乙肝病毒x蛋白坏死性凋亡肝细胞脂质蓄积线粒体脂质代谢

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Novel Human Cell Models for Evaluation of Toxicity

Novel Human Cell Models for Evaluation of Toxicity

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Xuefeng Liu Department of Pathology Center for Cell ReprogrammingGeorgetown University Medical Center

Historically,it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy ***,ex vivo and in vitro preclinical models are essential tools to study both basic cancer biology and to promote translational research,including drug discovery and safety ***,we described the conditional reprogramming(CR)which allows to rapidly establish primary cultures from normal and tumor *** method was published in Nature Protocols(Feb.2017).The Combination of CR and organoids((Matrigel,air-liquid interface(ALI)) cultures represents next generation human cell models and functional diagnostics for cancer precision medicine as described in the NCI precision medicine initiative(August,2015) and four recent nature review *** technique is relatively simple and has been reproduced in more than 100 *** cells can be used for various applications,including regenerative medicine,drug sensitivity testing,gene expression profiling and xenograft *** epithelial cells(skin,cornea,liver,kidney,airway,breast,prostate,etc.) can be propagated indefinitely in vitro,yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural *** of cell cultures from both normal and tumor tissue is highly *** most important,CR cultures can be generated readily from needle/core biopsies(even nasal brushes) as well as frozen *** robust nature of the technique is exemplified by the ability to produce 2 × 10～6 cells in 5 days from a core biopsy of rat breast *** breast and prostate cultures retain a normal karyotype and differentiation potential and cell lines derived from tumors retain their tumorigenic *** ability to produce inexhaustible cell populations from small biopsies and frozen tissue has the potential to transform biobanking repositories by enabling genetic,biochemical,metabolomic,proteomic,and biolo

关键词： Novel Human Cell Models for Evaluation of Toxicity

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miR-3940-5 palleviated genetic damage by enhancing homologous recombination after double strand break of Cr(Ⅵ) exposure

miR-3940-5 palleviated genetic damage by enhancing homologou...

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Guiping Hu Yang Li Guang Jia Department of Occupational and Environmental Health Sciences School of Public HealthPeking University

Hexavalent chromium[Cr(Ⅵ)]compounds are widely used in industry and agriculture and also ubiquitous environmental contaminant,that widely recognized as one kind of carcinogen,yet the molecular mechanism of carcinogenesis is still not well ***(miRNAs),the small non-coding RNAs,are involved in carcinogenesis and DNA damage *** understand the role of epigenetics in DNA repair genes mediated by miRNA,occupational epidemiological investigation and 16 HBE cells in vitro were performed to determine different expressed miRNA and effects of DNA *** Cr level showed significant association with plasma miR-3940-5 p level(r=-0.33,P=0.001) and non-linear relationship with micronuclei frequency and serum 8-OHdG level(βstd=0.29,P=0.039;βetd=0.35,P=0.001).XRCC2 and BRCC3 protein levels were statistically associated with miR-3940-5 p level respectively(βstd=0.31,P=0.010;βstd=0.24,P=0.037).Meanwhile,a weak but statistically negative association between XRCC2 level and micronuclei frequency was found(βstd=0.15,P=0.027).What's more,it is found that Cr(VI) treatment downregulated miR-3940-5 p level in16 HBE cells in ***-3904-5 p mimic downregulated XRCC2 *** a result,the formation of Rad51-foci was inhibited and double strand break repair was *** results indicate that miR-3940-5 p can alleviate genetic damage by enhancing homologous recombination and play a protective effect in Cr(Ⅵ) induced DNA damage.

关键词： DNA miR-3940-5 palleviated genetic damage by enhancing homologous recombination after double strand break of Cr exposure

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Exposure Science in the 21st Century:Current Progress and Challenges

Exposure Science in the 21st Century:Current Progress and Ch...

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Gao,Renjun Landenberger,Bryce Dow Chemical(China) Investment Company Limited The Dow Chemical Company

The National Research Council(NRC) report Exposure Science in the 21 st Century:A Vision and a Strategy(ES21) articulated a long-term vision for exposure science motivated by the advances in analytical methods,sensor systems,molecular technologies,informatics,and computational *** vision was to inspire a transformational change in the breadth and depth of exposure assessment that would improve integration with and responsiveness to toxicology and epidemiology.A primary objective for improving exposure science is to build confidence in the exposure estimates used to support risk-based decision-making by enhancing quality,expanding coverage,and reducing *** many scientific and technological advances that are transforming exposure science should help to meet that *** endeavors,in which progress has been made since the ES21 report,are:remote sensing,personal sensors,and other sampling techniques;computational exposure tools;targeted and non-targeted analyses;"-Omics" technologies;exposure matrices for life-span research;and physiologically based pharmacokinetic(PBPK) *** created the Exposure Forecasting(ExpoCast) program which focuses on developing high-throughput methods for estimating exposure and so far it has been used to make exposure predictions related to over 1,900 *** Institute of Environmental Health Sciences(NIEHS) is also interested in advancing exposure science and has supported research to develop new sensor systems and to identify biomarkers of response to exposure(NIEHS 2015).It has created the Children's Health Exposure Analysis Resource(NIEHS 2016),an infrastructure designed to enable and expand incorporation of environmental exposures into studies of children's health;there are international efforts,such as the Human Early-Life Exposome(HELIX) project and the EXPOsOMICS *** has the ambitious goal of characterizing early-life exposures and ultimately linking exposures with children's heal

关键词： Exposure Science physiologically based pharmacokinetic(PBPK) models ExpoCast HELIX EXPOsOMICS

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Contribution of bone marrow-derived fibrocytes to silicosis

Contribution of bone marrow-derived fibrocytes to silicosis

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Juan Li Wu Yao Lin Zhang Lei Bao Hutting Chen Di Wang Zhongzheng Yue Yiping Li Mlao Zhang Xinghao Yu Jianhui Chen Yaqian Qu Changfu Hao Department of Public Health Zhengzhou University

Background:Exposure to free silica induces silicosis and its mechanism is less *** is regarded as a primary effector cell which is highly synthetic for collagen and lead to extensive fibrosis in ***,its origin is still *** is one source of myofibroblast and proved to play a pivotal role in lung fibrogenesis,but whether fibrocyte participates in the process of silicosis is rarely ***,the present study was designed to ***:The rat model of silicosis was established by single intratracheal instillation of SiO solution（100 mg/0.5 ml/rat）.HE and Masson staining were used to evaluate the histopathology and collagen *** cytometry and immunofluorescence were performed to detect number of fibrocytes and contribution to ***:During experimental silicosis（from week 1,2,3,6,9,12）,the number of fibrocyte is markedly increased in peripheral blood and lung tissue by using flow *** study found that CD45 collagen Ifibrocyte is existed in lung tissue by using double-color immunofluorescence ***,fibrocyte and lung type Ⅱ epithelial cells contribute 15%～35%and 9%1%of myofibroblasts,*** trend analysis of different sources of myofibroblast during silicosis indicates that fibrocyte and lung type Ⅱ epithelial cell derived myofibroblast play an important role at the early stage of silicosis（week 1 to week 3）,while resident lung fibroblast-derived myofibroblast mainly do a predominant role during fibrosis formative period（week 6 to week 12）.Conclusion:Taken together,these data suggest that fibrocyte is involved in the pathogenesis of silicosis and it may be useful as an indicator for disease *** sources of myofibroblasts play roles in different phases of silicosis.

关键词： SiO2 Fibrocyte Myofibroblast Contribution Silicosis

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The identification of Epithelial mesenchymal transdifferentiation of Alveolar type Ⅱ epithelial cell induced by free Silicon dioxide

The identification of Epithelial mesenchymal transdifferenti...

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Huiting Chen Wu Yao Changfu Hao Department of Public Health Zhengzhou University

Background and objective:The studies show that fibroblasts are the main effector cells that secrete collagen fibers in liver,kidney and other fibrotic diseases,and the epithelial mesenchymal transition is one of the major *** pathogenesis of silicosis is not entirely clear,but it is also known that fibroblasts are the major effector cells in the synthesis and secretion of collagen fibers during the process of *** sources of myofibroblast,as well as endogenous fibroblasts,are equally *** type Ⅱ epithelial cells have certain ability of transdifferentiation and may be one of the potential sources of *** far,how to transdifferentiate into myofibroblast,transdifferentiation efficiency and the time-effect relationships of transdifferentiation markers induced by SiO in the process of silicosis are still ***,this study constructs the co-culture model and the experimental rat silicosis model,using transmission electron microscopy,fluorescence quantitative real-time PCR and confocal imaging and Western blot experimental technology,to explore the process and the time-effect of type Ⅱ alveolar epithelial cells,which may provide a new idea of understanding the mechanism of silicosis,and provides a reference for monitoring and intervention of ***:***-6 TN was purchered from ATCC and identified by electron *** were isolated from Specific pathogen-free（SPF） Sprague-Dawley（SD） ***-8 assay was used to determine the proper SiO dose by treating cells（AM and RLE-6 TN） and with SiO at the concentration of 0～140μg/mL for 24,48 and 72 ***-culture models of RLE-6 TN were divided into four groups（the direct control group,the direct exposure group,the indirect control group and the indirect exposure group） in *** detect the expression of surface markers,including GAPDH,E-cad and a-SMA in co-culture cells by the Western blot and qRT-PCR *** the enzyme linked immunosorbent as

关键词： Alveolar type Ⅱ epithelial cell Macrophage Silicosis E-cad α-smooth muscle actin

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Effective Use of Human Exposure Data in Aggregate Risk Assessment of Chemicals

Effective Use of Human Exposure Data in Aggregate Risk Asses...

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Gao,Renjun Landenberger,Bryce Dow Chemical(China) Investment Company Limited The Dow Chemical Company

Aggregate exposure considers all sources of exposure to a single chemical(*** care products,cosmetics,detergents,foods,environmental media,etc.) via all routes(oral,dermal,and inhalation).Exposure assessments should involve an iterative process,and should be conducted using a tiered strategy,where the lowest tier involves a semi-quantitative assessment of the all sources,pathways and routes contributing to aggregate exposure to a substance,the mid-tier tends to be a deterministic estimate with conservative assumptions,the higher tier is a more realistic estimation of population exposure with increased use of measured data using probabilistic methods,and at the highest tier exposure is modelled with a person-orientated approach using raw data *** consumer exposures tools are designed to evaluate single substance,single use *** tier exposure assessments require more realistic and representative data to the situation being assessed and additional understanding of data *** oriented aggregate tools(PACEM,Creme Care & Cosmetics) are available that allow aggregate exposure assessment within some consumer product *** example,in cosmetics and personal care products,the availability of robust tools and data sets(habits and practices data with product co-use,and the use of presence probabilities) allow refined estimates of aggregate exposure.A major challenge in estimating aggregate exposure in many product categories is obtaining representative information on exposure factors(Habits and Practices Data,Co-use Data,Chemical Concentration Data and Chemical Occurrence Data),as well as potential correlations between these *** some domains,such as household care products,the available data are *** should be developed to indicate when higher tier aggregate assessments might be a *** include relative contributions of different sources,level of conservatism in a screening single source assessme

关键词： Aggregate Exposure Assessment Tier approach Biomonitoring Exposure Models

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Using big data to develop greener chemicals through predictive toxicology

Using big data to develop greener chemicals through predicti...

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Jianming Yang UL Environment

Grouping of substances and utilizing read-across of data are the most commonly used alternative approaches for data gap filling in US EPA new chemical pre-manufacturing notices,European REACH registrations,and REACH Analogs such as China New Chemical Substance Notification "China REACH," and K-REACH.A new web-based tool called REACHAcross? software,a collaboration of UL with researchers from Johns Hopkins University,aims to support and automate structure-based read-across,which is aligned with existing Read-Across Assessment *** tool is based on pioneering work by Thomas Hartung,MD PhD,the Chair of The Bloomberg School of Public Health at Johns Hopkins University and Thomas Luechtefeld,a PhD student,who made the publicly available data registered in the ECHA's database machine-readable and-searchable using natural language processing and created a structured *** data can be used to explore toxicological data on a new scale for computational ***? software was initially created utilizing the publically available toxicology data from nearly 10,000 chemicals registered under REACH and has since been expanded to include in vivo and in vitro toxicology data from several other public *** biological data available in these datasets combined with in vivo endpoints from REACH represent an enormous modeling potential:both chemical and biological similarity can be used to interpolate properties of *** approach is based strictly on the local validity of similar chemicals but offers coverage of large parts of the chemical universe similar to a *** also allows to express the certainty of a prediction based on the local and regional data around the substance of *** software can be used to explore toxicological data on a new scale for computational *** web-based tool could help evaluate the risks posed by new chemicals even before synthesizing them,identifying safer alternatives for greener

关键词： Using big data to develop greener chemicals through predictive toxicology

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