吴茱萸碱对小鼠树突状细胞分泌细胞因子的影响

目的:利用抗体芯片技术和ELISA检测吴茱萸碱(EVO)对小鼠骨髓来源树突状细胞(DC)分泌相关细胞因子的影响,研究EVO的生物学调节功能。方法:体外诱导培养未成熟DC细胞(iDC),经LPS和EVO处理后,通过混合淋巴反应检测DC诱导异源T细胞的应答能力,ELISA检测上清中IL-12p40、IL-12p70和IL-10,小鼠抗体芯片检测细胞上清中细胞因子。结果:EVO促进了iDC和成熟DC(mDC)抗原提呈功能(P0.05),对iDC、mDC的细胞因子IL-10、IL-12分泌功能无影响(P0.05),另外EVO处理iDC后上调大于2倍的细胞因子有7个(VEGF、DPPIV/CD26、IGF-Ⅰ、IL-17BR、MDC、Pro-MMP-9、Eotaxin-2),处理mDC后上调大于2倍的细胞因子有2个(Eotaxin-2、IL-13)。结论:EVO处理mDC和iDC后,细胞因子的分泌功能发生了明显改变,这些细胞因子影响着DC的抗原摄取、迁移、抗原提呈,为进一步寻找药物靶点提供了线索。     

利用生物芯片技术检测人参皂苷Rg1对小鼠树突状细胞功能调控相关基因表达的影响

目的:利用基因芯片技术检测人参皂苷Rg1对小鼠骨髓来源DC细胞功能调控相关基因表达的影响.方法:分离C57小鼠骨髓细胞,经GM-CSF、IL-4体外诱导培养6天的未成熟DC细胞(iDC)经不同因素处理并分为:对照组(Ⅰ)、人参皂苷Rg1组(Ⅱ)、内毒素(LPS)组(Ⅲ)、Rg1+LPS组(Ⅳ),24小时后收集各组细胞,抽提总RNA,利用树突状和抗原呈递细胞基因芯片对细胞功能相关基因进行检测.结果:(Ⅱ/Ⅰ)上调≥2倍基因23个,下调≥2倍基因45个;(Ⅲ/Ⅰ)上调≥2倍基因15个,下调≥2倍基因47个;(Ⅳ/Ⅱ)上调≥2倍基因35个,下调≥2倍基因15个;(Ⅳ/Ⅲ)上调≥2倍基因37个,下调≥2基因10个.这些基因功能主要涉及细胞因子分泌及其受体表达、抗原摄取、抗原提呈、细胞表面受体、信号传导.结论:人参皂苷Rg1对DC作用涉及多个基因的表达调控,这些基因控制并影响着DC功能、分化和成熟,为进一步寻找药物靶点提供了线索.     

吴茱萸碱抑制人骨肉瘤HOS细胞的增殖并促进其凋亡

目的探讨吴茱萸碱对骨肉瘤HOS细胞株增殖凋亡的影响及其可能的机制。方法通过利用0、1、2、4、8μmol/L浓度吴茱萸碱处理HOS细胞24、48 h后,利用CCK-8法检测HOS细胞活性。用3μmol/L的吴茱萸碱处理HOS细0、24、48 h后,利用细胞凋亡-Hoechst染色试剂盒染色观察HOS细胞细胞核染色质的形态。用3μmol/L吴茱萸碱处理0、24、48 h后,利用流式细胞仪检测HOS细胞的凋亡率。3μmol/L吴茱萸碱处理0、24、48 h后,Westernblot检测HOS细胞内Caspase 3、Bcl-2蛋白的表达变化情况。结果 2~8μmol/L的吴茱萸碱可抑制HOS细胞的细胞活性,抑制其增殖,呈剂量-时间依赖性。8μmol/L处理48 h后细胞存活率为0.453±0.071,与对照组比较,差异有统计学意义(P<0.01)。Hoechst-33258染色观察可见凋亡细胞染色质颜色发白,呈固缩状或者碎裂状染色质,染色质着色不均匀,核形态各异。流式细胞仪检测3μmol/L吴茱萸碱处理HOS细胞0、24、48 h后的凋亡率分别为(5.32±1.62)%、(10.85±1.49)%和(12.47±0.59)%,与对照组比较,差异有统计学意义(P<0.01)。吴茱萸碱可上调HOS细胞内的Caspase 3蛋白的表达,同时下调Bcl-2蛋白的表达,与对照组相比较,差异有统计学意义(P<0.05)。结论吴茱萸碱可降低人骨肉瘤HOS细胞的细胞活性,抑制其体外增殖,诱导其发生凋亡,其机制可能与其上调Caspase 3蛋白的表达,下调Bcl-2蛋白的表达有关。     

吴茱萸碱抑制Huh7细胞生长并增强细胞对TRAIL的敏感性

目的:探讨吴茱萸碱对人肝癌Huh7细胞生长和凋亡的影响,阐明吴茱萸碱促进肿瘤坏死因子凋亡诱导配体(TRAIL)抗肿瘤活性的分子机制。方法:MTT法检测吴茱萸碱对Huh7细胞活力的影响;流式细胞术观察吴茱萸碱对细胞周期的阻滞;TUNEL染色法检测细胞凋亡的变化;Western blot实验测定细胞内细胞周期和凋亡相关蛋白的表达水平。结果:Huh7细胞经吴茱萸碱处理后,细胞活力明显下降(P0.05);同时,细胞发生G_2/M期阻滞,p27、cyclin B1、细胞分裂周期蛋白2(Cdc2)和p-Cdc2的蛋白水平上调(P0.05);吴茱萸碱能够诱导Huh7细胞发生凋亡,促进多聚ADP核糖聚合酶(PARP)和caspase-3的切割。当吴茱萸与TRAIL联用后,Huh7细胞活力明显下降,PARP和caspase-3的切割增加;另外,吴茱萸上调Huh7细胞中死亡受体5(DR5)的蛋白水平。结论:吴茱萸碱通过抑制细胞活力和阻滞细胞周期于G_2/M期而抑制细胞生长,并诱导Huh7细胞发生凋亡;上调DR5的表达水平可能与吴茱萸碱增强Huh7细胞对TRAIL的敏感性相关。     

吴茱萸碱抑制荷瘤小鼠结肠癌HCT-116细胞增殖

目的研究吴茱萸碱(Evo)对人结肠癌荷瘤Balb/c裸鼠HCT-116细胞增殖的影响,并探讨其可能机制。方法用HCT-116细胞接种到4周龄的Balb/c裸小鼠右下腋部,待荷瘤直径约0.5 cm后用灌胃针灌入Evo(3 mg/kg)进行治疗,每3 d测瘤体直径及小鼠质量,绘制质量曲线及瘤体体积曲线,灌喂22 d后处死,取瘤体组织;HE染色法验证Balb/c裸鼠瘤体的成瘤情况;免疫组化检测瘤体HDAC3、NF-κB、p53的表达;Westernblot检测瘤体组蛋白去乙酰化酶HDAC3、NF-κB、p53的变化。结果 Evo灌喂组小鼠的瘤体体积和瘤体质量明显小于对照组,质量较对照组重;Evo灌喂组肿瘤细胞皱缩,胞核深染,较对照组核分裂像明显减少;经吴茱萸碱处理的裸鼠的瘤体中NF-κB、p53表达量较对照组增高,HDAC3则反之(P0.05)。结论吴茱萸碱可以通过下调HDAC3来影响NF-κB及p53蛋白的表达,抑制人结肠细胞系HCT-116的体内增殖。     

吴茱萸碱诱导A375-S2细胞死亡过程中对ERK激酶的调控

目的:对比吴茱萸碱与化疗药物对A375-S2细胞的细胞毒活性,并且研究PKC及ERK激酶在吴茱萸碱诱导A375-S2细胞死亡中的作用。 方法: MTT法,Western印迹法。 结果:虽吴茱萸碱对A375-S2细胞毒作用比化疗药物放线菌素D、顺铂和5-氟尿嘧啶弱,但对药物撤除后细胞继续增殖能力的影响远胜于3种化疗药物。低浓度(10 μg/L) 佛波酯 (PMA) 引起的PKC的激活可部分抑制吴茱萸碱引起的细胞死亡,PKC及ERK抑制剂可逆转这种作用,而且吴茱萸碱减少了ERK蛋白表达,并降低了ERK的磷酸化水平。结论: 吴茱萸碱对A375-S2细胞的细胞毒作用即使在药物撤除后仍对细胞继续增殖能力产生抑制作用。吴茱萸碱可通过减少ERK及磷酸化ERK的蛋白表达而阻断ERK激酶对细胞的保护作用。     

树突状细胞成熟及其信号传导基因表达谱的研究

目的利用多种细胞因子及肿瘤细胞裂解物刺激诱导树突状细胞(dendritic cell,DC)成熟,并利用基因芯片技术对其信号传导基因表达谱进行研究。方法外周血单个核细胞在体外用细胞因子GM-CSF(100 ng/m l),IL-4(1 000 U/m l)诱导下获取DC,以乳腺癌MCF-7细胞反复冻融的裂解物冲击DC,通过形态学和FCM检测来确定DC。再用基因芯片技术检测其细胞因子及信号传导相关基因表达的变化。结果DC在细胞因子的诱导下,形态上伸出许多伪足样突起,在摄取抗原后可见内吞样小泡。FCM检测呈现成熟DC的标志:CD40、CD83、CD80、CD86、HLA-DR等高表达。芯片扫描发现16种信号传导因子发生5倍以上的改变,其中包括钙调磷酸酶结合蛋白1、TGF-β3受体、FKBP-雷帕霉素相关蛋白、小分子细胞因子A(Cys-Cys)、人T细胞特异蛋白(RANTES)等。结论通过体外细胞因子诱导及抗原冲击的成熟DC不但在形态和表型上存在明显差异,而且在功能和信号传导方面也存在显著变化。     

小鼠树突状细胞肉瘤细胞系的细胞功能鉴定

本实验目的是通过功能试验进一步鉴定ＤＣＳ细胞的组织来源，我们建立的ＤＣＳ细胞系经酶组织化学及免疫组织化学鉴定初步确定来源于小鼠脾交错树突状细胞。本实验研究了ＤＣＳ细胞吞噬、胞饮和诱导淋巴细胞增殖反应等功能试验，发现ＤＣＳ细胞不具有吞噬及胞饮功能。这些功能试验进一步表明该细胞系来源于小鼠脾交错树突状细胞。     

利什曼原虫感染树突状细胞早期基因表达差异分析及关键基因筛选

目的:分析利什曼原虫感染树突状细胞(DCs)早期的基因表达与信号通路变化,探究DCs感染后应答,寻找利什曼原虫感染后基于DCs的免疫治疗方法。方法:GEO数据库下载利什曼原虫感染前后DCs基因芯片数据,RStudio软件筛选差异表达基因(DEGs),STRING构建DEGs蛋白质相互作用网络(PPI),Cytoscape筛选差异表达蛋白质的核心模块,RStudio软件对DEGs进行GO和KEGG富集分析。结果:共筛选出DEGs 129个,其中IL12B与CXCL10差异最为显著,GO分析共富集23个过程,主要涉及病毒感染过程相关细胞反应及Ⅰ-IFN相关免疫反应;KEGG分析共富集3条信号通路,分别为甲型流感、麻疹及DNA复制信号通路。结论:利什曼原虫感染DCs前后Ⅰ-IFN信号通路和TLR4/NF-κB信号通路激活,影响IL12表达,提示Ⅰ-IFN/IL12信号通路与TLR4/NF-κB/IL12信号通路可作为利什曼原虫感染治疗的靶点,CXCL10也有望成为潜在的治疗靶点;利什曼原虫感染后,出现类似病毒感染现象,推测抗病毒免疫疗法可能在对抗利什曼原虫感染中具有一定疗效。     

树突状细胞对自然杀伤细胞功能调控的研究进展

树突状细胞（DC）和自然杀伤细胞（NK）分别在固有免疫和适应性免疫中发挥着关键作用,二者之间还存在着复杂的交互作用.就DC对NK细胞功能的影响而言,前者可以通过膜表面分子直接激活静息的NK细胞,也可以在趋化因子的作用下将NK细胞招募至炎症部位或次级淋巴结,通过分泌可溶性细胞因子促进NK细胞活化、增殖,增强产生IFN-γ的能力,提高细胞毒活性,进而增强其抗病毒、抗肿瘤等效应.DC对NK细胞功能调控的研究在感染、肿瘤和免疫排斥等的防治中具有重要意义.     

树突状细胞对调节性T细胞的调控作用

树突状细胞(DCs)是目前已知的体内功能最强大的专职性抗原提呈细胞,具有启动免疫应答和诱导免疫耐受的双重特性.近年来树突状细胞对调节性T细胞的调控作用是免疫学领域的一个研究热点.Foxp3+ Tregs是一群同时具有免疫低反应性和免疫抑制性功能两大特征的T淋巴细胞,它在维持机体内环境稳定、预防自身免疫性疾病、抑制移植排斥反应等病理生理过程中发挥着重要作用.越来越多的研究结果证实DCs和Tregs二者在维持外周免疫耐受中存在着紧密联系,DCs可以诱导抗原特异性Tregs的生成并增加后者的抑制活性,其中参与该调节机制的分子主要包括相关细胞因子、Toll样受体、共刺激分子及维甲酸等.对DCs在接触共生和致病微生物时诱导和调控Tregs细胞有了一些新发现.     

Cytokine-dependent regulation of growth and maturation in murine epidermal dendritic cell lines

We have recently established dendritic cell (DC) lines (XS series) from the epidermis of newborn mice by repeated feeding with granulocyte/macrophage-colony-stimulating factor (GM-CSF) and culture supernatants from skin-derived stromal cell lines (NS series). XS lines resemble resident Langerhans cell (LC), which are immature DC that reside in epidermis, by their surface phenotype and antigen-presenting profile. XS lines further resemble resident LC in that they express mRNA for interleukin-1β and macrophage inflammatory protein (MIP)-1α, and by the absence of mRNA for IL-6. Their growth is promoted by GM-CSF, colony-stimulating factor-1 (CSF-1), or NS culture supernatant, and inhibited by interferon-γ or tumor necrosis factor-α. The expression by the XS lines of la molecules is up-regulated by GM-CSF, and down-regulated by NS supernatant. These results suggest the existence of negative regulatory mechanisms in which the growth and/or maturation of DC is suppressed by selected cytokines.     

Expression of functional kappa-opioid receptors on murine dendritic cells

We have shown that two of the matrix metalloproteinases (MMPs), matrilysin and stromelysin-1, are capable of cleaving all of the human IgG subclasses. The cleavage occurs at a conserved site in the CH(2) domain of the heavy chain of IgG, releasing a single chain Fc-like fragment. We have not been able to demonstrate cleavage of IgA, IgD, IgM or IgE classes, which lack the cleavage site, nor could we show cleavage of IgG by collagenase, gelatinase, macrophage metalloelastase or membrane-type (MT)-MMP. This cleavage of IgG, by separating the antigen-binding (Fabprime prime or minute)(2) from the Fc portion, will remove much of the immunoglobulins' functionality, e.g. complement fixation, Fc receptor binding. In the context of a tumour producing matrilysin or stromelysin, this may represent a way in which the tumour protects itself from ADCC. In inflamed or damaged tissues where plasma protein leakage occurs, degradation by MMPs may be a mechanism for clearance of IgG.     

Identification of a novel miRNA-target gene regulatory network in osteosarcoma by integrating transcriptome analysis

Osteosarcoma remains a leading cause of cancer death in children and young adolescents. Although the introduction of multiagent chemotherapy, survival rates have not improved in two decades. Therefore, it is urgently needed to know the details regarding molecular etiology to driving therapeutic inroads for this disease. In this study we performed an integrated analysis of miRNA and mRNA expression data to explore the dysregulation of miRNA and miRNA-target gene regulatory network underlying OS. 59 differentially expressed miRNAs were identified, with 28 up-regulated and 31 down-regulated miRNAs by integrating OS miRNA expression data sets available. Using miRWalk databases prediction, we performed an anticorrelated analysis of miRNA and genes expression identified by a integrated analysis of gene expression data to identify 109 differently expressed miRNA target genes. A novel miRNA-target gene regulatory network was constructed with the miRNA-target gene pairs. miR-19b-3p, miR-20a-5p, miR-124-3p and their common target CCND2, the nodal points of regulatory network, may play important roles in OS. Bioinformatics analysis of biological functions and pathways demonstrated that target genes of miRNAs are highly correlated with carcinogenesis. Our findings may help to understand the molecular mechanisms of OS and identify targets of effective targeted therapies for OS.     

The effect of ionizing radiation on the homeostasis and functional integrity of murine splenic regulatory T cells

Objective

Radiotherapy affects antitumor immune responses; therefore, it is important to study radiation effects on various compartments of the immune system. Here we report radiation effects on the homeostasis and function of regulatory T (Treg) cells, which are important in down-regulating antitumor immune responses.

Methods

C57Bl/6 mice were irradiated with 2 Gy and alterations in splenic lymphocyte fractions analyzed at different intervals.

Results

Total CD4+ numbers showed stronger decrease after irradiation than CD4+Foxp3+ Tregs. Tregs were less prone to radiation-induced apoptosis than CD4+Foxp3? T cells. The ratio of CD4+Foxp3? and CD4+Foxp3+ fractions within the proliferating CD4+ pool progressively changed from 74:26 in control animals to 59:41 eleven days after irradiation, demonstrating a more dynamic increase in the proliferation and regeneration of the Treg pool. The CD4+Foxp3+ fraction expressing cell-surface CTLA4, an antigen associated with Treg cell activation increased from 5.3 % in unirradiated mice to 10.5 % three days after irradiation. The expression of IL-10 mRNA was moderately upregulated, while TGF-β expression was not affected. On the other hand, irradiation reduced Treg capacity to suppress effector T cell proliferation by 2.5-fold.

Conclusion

Tregs are more radioresistant, less prone to radiation-induced apoptosis, and have faster repopulation kinetics than CD4+Foxp3? cells, but irradiated Tregs are functionally compromised, having a reduced suppressive capacity.     

Selective isolation of poliovirus in recombinant murine cell line expressing the human poliovirus receptor gene.

Sixty-eight laboratory strains representing 49 enterovirus, 10 adenovirus, and 3 reovirus serotypes were inoculated in a recombinant murine cell line expressing the human poliovirus receptor gene (L alpha cells). Only polioviruses caused cytopathic effect over a 10-day period. Likewise, only polioviruses were isolated, by use of L alpha cells, from 168 fecal specimens from children from developing countries. These results suggest that the recombinant L alpha cells can be used for selective isolation of poliovirus from clinical specimens.