Cyto and genotoxicity of gold nanoparticles in human hepatocellular carcinoma and peripheral blood mononuclear cells

Engineered nanomaterials have been extensively applied as active materials for technological applications. Since the impact of these nanomaterials on health and environment remains undefined, research on their possible toxic effects has attracted considerable attention. It is known that in humans, for example, the primary site of gold nanoparticles (AuNps) accumulation is the liver. The latter has motivated research regarding the use of AuNps for cancer therapy, since specific organs can be target upon appropriate functionalization of specific nanoparticles. In this study, we investigate the geno and cytotoxicity of two types of AuNps against human hepatocellular carcinoma cells (HepG2) and peripheral blood mononuclear cells (PBMC) from healthy human volunteers. The cells were incubated in the presence of different concentrations of AuNps capped with either sodium citrate or polyamidoamine dendrimers (PAMAM). Our results suggest that both types of AuNps interact with HepG2 cells and PBMC and may exhibit in vitro geno and cytotoxicity even at very low concentrations. In addition, the PBMC were less sensitive to DNA damage toxicity effects than cancer HepG2 cells upon exposure to AuNps.     

细脚拟青霉总多糖对人外周血单核细胞TNF-α水平及增殖活性的影响

目的提取分离细脚拟青霉总多糖 (PtPs) ,探讨其对人外周血单核细胞 (PBMC)的调节作用。方法采用水提醇沉淀法提取PtPs成分 ;用不同浓度的PtPs单独或协同脂多糖 (LPS)在体外刺激PBMC ,酶联免疫法测定TNF α的水平 ,溴化四唑蓝法测定增殖活性。结果所得PtPs制品的纯度为 76 .1% ;适当剂量的PtPs (10 0～ 5 0 0 μg/ml)能够单独或协同LPS提高PBMC对TNF α的分泌 ;还可剂量依赖性地促进PBMC的增殖活性。结论PtPs对体外培养的PBMC具有激活作用 ,可能是发挥免疫药理作用的主要活性成分。     

TiO2 nanoparticles and bulk material stimulate human peripheral blood mononuclear cells

Nanomaterials are increasingly produced and used throughout recent years. Consequently the probability of exposure to nanoparticles has risen. Because of their small 1–100 nm size, the physicochemical properties of nanomaterials may differ from standard bulk materials and may pose a threat to human health. Only little is known about the effects of nanoparticles on the human immune system. In this study, we investigated the effects of TiO₂ nanoparticles and bulk material in the in vitro model of human peripheral blood mononuclear cells (PBMC) and cytokine-induced neopterin formation and tryptophan breakdown was monitored. Both biochemical processes are closely related to the course of diseases like infections, atherogenesis and neurodegeneration. OCTi60 (25 nm diameter) TiO₂ nanoparticles and bulk material increased neopterin production in unstimulated PBMC and stimulated cells significantly, the effects were stronger for OCTi60 compared to bulk material, while P25 TiO₂ (25 nm diameter) nanoparticles had only little influence. No effect of TiO₂ nanoparticles on tryptophan breakdown was detected in unstimulated cells, whereas in stimulated cells, IDO activity and IFN-γ production were suppressed but only at the highest concentrations tested. Because neopterin was stimulated and tryptophan breakdown was suppressed in parallel, data suggests that the total effect of particles would be strongly pro-inflammatory.     

Toxicity assessment of six titanium dioxide nanoparticles in human epidermal keratinocytes

Purpose: The aim of this study was to evaluate and compare the toxicity of six different types of titanium dioxide (TiO₂) nanoparticles (NP) on human epidermal keratinocytes (HEK).

Materials and methods: Six TiO₂ NP (A (10?nm), A*(32?nm), B (27.5?nm), C (200?nm), C*(30–40?nm), and D*(200–400?nm)) were suspended in water or culture medium and characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). In addition, these NP were assayed with cell viability, cytokine release and cellular uptake in HEK.

Results: TiO₂NP did not change in shape in the culture medium when visualized by TEM. There was an increase in agglomeration with all TiO₂NP in the medium when measured by DLS. Since TiO₂NP interfered with the CellTiter 96®AQueous One and MTT assays but had a minimal effect on alamar Blue (aB). The aB viability assay was selected to assess all six types of TiO₂NP and sample B had a statistically significant decrease in viability at 0.4?mg/ml. A slight increase in TNF-α was noted in sample A*, C, and D* at as low as 0.05?mg/ml. Sample A* and B at certain concentrations showed an increase in Interleukin (IL)-6. IL-10 and IL-1β release for all TiO₂NP were noted around the detection limit with no significant changes compared to control. A statistically significant decrease in IL-8 was noted for all TiO₂NP at the highest concentrations due to the adsorption of IL-8 by TiO₂. All TiO₂NP were localized within cytoplasmic vacuoles of HEK and the element Ti was detected by energy-dispersive x-ray spectroscopy analysis.

Conclusions: Based on cell viability, only sample B was slightly cytotoxic to HEK and samples B and A* have the potential to cause inflammation indicated by an increase in IL-6.     

川芎嗪对类风湿性关节炎患者PBMC免疫球蛋白合成的影响

目的：探讨川芎嗪对培养的类风湿性关节炎(RA)活动期、静止期外周血单个核细胞(PBMC)产生免疫球蛋白(Ig)的影响。方法：应用细胞培养技术，采用ELISA检测方法，检测细胞培养上清液IgG、IgM的含量。结果：川芎嗪明显抑制类风湿性关节炎活动期及静止期IgG、IgM的合成。结论：川芎嗪可以抑制RA患者中IgG、IgM的合成，可减轻RA的发展。     

重症AP患者外周血中单个核细胞Toll样受体4的表达及与血浆内毒素的关系

目的动态观察重症急性胰腺炎（SAP）患者外周血中单个核细胞Toll样受体4（TLR4）表达的变化,并探讨其与内毒素血症的关系。方法收集31例SAP患者和25例轻型急性胰腺炎（MAP）患者,SAP患者于入院后1、3、5、7、14、21d,MAP患者于入院后1、3、5、7d分别抽取外周静脉血,用RT-PCR法检测TLR4基因表达水平,同时用鲎试剂测定血浆内毒素水平。结果所有患者的血浆中均检出内毒素,与MAP组比较,SAP组内毒素在各个时期浓度都明显升高,差异有统计学意义（P〈0.05）。与正常对照组比较,SAP患者在入院后1d外周血单个核细胞TLR4基因水平显著升高（P〈0.05）,达正常对照组的1.55倍,并持续较长时间。相关分析显示,SAP患者在入院后第3、5、7天TLR4mRNA与血浆内毒素水平变化趋势一致,两者呈正相关（P〈0.05）。结论 TLR4通路的异常可能在SAP的发病及炎性反应中均发挥一定作用,其诱生与内毒素刺激密切相关。     

Investigation of metabolic properties and effects of 17β‐carboxamide glucocorticoids on human peripheral blood leukocytes

The biological activity of three previously synthesized 17β‐carboxamide glucocorticoids (BG, BEG, and MPEA) was tested in vitro on mitogen stimulated and non‐stimulated peripheral blood mononuclear cells (MNCs) and granulocytes from human healthy donors, and the results were compared to the conventional glucocorticoid dexamethasone. The tested 17β‐carboxamide glucocorticoids did not induce decreases in MNC viability and proliferation, while modulation of reactive oxygen species (ROS) synthesis in granulocytes was dependent on the cell donor. The obtained results indicate the possibility of avoidance of strong lymphocyte suppression, which is generally recognized during administration of conventional glucocorticoids. Furthermore, the metabolism of the tested derivatives was predicted in silico. The predicted metabolites were synthesized and the in silico results were confirmed by in vitro evaluation of the metabolism of BG, BEG, and MPEA in human serum and in cultures of peripheral blood MNCs. The results of the biological activity and metabolism evaluation and of previous in vivo evaluations of biological activity indicate the soft drug nature of BG, BEG, and MPEA. In order to be fully considered as soft glucocorticoids, further investigations on the toxicity and activity of the formed metabolites are required.

     

川芎嗪对类风湿性关节炎患者外周血单个核细胞IL-12mRNA表达的影响

目的:探讨川芎嗪对培养的类风湿性关节炎(RA)活动期、静止期外周血单个核细胞(PBMC) IL- 12 m RNA表达的影响。方法:应用细胞培养技术,采用RT- PCR方法,检测细胞培养液IL - 12 m RNA表达水平。结果:RA患者活动期、静止期IL - 12高度表达(P <0 .0 5 ) ,川芎嗪可以抑制RA活动期PBMC IL - 12合成,并对活动期PBMCIL - 12 m RNA有抑制作用(P<0 .0 5 )。结论:川芎嗪可通过抑制RA患者中PBMCIL - 12 m RNA的表达,减轻RA进展。     

Differential gene expression in human peripheral blood mononuclear cells induced by cigarette smoke and its constituents.

In current molecular epidemiology studies, a wide range of methods are used to monitor early biological effects after exposure to xenobiotic agents. Gene expression profiling is considered a promising tool that may provide more sensitive, mechanism-based biomarkers. As a first step toward obtaining information on the applicability of gene expression profiles as a biomarker for early biological effects of carcinogen exposure, we conducted in vitro studies on human peripheral blood mononuclear cells (PBMC). We used cigarette smoke condensate (CSC) and a selection of its genotoxic constituents as model agents, applying cDNA microarray technology to investigate modulated gene expression. In independent experiments using cells from several donors, quiescent PBMC were exposed for 18 h, followed by gene expression analyses on a microarray containing 600 toxicologically relevant genes. The search for candidate biomarker genes was binomial: first we looked for genes responding similarly to all agents; second, for agent-specific genes. Many genes were significantly deregulated by all compounds, but as the direction of deregulation frequently differed per agent, they are not useful as generic biomarkers. Cigarette smoke condensate modulated the expression of many more genes than any of its constituents, with the largest effect in SERPINB2. The affected genes are involved in immune or stress responses, but surprisingly no genes involved in DNA damage response were modulated, and only a few in DNA repair. In conclusion, several genes have been identified as potential biomarkers for population studies on early biological effects caused by cigarette smoke exposure, but no genes were identified that represent a generic biomarker.     

超抗原SEA原核表达及诱导淋巴细胞杀伤人肺腺癌细胞效果观察

目的探讨重组质粒pET22b-SEA经原核表达后,金黄色葡萄球菌肠毒素A（SEA）对人外周血单核细胞（PBMC）杀瘤活性的影响。方法将重组质粒pET22b—SEA转化至E．coli BL21（DE3）扩大培养,IPTG诱导后,进行SDS-PAGE分析;密度梯度离心分离PBMC,不同浓度SEA诱导PBMC,12、36、60h后用MTT法测定PBMC对人肺腺癌细胞A549的杀伤活性。结果经诱导,pET22b-SEA得到表达,产物相对分子质量约为30kDa,与理论值大小一致;随着SEA浓度的升高PBMC的杀瘤活性显著增强,在36h、高效靶比、高SEA浓度的情况下,杀伤率最高可达57．21％。结论重组质粒pET22b-SEA在大肠杆菌中成功表达;SEA可诱导PBMC对人肺腺癌细胞A549产生更强的杀瘤活性。     

裙带菜多糖对人外周血淋巴细胞因子分泌的影响

目的分析裙带菜多糖对人外周血淋巴细胞亚群、细胞因子分泌的影响,探讨裙带菜多糖的免疫调节机制。方法用流式细胞术检测淋巴细胞亚群的变化,用酶联免疫方法测定裙带菜多糖作用后淋巴细胞培养上清中细胞因子的浓度。结果不同浓度的裙带菜多糖可使CD+8T细胞比例明显增加(P<0.01);并可使CD+4T细胞比率增加,但与阴性对照组比较差异无统计学意义(P>0.05);裙带菜多糖对B细胞增殖反应无明显影响(P>0.05)。不同浓度的裙带菜多糖作用后,NK细胞比例明显增高(P<0.01),并呈浓度依赖性,可明显刺激外周血淋巴细胞产生白细胞介素-2(IL-2)(P<0.01),但对IL-6及IL-10的产生作用无明显影响(P>0.05)。结论裙带菜多糖可促进CD+8、NK细胞增殖,产生Th1类细胞因子发挥免疫调节作用。     

纳米活性炭,纳米二氧化硅和纳米二氧化钛对人胃肿瘤BGC-823细胞的毒性作用

目的探讨不同化学组成的纳米颗粒对人胃癌BGC-823细胞的毒性作用及其机制。方法分别以纳米活性炭(ACNP)、纳米二氧化硅(SiO2)和纳米二氧化钛(TiO2)100,200,400,800和1600mg·L-1悬液作用BGC-823细胞24,48和72h,MTT法检测细胞增殖,比色法检测乳酸脱氢酶(LDH)漏出量。ACNP100mg·L-1,纳米SiO2200mg·L-1,纳米TiO2200mg·L-1作用BGC-823细胞24h,透射电镜观察细胞形态及超微结构的影响。纳米SiO2和纳米TiO2100,200,400mg·L-1作用细胞24h后,AnnexinⅤ-FITC/PI双染法检测细胞凋亡。ACNP、纳米SiO2和纳米TiO2100,200mg·L-1作用细胞48h后,用PI染色法检测细胞周期。结果 ACNP,纳米SiO2和纳米TiO2均能明显抑制BGC-823细胞的增殖,作用72h后的IC50分别为874.2,676.2和883.5mg·L-1。与正常对照组相比,纳米SiO2100～800mg·L-1组LDH漏出量均显著升高,并呈浓度依赖性(r=0.9751,P<0.01),而纳米TiO2100mg·L-1作用细胞24h,LDH漏出量与对照组相比没有显著差异,但随着作用浓度增加和作用时间延长,各组LDH漏出量明显高于对照组(P<0.05)。ACNP100mg·L-1作用24h后,细胞出现细胞质浓缩、细胞核固缩和裂解。纳米SiO2200mg·L-1和纳米TiO2200mg·L-1作用24h后均出现细胞坏死。纳米颗粒ACNP,SiO2和TiO2作用组均可见纳米颗粒进入细胞及线粒体损伤。纳米SiO2100mg·L-1和纳米TiO2100mg·L-1作用24h,细胞坏死率与正常对照组(4.59±1.20)%相比显著升高(P<0.01),分别为(39.40±1.72)%和(14.12±0.90)%(P<0.05);细胞凋亡率与对照组相比没有显著差异。ACNP,纳米SiO2和纳米TiO2100和200mg·L-1作用细胞48h后,S期细胞增多,G0/G1期细胞减少,细胞碎片增多;ACNP组亚二倍体细胞增多。结论 ACNP、纳米SiO2和纳米TiO2能够抑制BGC-823细胞的增殖。ACNP可诱导细胞凋亡。纳米SiO2和纳米TiO2能损伤细胞膜,造成以细胞坏死为主的毒性损伤。     

扩张型心肌病患者外周血单个核细胞IL-34表达及分泌的初步研究

目的观察白介素-34（IL-34）在扩张型心肌病（DCM）患者外周血单个核细胞（PBMCs）中表达和血浆水平变化,探讨其在DCM发病中的作用。方法选择30例明确诊断为DCM的患者（DCM组）,24例心力衰竭的患者（HF组）及30例健康体检者为正常对照组（NC组）。用逆转录-聚合酶链反应（RT—PCR）测定PBMCS的IL-34 mRNA的表达。用酶联免疫吸附法（ELISA）检测血浆中IL-34水平。结果DCM组PBMCs表达IL-34 mRNA水平明显高于HF组及NC组,差异有统计学意义（P〈0．01）;而HF组及NC组间差异无统计学意义（P〉0．05）。DCM组IL-34的灰度值比和血浆蛋白含量明显高于HF组和NC组,差异有统计学意义（P〈0．01）;而HF组及NC组间差异无统计学意义（P〉0．05）。DCM组的IL-34 mRNA表达和蛋白水平与射血分数（EF值）呈明显负相关（r=-0．517,P〈0．01;r=-0．512,P〈0．01）。结论IL-34可能在DCM的免疫发病机制中起了一定作用。     

Ochratoxin A induces oxidative DNA damage and G1 phase arrest in human peripheral blood mononuclear cells in vitro

Ochratoxin A is one of the most abundant food-contaminating mycotoxins worldwide, and its immunosuppressive effects in human caused more and more concern in biomedical field. In the present study, the toxicity of OTA on human peripheral blood mononuclear cells (hPBMC) was explored by analyzing the involvement of oxidative pathway. It was found that OTA treatment led to the release of reactive oxygen species (ROS) and the increase of 8-hydroxydeoxyguanosine (8-OHdG), an important biomarker of oxidative DNA stress. Moreover, we found that OTA treatment induced DNA strand breaks in hPBMC as evidenced by DNA comet tails formation and increased γ-H2AX expression. In addition, OTA could induce cell cycle arrest at G1 phase by down-regulating the expression of CDK4 and cyclinD1 protein, as well as apoptosis in hPBMC in vitro. Pre-treatment of hPBMC with antioxidant, N-acetyl-L-cysteine (NAC), could reduce OTA-induced ROS release and DNA damage, thus confirming the involvement of oxidative DNA damage in the OTA genotoxicity in hPBMC. NAC pre-treatment could also significantly prevent OTA-induced down-regulation of CDK4 and cyclinD1 expression in hPBMC. All the results demonstrated the involvement of oxidative pathway in OTA mediated cytotoxicity in human immune cells, which including the ROS accumulation-oxidative DNA damage-G1 arrest and apoptosis. Our results provide new insights into the molecular mechanisms by which OTA might promote immunotoxicity.     

Cytotoxicity of vanadium oxide nanoparticles and titanium dioxide-coated vanadium oxide nanoparticles to human lung cells

Due to excellent metal–insulator transition property, vanadium dioxide nanoparticles (VO₂ NPs)-based nanomaterials are extensively studied and applied in various fields, and thus draw safety concerns of VO₂ NPs exposure through various routes. Herein, the cytotoxicity of VO₂ NPs (N-VO₂) and titanium dioxide-coated VO₂ NPs (T-VO₂) to typical human lung cell lines (A549 and BEAS-2B) was studied by using a series of biological assays. It was found that both VO₂ NPs induced a dose-dependent cytotoxicity, and the two cell lines displayed similar sensitivity to VO₂ NPs. Under the same conditions, T-VO₂ NPs showed slightly lower cytotoxicity than N-VO₂ in both cells, indicating the surface coating of titanium dioxide mitigated the toxicity of VO₂ NPs. Titanium dioxide coating changed the surface property of VO₂ NPs and reduced the vanadium release of particles, and thus helped lowing the toxicity of VO₂ NPs. The induced cell viability loss was attributed to apoptosis and proliferation inhibition, which were supported by the assays of apoptosis, mitochondrial membrane damage, caspase-3 level, and cell cycle arrest. The oxidative stress, i.e., enhanced reactive oxygen species generation and suppressed reduced glutathione , in A549 and BEAS-2B cells was one of the major mechanisms of the cytotoxicity of VO₂ NPs. These findings provide safety guidance for the practical applications of vanadium dioxide-based materials.     

急性脑卒中患者外周血单个核细胞来源的AChE相关的microRNAs的表达变化

目的 探讨急性脑卒中患者外周血单个核细胞内乙酰胆碱酯酶（AChE）相关的 microRNAs 的表达变化。方法 利用 microRNAs 的预测软件并结合文献, 筛选靶向 AChE 的 microRNAs。 收集发病 24 h 以内的急性脑卒中患者和与之相匹配的对照组人群, 留取静脉血提取单个核细胞。 利用实时荧光定量 PCR（qRT-PCR） 技术检测 microRNAs 和 AChE mRNA 的表达, 利用 Western blot 技术检测 AChE 蛋白的表达。 结果 预测的靶向 AChE 的 microRNAs 包括 microRNA（miR）-24、-28、-124、-132、-182*、-194、-484。 与对照组相比, 脑卒中患者外周血单个核细胞内 miR-24、-124、-132 和-194 表达升高（P＜ 0.05）, miR-28、-182*及-484 无明显变化, AChE mRNA 和蛋白表达水平降低（P < 0.05）。 结论 脑卒中时 miRNAs 可能通过靶向 AChE 增强胆碱能抗炎通路。     

糖皮质激素对哮喘大鼠血单个核细胞及肺组织ICAM-1表达的影响

目的　探讨细胞间粘附分子 1(ICAM 1)在哮喘气道炎症粘附机制中的作用及糖皮质激素对哮喘大鼠血单个核细胞及肺组织ICAM 1表达的影响。方法　将SD大鼠随机分成哮喘组、地塞米松治疗组和正常对照组 ,采用免疫细胞化学和免疫组织化学研究各组大鼠血单个核细胞和肺组织ICAM 1表达情况。结果　 (1)ICAM 1主要表达在肺小血管、毛细血管内皮细胞、支气管、肺泡上皮细胞、粘膜下基底膜及浸润的炎细胞上 ,哮喘组大鼠肺组织ICAM 1表达显著高于地塞米松治疗组和正常对照组 (P <0 0 5 )。 (2 )地塞米松组大鼠单个核细胞ICAM 1表达较哮喘组减轻 (P <0 0 5 )。 (3)治疗组大鼠肺组织和气管炎症反应以及气道反应性较哮喘组减轻。结论　证实了I CAM 1在哮喘炎性反应中的作用 ,地塞米松抑制ICAM 1表达。     

Synthetic peptides from heat‐shock protein 65 inhibit proinflammatory cytokine secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients

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Phthalate esters modulate the differentiation and maturation of mouse peripheral blood mononuclear cell-derived dendritic cells

Phthalate esters, such as di‐(2‐ethylhexyl) phthalate (DEHP), are widespread environmental contaminants. Previously, we have observed that DEHP exacerbates dermatitis elicited by mite antigen in NC/Nga mice. Also, DEHP enhances the functions of bone marrow‐derived dendritic cells (DCs) in vitro. The present study sought to investigate whether phthalate esters affect peripheral blood mononuclear cell (PBMC)‐derived DCs of NC/Nga mice. First, we studied the time course of DC generation from PBMCs and the dose dependency of granulocyte macrophage colony‐stimulating factor and interleukin‐4, and then determined the conditions under which DC differentiation and maturation are moderately induced from PBMCs. Under the conditions determined above, DEHP at 10 μ m significantly inhibited the expression of DC differentiation and maturation markers, such as CD11c, CD40, CD80, CD86 and CD205, whereas mono‐(2‐ethylhexyl) phthalate, a metabolite of DEHP, did not. Furthermore, the effects of DEHP on PBMC‐derived DCs were partially rescued by treatment with ICI 182,780, an estrogen receptor antagonist. Taken together, these results suggest that DEHP can modulate the differentiation and maturation of mouse PBMC‐derived DCs at least partially through activation of the estrogen receptor under our experimental conditions.     

外周血单个核细胞内免疫抑制剂的浓度与实体器官移植术后不良反应相关性的研究进展

目的：随着临床治疗中器官移植技术的发展和广泛应用，免疫抑制剂的正确使用是预防器官移植术后的排斥反应和提高移植器官存活率的关键环节。目前，临床采用常规全血中免疫抑制药物浓度监测联合药物遗传学监测能够提高疗效，避免药物不良反应，但部分患者仍出现全血浓度处于治疗窗，发生排斥反应的情况。由于外周血单个核细胞是免疫抑制剂的作用靶细胞，越来越多的研究显示监测外周血单个核细胞中药物浓度可能比全血中的药物浓度更有意义。本综述以外周血单个核细胞为研究模型，总结了免疫抑制剂在全血和外周血单个核细胞中浓度与急性排斥反应之间的潜在的相关性的最新进展。