THP-1分化巨噬细胞用于检测细胞免疫的体外方法的建立

目的建立THP-1分化巨噬细胞用于检测细胞免疫的体外方法。方法将HPV治疗性疫苗原液参考品及样品用含佛波酯(phorbol myristate acetate,PMA)的RPMI1640培养基稀释后,加入THP-1细胞,设PMA对照(只加PMA),37℃,5%CO2培养箱孵育,取上清,检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的含量,测定样品体外相对效力。按上述方法对细胞密度(2.0×105、5.0×105、10.0×105个/ml)、PMA终浓度(2.8、8.3、25、75、225 ng/ml)和培养时间(2、3、4 d)进行优化,确定最佳检测条件。用HPV预防性疫苗原液及ELISPOT法验证该方法的有效性。结果优化的最佳检测条件为:细胞密度5.0×105个/ml,PMA终浓度25 ng/ml,培养时间3 d。验证结果表明该方法有效可行。结论建立了THP-1分化巨噬细胞用于检测细胞免疫的体外方法,为检测治疗性疫苗的细胞免疫提供了一个体外检测平台。     

一种体外评价佐剂诱导人急性白血病单核细胞THP-1炎症因子方法的建立

目的 以人急性白血病单核细胞THP-1为模型,建立一种体外评价佐剂诱导人源化细胞炎症因子水平的方法。方法 以细菌内毒素标准品为阳性对照,与佛波酯(phorbol myristate acetate,PMA)共刺激THP-1细胞,检测炎症因子IL-8、干扰素诱导蛋白-10(interferon-induced protein,IP-10)、IL-1β及单核细胞趋化蛋白(monocyte chemotactic protein,MCP-1)的分泌水平,分别对PMA终浓度(25、5、1、0.1 ng/mL)、刺激时间(4、16、24 h)和细胞密度(1×10⁵、5×10⁵、1×10⁶个/mL)进行优化,确定最佳条件。用不同佐剂[(Toll样受体(toll-like receptors,TLR)激动剂类佐剂Pam2CSK4和TLR4以及乳剂类佐剂MF59和AS03]与PMA刺激THP-1细胞,考察不同佐剂刺激产生炎症因子的水平。结果 炎症因子IL-8、IP-10、IL-1β、MCP-1的最佳诱导条件分别为：PMA终浓度0.1、1...     

皮肤致敏替代方法LuSens的建立与应用

建立皮肤致敏性体外检测方法 LuSens试验,同时评估该方法对我国化妆品原料致敏反应应用的可行性。参考OECD TG442D(Test Guideline No. 442D)标准指南建立方法,确定试验可转移性。对13种化妆品原料组分进行检测,其中10种受试物结果与小鼠局部淋巴结试验（local lymph node assay,LLNA）方法的结果一致,与LLNA相比总体准确性为83%(10/12);11种受试物结果与人体数据一致,与人体数据相比总体准确性为100%(11/11)。成功建立了LuSens检测方法并用于部分化妆品原料的致敏性检测,可较好地筛选皮肤化学致敏原。     

Msi2对急性髓系白血病THP-1细胞体外增殖能力的影响

目的观察Musashi(Msi)2对急性髓系白血病(acute myelogenous leukemia,AML)THP-1细胞体外增殖能力的影响,初步探讨Msi2在AML中可能的调控作用。方法利用RNA干扰技术沉默Msi2表达,实时荧光定量PCR法检测Msi2、cyclin D1、p21、cdk2基因mRNA转录水平;Western blot法检测Msi2蛋白表达水平;生长曲线观察THP-1细胞体外增殖能力;流式细胞仪检测细胞周期进程。结果与scramble组及空白对照组相比,Msi2-siRNA组细胞cyclin D1、cdk2基因mRNA转录水平明显降低(P0.05),p21基因mRNA转录水平明显增高(P0.05),Msi2 mRNA转录及蛋白表达水平均明显降低(P0.05);Msi2-siRNA组细胞体外增殖能力降低(P0.01);G1期细胞比例明显增高(P0.05),S期细胞比例显著降低(P0.05)。结论 Msi2可能通过调控细胞周期进程,促进白血病细胞恶性增殖,在AML的发生发展中发挥重要作用。     

皮肤致敏测试整合策略的现状与展望

近年来,化妆品接触性皮炎、化妆品痤疮等化妆品皮肤病发病率呈明显上升趋势,日益严峻的化妆品的安全问题引起了社会的广泛关注。因此,国家陆续出台了一系列法律法规进一步规范化妆品的安全性问题,企业也必须对化妆品原料及成品进行充分的安全性评价和风险物质评估。皮肤致敏性检测是化妆品安全评价的毒理学终点之一。由于欧盟已全面禁止化妆品成分动物实验和禁止销售经过动物实验的化妆品原料及成品,因此,传统的动物实验尽管能满足皮肤致敏性测试要求,但不符合3R原则,所以替代实验逐渐取代动物实验成为化妆品安全性评价的重要工具。目前,通过OECD认证的单一致敏替代实验主要包括直接肽链反应检测实验、KeratinoSens~(TM)实验、人细胞系活化实验等等。但单一的致敏替代实验通常不足以准确判断化合物的致敏性。因此,常将不同的单一实验结果根据AOP通路组合成整合策略,综合判定化合物的皮肤致敏性,从而提高预测准确性。目前,现有的整合策略主要由巴斯夫、欧莱雅、资生堂等几大日化企业自主开发,包括3选2原则、stacking-meta模型、ANN-EC3模型等。由于中国对动物替代实验方法引入较晚,国内日化企业及机构尚未开发自有的整合模型,但我们正在努力开发新型的皮肤致敏替代技术,例如在基因实时表达报告细胞构建、新QSAR计算机系统开发及THP-1/KC共培养系统构建等方向均已取得了较好成果。文章就日化相关企业目前使用的皮肤致敏测试的整合策略方法进行概括与阐述,并总结了中国自主开发的皮肤致敏替代方法的发展现状。     

皮肤致敏测试动物替代方法DPRA的建立和应用

皮肤致敏是化妆品原料安全性评估中的一项重要内容。按照ECVAM(European Centre for the Validation of Alternative Methods)的标准,通过对16个标准品进行检测,成功建立了DPRA(Direct Peptide Reaction Assay,直接肽链反应分析)实验方法。其检测结果的敏感性,特异性,准确性,S/NS的判定以及4 Class分别为81.82%,100%,77.8%,100%和94.7%。目前该方法已成功运用在实际工作中,并检测出多款具有引起皮肤致敏潜在能力的化妆品原料。     

二磷酸氯喹对THP-1细胞增殖及凋亡的影响

目的探讨不同浓度二磷酸氯喹对急性单核细胞性白血病细胞THP-1增殖及凋亡的影响。方法分别用10、20、40、80、100μmol/L氯喹处理THP-1细胞,分别于处理12、24、48和72 h收集细胞,MTS法检测细胞增殖情况,同时采用Hoechst33342荧光染色法及流式细胞仪检测二磷酸氯喹对THP-1细胞凋亡的影响。结果处理细胞12、24 h时,10、20、40、80、100μmol/L二磷酸氯喹对细胞增殖无明显影响,各组间差异无统计学意义(P0.05);处理细胞48、72 h时,各浓度二磷酸氯喹对细胞增殖均有抑制作用,细胞活力均明显下降,各组间差异有统计学意义(P0.01),并具有剂量依赖性。不同浓度二磷酸氯喹对THP-1细胞凋亡均具有诱导作用,且随着浓度的增加,细胞凋亡愈加明显。结论二磷酸氯喹在体外可抑制THP-1细胞的增殖,并诱导细胞凋亡,其作用具有剂量依赖性。     

佛波酯对人单核白血病细胞THP-1中颗粒溶素表达的激活作用

目的探讨佛波酯(phorbol 12-myristate 13-acetatefor,PMA)对人单核白血病细胞THP-1中颗粒溶素(granulysin,GLS)表达的激活作用。方法分别用不同浓度的PMA(130、160、180 nmol/L)诱导THP-1细胞24 h,RT-PCR法检测细胞中GLS基因mRNA的转录,筛选PMA最佳诱导浓度;用PMA最佳诱导浓度分别诱导THP-1细胞12、24和48 h,RT-PCR法检测GLS基因mRNA的转录,筛选最佳诱导时间;用PMA最佳诱导浓度分别诱导THP-1细胞12、24、48和72 h,Western blot法检测GLS蛋白的表达,免疫细胞化学法检测48 h时GLS蛋白的表达。结果以160 nmol/L的PMA诱导THP-1细胞24 h,细胞中GLS基因mRNA的转录水平最高;诱导48 h后可检测到GLS蛋白大量表达。结论 PMA可激活THP-1细胞中GLS的表达,本实验为后续进一步研究GLS表达的调控机制奠定了基础。     

基于荧光定量的人细胞系激发试验评价皮肤致敏性的研究

建立基于荧光定量(QPCR)的人细胞系激发试验(h-CLAT),探讨其在化学物和化妆品原料皮肤致敏性评价中的适用性。体外培养人急性单核细胞白血病细胞株THP-1,选用8种已知皮肤致敏特性的参照物处理THP-1细胞,通过QPCR检测CD86和CD54共刺激分子的m RNA水平,判定受试物是否具有潜在皮肤致敏性。同时对正在开发的两个化妆品新原料以及致敏物加标的一个植物提取物原料进行了预测。结果显示,当判断标准设置为当THP-1细胞存活率高于50%,MIF CD86大于等于120%和/或MIF CD54大于等于150%,h-CLAT能准确区分致敏物和非致敏物。此外,对致敏物加标的植物提取物原料也能提示其致敏风险。     

基于超高效液相色谱的改良直接肽反应试验预测化学物皮肤致敏性

建立基于超高效液相色谱改良直接肽反应试验预测化学物致敏性并提高检测效率和通量。选择8种已知皮肤致敏性化合物与半胱氨酸多肽或赖氨酸多肽反应24 h,通过基于超高效液相色谱或高效液相色谱的检测系统计算得到多肽消耗率预测受试物皮肤致敏性。结果显示,基于超高效液相色谱的改良直接多肽反应试验能正确区分8种化学物为皮肤致敏物或非致敏物,且节省检测时间,提高检测通量,同时色谱峰分离度更高。     

Quantitative Proteomics to Characterize Specific Histone H2A Proteolysis in Chronic Lymphocytic Leukemia and the Myeloid THP-1 Cell Line

Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation.     

基于皮肤本态数据库的中青年女性面部皮肤综合状态评价模型构建

从中国女性皮肤本态数据库(北京工商大学)中随机抽取22～42岁女性志愿者1 085例,采集志愿者的皮肤数据,通过相关性分析初步降噪,得到皮肤状态核心指标,并采用KMO检验和巴利特球形检验方法进行检验。根据主成分分析法计算评价模型的各项系数,最终构建皮肤状态综合评价模型。皮肤状态相关的核心指标包括:年龄、皮肤总弹性、皮肤光泽度、黑色素含量、血红素含量、水分含量、经皮水分散失量、肤色ITA°、油脂含量、毛孔数量等10项。该模型对女性的综合皮肤状态给予客观的量化判定,弥补了肉眼判断受到的周围环境以及个人主观意识的影响,解决了皮肤评价的时空限制,采集相关指标数据即可对女性皮肤进行有效评价。研究结果可为女性皮肤状态综合判定提供技术支持。     

Polyunsaturated Fatty Acids and Modulation of Cholesterol Homeostasis in THP-1 Macrophage-Derived Foam Cells

Transformation of macrophages to foam cells is determined by the rates of cholesterol uptake and efflux. This study uses a real time RT-PCR technique to investigate the role of conjugated linoleic acid (CLA), α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) in the regulation of the ATP-binding cassette A1 (ABCA1) and liver X receptor α (LXR) genes, which are involved in cholesterol homeostasis. Accordingly, these fatty acids significantly reduced the total, free and esterified cholesterols within the foam cells. While the expression of the ABCA1 and LXRα genes was increased in the presence of the pharmacological LXRα ligand, T0901317, their mRNA expression was not significantly affected by CLA, ALA and EPA. These results suggest that although polyunsaturated fatty acids have an effect on cholesterol homeostasis, they cannot change the expression of the ABCA1 and LXRα genes. Alternatively, several other genes and proteins may be involved.     

A Global Picture of Molecular Changes Associated to LPS Treatment in THP-1 Derived Human Macrophages by Fourier Transform Infrared Microspectroscopy

Macrophages are among the first immune cells involved in the initiation of the inflammatory response to protect the host from pathogens. THP-1 derived macrophages (TDM) are used as a model to study the pro-inflammatory effects of lipopolysaccharide (LPS) exposure. Intact TDM cells were analysed by Fourier transform infrared (FTIR) microspectroscopy, supported by multivariate analysis, to obtain a snapshot of the molecular events sparked by LPS stimulation in macrophage-like cells. This spectroscopic analysis enabled the untargeted identification of the most significant spectral components affected by the treatment, ascribable mainly to lipid, protein, and sulfated sugar bands, thus stressing the fundamental role of these classes of molecules in inflammation and in immune response. Our study, therefore, shows that FTIR microspectroscopy enabled the identification of spectroscopic markers of LPS stimulation and has the potential to become a tool to assess those global biochemical changes related to inflammatory and anti-inflammatory stimuli of synthetic and natural immunomodulators different from LPS.     

Comparing α-Quartz-Induced Cytotoxicity and Interleukin-8 Release in Pulmonary Mono- and Co-Cultures Exposed under Submerged and Air-Liquid Interface Conditions

The occupational exposure to particles such as crystalline quartz and its impact on the respiratory tract have been studied extensively in recent years. For hazard assessment, the development of physiologically more relevant in-vitro models, i.e., air-liquid interface (ALI) cell cultures, has greatly progressed. Within this study, pulmonary culture models employing A549 and differentiated THP-1 cells as mono-and co-cultures were investigated. The different cultures were exposed to α-quartz particles (Min-U-Sil5) with doses ranging from 15 to 66 µg/cm² under submerged and ALI conditions and cytotoxicity as well as cytokine release were analyzed. No cytotoxicity was observed after ALI exposure. Contrarily, Min-U-Sil5 was cytotoxic at the highest dose in both submerged mono- and co-cultures. A concentration-dependent release of interleukin-8 was shown for both exposure types, which was overall stronger in co-cultures. Our findings showed considerable differences in the toxicological responses between ALI and submerged exposure and between mono- and co-cultures. A substantial influence of the presence or absence of serum in cell culture media was noted as well. Within this study, the submerged culture was revealed to be more sensitive. This shows the importance of considering different culture and exposure models and highlights the relevance of communication between different cell types for toxicological investigations.     

Benzyl Isothiocyanate Attenuates Inflammasome Activation in Pseudomonas aeruginosa LPS-Stimulated THP-1 Cells and Exerts Regulation through the MAPKs/NF-κB Pathway

Inflammasomes are a group of intracellular multiprotein platforms that play important roles in immune systems. Benzyl isothiocyanate (BITC) is a constituent of cruciferous plants and has been confirmed to exhibit various biological activities. The modulatory effects of BITC on inflammasome-mediated interleukin (IL)-1β expression and its regulatory mechanisms in Pseudomonas aeruginosa (P. aeruginosa) LPS/ATP-stimulated THP-1 cells was investigated. Monocytic THP-1 cells were treated with phorbol myristate acetate (PMA) to induce differentiation into macrophages. Enzyme-linked immunosorbent assays (ELISA) were performed to measure the levels of IL-1β produced in P. aeruginosa LPS/ATP-exposed THP-1 cells. Western blotting was performed to examine the BITC modulatory mechanisms in inflammasome-mediated signaling pathways. BITC inhibited IL-1β production in P. aeruginosa LPS/ATP-induced THP-1 cells. BITC also inhibited activation of leucine-rich repeat protein-3 (NLRP3) and caspase-1 in P. aeruginosa LPS/ATP-induced THP-1 cells. Furthermore, we show that mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation in P. aeruginosa LPS was attenuated by BITC. These BITC-mediated modulatory effects on IL-1β production may have therapeutic potential for inflammasome-mediated disorders such as a nasal polyp.     

A multi-scale analysis of the effect of complex viscoelastic models on Listeria dynamics and adaptation in co-culture systems

Natural antimicrobials are of interest to replace traditional food decontamination methods: they are milder and maintain desirable sensory characteristics. However, efficacy can be affected by food structure/composition, thus structural effects in a co-culture pathogen/microflora system are investigated. Listeria was grown planktonically (liquid broth) or on a biphasic viscoelastic system, in monoculture with/without artificial nisin, or in co-culture with Lactococcus lactis (nisin/non-nisin producing). Microbial growth kinetics were monitored and advanced microscopy techniques were utilized to quantify cellular interactions and spatial organization. Microstructural effects are observed on the kinetics, with differences in monoculture/co-culture. Significant microscopic differences are observed in spatial organization and colony size. We are the first to observe changing growth location for all species in monoculture/co-culture, with differences in colony size/organization through stationary phase. This study provides insight into the environmental stress response/adaptation of Listeria grown on structured systems in response to L. lactis and natural antimicrobials.     

基于LPV模型的燃料电池空气进气系统控制

质子交换膜燃料电池是一种通过氢气和氧气的电化学反应将化学能直接转化为电能的装置。提出一种改进的四阶燃料电池进气系统模型,分析了系统的约束性。针对系统模型所具有的非线性特性,提出建立线性变参数（LPV）模型用于对系统的控制。针对状态变量不可测的问题引入卡尔曼滤波器,同时通过可观性分析得出系统所需测量的最佳变量。在符合约束条件下设计基于线性变参数模型的状态空间模型预测控制器,控制空压机的工作电压保证氢气燃料的充分反应。仿真结果表明,基于LPV模型的模型预测控制器能够对空气进气系统进行有效的控制,且满足空压机喘振和阻塞边界等约束条件,与单模型预测控制相比具有更好的控制效果。