乙型肝炎病毒转基因小鼠树突状细胞提呈功能的初步研究

目的;探讨HBV转基因小鼠树突状细胞（DC）功能与其免疫耐受状态的相羞生。方法：按改良Steinman方法,对正常非转基因小鼠和HBV转基因小鼠脾脏树突状细胞进行分离,DC经HBeAg预刺激后,再用此DC与ConA共刺激淋巴细胞增殖反应。结果：HBV转基因小鼠的DC细胞数为10^2／孔、10^3／孔、10^4／孔,分别刺激各组小鼠淋巴细胞时,HBV转基因小鼠及非转基因小鼠的淋巴细胞^3H-TdR掺入量均显著低于对照组。结论：提示HBV转基因小鼠的DC提呈功能低下。     

ADMA对单核细胞来源的树突状细胞成熟和免疫的影响

目的:通过观察非对称性二甲基精氨酸(ADMA)对树突状细胞(dendritic cells,DCs)成熟及免疫的影响来探讨AS形成的可能机制。方法:贴壁法分离人外周血单核细胞,在含重组人粒-巨噬细胞集落刺激因子(rhGM-CSF 20ng/ml)和重组人白细胞介素-4(rhIL-4,10ng/ml)的完全培养基中培养,五天后收集imDC,用1、8、16umol/L的ADMA干预未成熟DC 24h。用流式细胞术检测DC细胞表面分子的表达、吞噬能力及DC的凋亡,用混合淋巴细胞反应检测成熟DC刺激T淋巴细胞增殖的能力,用ELISA检测DC细胞因子的分泌。结果:生理浓度ADMA并不刺激DC成熟及分化;但病理浓度ADMA抑制DC成熟;抑制DC诱导的T淋巴细胞增殖;诱导DC凋亡:抑制DC分泌IL-12细胞因子、TNF-α及IL-10细胞因子。结论:生理浓度ADMA并不刺激DC成熟及分化;但病理浓度AD-MA抑制DC成熟和免疫。     

Hemozoin Enhances Maturation of Murine Bone Marrow Derived Macrophages and Myeloid Dendritic Cells

Background: Falciparum malaria is a severe health burden worldwide. Antigen presenting cells are reported to be affected by erythrocytic stage of the parasite. Malarial hemozoin (HZ), a metabolite of malaria parasite, has adjuvant properties and may play a role in the induction of immune response against the parasite. Objective: To determine the immunological impact of hemozoin on the capacity of innate immune cells maturation. Methods: Plasmodium falciparum (F32 strain) was cultured in O+ blood group up to 18% parasitemia. Natural hemozoin was extracted from infected red blood cells. Murine bone marrow derived macrophages and myeloid dendritic cells were stimulated with 4 ߤg/mL or 40 ߤg/mL of synthetic hemozoin (β-hematin) or natural hemozoin. We assessed the immunomodulatory role of synthetic or natural hemozoin in vitro by flowcytometric analysis. Results: The maturation markers MHCII, CD80 and CD86 were significantly upregulated (p<0.05) on the surface of murine bone marrow derived macrophages or myeloid dendritic cells. Data confirmed the potential of macrophages or myeloid dendritic cells, through hemozoin activation, to establish an innate immune response against malaria parasites. Conclusion: Both synthetic and natural hemozoin are potent inducers of cellular immunity against malaria infection. However, natural hemozoin is a stronger inducer as compared to synthetic hemozoin.     

Evaluation of the Immunomodulatory Effect of Curdlan on Maturation and Function of Mouse Spleen-Derived Dendritic Cells

Background: T helper 1 and T helper 17 cells play important roles in immunity against foreign invaders. Differentiation of these Th subsets is affected by state of maturation and cytokines that are produced by dendritic cells (DCs). Curdlan is a linear (1→3)-β- glucan and has shown activity against tumors and infectious agents. Objective: This study aims to investigate whether curdlan plays its role through affecting the maturation and cytokine production by DCs. Methods: DCs were isolated from the spleen of BALB/c mice by MACS method. After an overnight culture of DCs in the presence of curdlan, the expression levels of CD40, CD86, and MHC-II molecules were determined by flow cytometry. The production of cytokines involved in Th1 and Th17 cell differentiation (IL-12 and IL-6, respectively) was also evaluated by ELISA. Lipopolysaccharide (LPS) treated and untreated cells were considered as positive and negative controls, respectively. Results: The results of this study did not show a significant difference in the levels of surface expression of CD40 (p=0.82), CD86 (p=0.79), and MHC class II (p=0.84) molecules upon exposure to curdlan. However, LPS increased the intensity of CD40 expression on dendritic cells (p=0.04). In addition, it was revealed that curdlan-exposed DCs are not able to produce a significant amount of IL-6 and IL-12 cytokines. Conversely, LPS-treated DCs were able to make a significant amount of IL-12 (p=0.005). Conclusion: The results of the present study suggest that curdlan has no effect on Th1 or Th17 differentiation while LPS may induce Th1 deviation by induction of CD40 expression and IL-12 production.     

Dendritic Cells: Sentinels of Immunity and Tolerance

The induction of effective antigen-specific T-cell immunity to pathogens without the initiation of autoimmunity has evolved as a sophisticated and highly balanced immunoregulatory mechanism. This mechanism assures the generation of antigen-specific effector cells as well as the induction and maintenance of antigen-specific tolerance to self-structures of the body. As professional antigen-presenting cells of the immune system, dendritic cells (DC) are ideally positioned throughout the entire body and equipped with a unique capability to transport antigens from the periphery to lymphoid tissues.There is growing evidence that DC, besides their well-known immunostimulatory properties, also induce and regulate T-cell tolerance in the periphery. This regulatory function of DC is strictly dependent on their different stages of maturation and activation. Additionally, immunosuppressive agents and cytokines further influence the functions of maturing DC. The regulatory properties of DC include induction of T-cell anergy, apoptosis, and the generation of T-cells with regulatory capacities. This brief review summarizes the current knowledge about the immunoregulatory role of DC as guardians for the induction of T-cell immunity and tolerance.     

乙型肝炎患者树突状细胞内HBV DNA存在状况的研究

目的:研究慢性乙型肝炎患者外周血培养的树突状细胞(DC)内乙型肝炎病毒DNA(HBV DNA)存在状况,分析HBV DNA与DC功能下降的内在联系.方法:从慢性乙型肝炎患者外周血中分离单个核细胞,在含rhGM-CSF,rhIL-4的培养基中诱导培养为DC,用流式细胞仪检测乙肝患者DC的表型变化,用流式细胞仪分选出树突状细胞,应用PCR技术检测DC内HBV DNA存在状况.结果:①表达于慢性乙型肝炎患者DC表面的共刺激分子(B7-1,B7-2,CD1α)及MHC-Ⅱ类分子(HLA-DR)的水平明显降低.②树突状细胞内HBV DNA检测结果阳性.结论:乙型肝炎患者树突状细胞内存在HBV DNA感染,慢性乙型肝炎患者树突状细胞与正常人相比,表型表达降低.     

LPS诱导的DCs成熟在小鼠免疫耐受中的作用研究

目的探讨LPS诱导DCs成熟对于过敏原致耐受中的作用。方法分离、培养、纯化DCs,加入LPS100 ng/m l诱导DCs成熟,然后加入OVA共同孵育24 h后收集DCs,按每鼠106/m l气管内接种。以PBS作为阴性对照,以未加LPS刺激、与OVA共同培养的DCs气管内接种作为阳性对照。接种次日后将小鼠置于雾化箱内以1%OVA雾化,连续5 d。结果 LPS刺激后DCs对OVA的吞噬能力下降（其吞噬能力在30 m in时,分别为75.7%和34%;60 m in分别下降为71%和29.7%）,将这些DCs接种于气管内不能诱发典型的肺部过敏性炎症,与注射PBS相似;而未经LPS处理的DCs与OVA共培养后再接种于气管,经雾化激发后可引起典型的过敏性炎症。结论 LPS诱导的DCs成熟后对过敏原的递呈能力下降可能是机体免疫耐受中的主要原因。     

高浓度胰岛素对急性冠状动脉综合征患者外周单核细胞源树突状细胞分化成熟及免疫功能的影响

目的 研究高浓度胰岛素对急性冠状动脉综合征患者外周单核细胞源树突状细胞分化、成熟及免疫功能的影响.方法 采用贴壁法分离急性冠状动脉综合征患者(ACS组)和正常人(正常组)外周血单核细胞,在含粒-巨噬细胞集落刺激因子(100 μg/L)和白细胞介素4(100 μg/L)的RPMI 1640完全培养基中培养.5天后收集细胞,作为未成熟树突状细胞,重新铺板后继续在胰岛素浓度分别为1、10 nmol/L和100 nmol/L的RPMI1640完全培养基中培养48 h后,收集细胞和上清液,此时细胞作为成熟树突状细胞,采用流式细胞术检测成熟树突状细胞表面 CD40、CD80和CD83的表达;用酶联免疫吸附法测定检测上清液中细胞因子白细胞介素12、白细胞介素10和肿瘤坏死因子α的浓度;用倒置显微镜动态观察树突状细胞形态变化.结果 树突状细胞表型CD40、CD80和CD83随着胰岛素浓度升高而升高(P＜0.05),培养上清液中细胞因子白细胞介素12、肿瘤坏死因子α的浓度也随着胰岛素浓度升高而升高(P＜0.05),而细胞因子白细胞介素10的浓度则随着胰岛素浓度升高而降低(P＜0.05).同等胰岛素浓度下,急性冠状动脉综合征患者较正常组的树突状细胞表面CD40、CD80和CD83的阳性表达率升高(P＜0.05),培养上清液中细胞因子白细胞介素12、肿瘤坏死因子α的浓度升高(P＜0.05),而细胞因子白细胞介素10的浓度则降低(P＜0.05).结论 高浓度胰岛素促进了急性冠状动脉综合征患者的树突状细胞表面标志物CD40、CD80和CD83的表达;促进了树突状细胞对细胞因子白细胞介素12和肿瘤坏死因子α的分泌;对白细胞介素10的分泌则起抑制作用.高浓度胰岛素通过促进树突状细胞免疫功能成熟,参与动脉粥样硬化免疫炎症反应的发生和发展,是急性冠状动脉综合征发生的可能机制之一.     

树突状细胞与冠状动脉严重狭窄的关系及辛伐他汀的干预作用

目的探讨冠状动脉严重狭窄患者动脉粥样硬化与树突状细胞关系及辛伐他汀免疫干预作用的可能机制。方法根据选择性冠状动脉造影结果,20例冠状动脉正常,无明显动脉粥样硬化斑块者为阴性对照组;20例冠状动脉严重狭窄,未服用他汀类药物者为动脉粥样硬化组;20例冠状动脉严重狭窄,服用辛伐他汀40mg/d,连续30天以上者为辛伐他汀组。分别抽取冠状动脉血20mL,采用密度梯度离心法分离单个核细胞,行树突状细胞体外培养扩增,采用流式细胞术检测树突状细胞免疫表型、平均荧光强度、刺激T淋巴细胞增殖能力的刺激指数;动脉粥样硬化组分为B1、B2两个亚组,B2组在树突状细胞培养第5天时加入100μmol/L辛伐他汀,其他培养与检测方法相同。结果冠状动脉血分离单个核细胞后经体外培养与扩增,均成功培养出典型树突状细胞,各组树突状细胞形态无差异;与阴性对照组比较,B1组CD1a阳性细胞比例、CD1a与CD80、CD83、CD86双阳性细胞比例、平均荧光强度、收获细胞总数、树突状细胞数量和刺激指数均明显增高(P<0.05);与B1组比较,B2组收获细胞总数无差异,但CD1a阳性细胞比例、CD1a与CD80、CD83、CD86双阳性细胞比例、平均荧...     

The Effect of Beta Interferon on Dendritic Cells and Cytokine Synthesis by CD4+ T Cells

Background: Dendritic cells (DC) are a key regulator of the immune response, and interferon- beta (IFN-β) is considered an immunomodulatory molecule for DC. Objective: The purpose of this study was to evaluate the ability of IFN-β treated DC to induce cytokine secretion by CD4+ T cells. Methods: Dendritic cells were generated from blood monocytes with granulocyte-monocyte colony-stimulating factor and interleukin-4 with or without IFN-β. We analyzed the production of CD4+ T helper cytokines (IL-17, IFN- γ and IL-10) in the supernatant of the dendritic cell-T cell co- cultures by ELISA. We also studied the effects of HLA-G and costimulatory molecules on immature and mature DC. Results: IFN-γ and IL-17 decreased significantly in the presence of HLA-Gbearing DC compared to control cultures (p<0.05). Conclusion: Using the mixed leukocyte reaction, we found that DC treated with IFN-β mediated the inhibition of T cell activation via cytokine production. We conclude that this is important for preventing overactivation of the immune system.     

高浓度胰岛素对人树突状细胞分化、成熟及免疫功能的影响

目的:研究高浓度胰岛素对树突状细胞(Dendritic Cell,DC)分化、成熟及免疫功能的影响。方法:采用连续贴壁法分离正常人外周血单核细胞,在含重组人粒-巨噬细胞集落刺激因子(GM-CSF,100ng/ml)和重组人白细胞介素-4(IL-4,100ng/ml)的完全培养基中培养。5天后收集细胞,重新铺板后继续在胰岛素浓度分别为0nmol/L、10nmol/L及100nmol/L的培养基中培养48小时,收集细胞和上清液,采用流式细胞术检测细胞表面CD40、CD80和CD83的表达;用ELISA法检测上清液中细胞因子IL-12、IL-10、TNF-α的浓度;用倒置显微镜动态观察DC形态变化。结果:胰岛素浓度为10nmol/L、100nmol/L组的DC表面标成CD40、CD80和CD83阳性表达率较含胰岛素0nmol/L的对照组升高,培养上清液中细胞因子IL-12、TNF-α的浓度也较对照组升高,而细胞因子IL-10的浓度则较对照组降低。结论:高浓度胰岛素促进了树突状细胞表型CD40、CD80和CD83的表达;促进了DC对细胞因子IL-12和TNF-α的分泌;对IL-10的分泌则起抑制作用。高浓度胰岛素通过促进树突状细胞免疫功能的成熟,可能是其参与动脉粥样硬化免疫炎症反应的发生、发展的机制之一。     

HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的树突细胞免疫功能的影响

背景：树突细胞（DC）是体内功能最强的抗原呈递细胞,可激活初始型T细胞,生成辅助性T细胞和杀伤性T细胞。DC具有特异性呈递肿瘤抗原的能力,在肿瘤免疫中发挥重要作用。目的：探讨HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的DC免疫功能的影响。方法：分离培养脐血CD34^＋造血干细胞后,加入细胞因子组合诱导生成DC并将其分成HepG2细胞抗原负载组和对照组．以流式细胞仪测定DC生成率和免疫表型,以酶联免疫吸附测定（ELISA）检测干扰素-γ（IFN-γ）含量,以MTT法检测细胞毒性T淋巴细胞（CTL细胞）对HepG2细胞的杀伤作用。结果：DC生成率为60．2％±9．4％。与对照组相比,HepG2细胞抗原负载组DC免疫表型CD1a^＋／CD40^＋、CD83^＋／CD86^＋、CD14^＋／HLA-DR^＋比例显著增高（57．6％±5．4％对33．2％±6．0％、32．5％±3．9％对26．0％±2．8％、38．1％±2．6％对29．1％±2．1％,P〈0．01）;IFN-γ含量呈时间依赖性增高;CTL细胞对HepG2细胞的杀伤作用显著增强（43.3％±11.3％对13．9％±4．6％,P〈0．01）。结论：应用HepG2细胞抗原孵育脐血CD34^＋造血干细胞可诱导分化成熟DC,DC可促进异基因淋巴细胞活化分泌IFN-γ,并产生特异性CTL细胞,杀伤肝癌HepG2细胞。     

扶正化瘀方对大鼠CCl4损伤肝Kupffer细胞功能的影响

目的：探讨扶正化瘀方对大鼠急性ＣＣｌ４损伤肝Ｋｕｐｆｆｅｒ细胞功能的影响。方法：体外分离培养大鼠急性ＣＣｌ４损伤肝Ｋｕｐｆｆｅｒ细胞,制备扶正化瘀方药物血清对其进行干预,观察药物对其ＰＤＧＦ和ＴＧＦβ活性以及对Ｋｕｐｆｆｅｒ细胞线粒体呼吸功能的影响。结果：药物组ＰＤＧＦ和ＴＧＦβ活性均显著降低（Ｐ〈０．０５）;药物组闰体呼吸功能明显降低（Ｐ〈０．０５）。结论：扶正化瘀方对大鼠急性ＣＣｌ４损伤肝Ｋｕｐｆｆｅｒ细胞活化有抑制作用,可能抑制肝星状细胞旁分泌激活,是该方抗肝纤维化的作用机理之一。     

肝细胞对星状细胞凋亡的影响和复方861的干预作用

目的：研究肝细胞对星状细胞凋亡和影响中药复方861的干预作用。方法：体外采用分离培养的正常大鼠原代肝细胞和星状细胞系,将培养的肝细胞和复方861作用于不同的培养基再作用于星状细胞,用电镜观察和流式细胞仪检测细胞凋亡。临床研究对象是用复方861治疗6个月行治疗前后肝穿的慢性乙型肝炎病人。结果：原代培养的正常肝血细胞促进了星状细胞的凋亡（P＜0．05）。复方861可以增加这种促凋亡作用（P＜0．05）。临床研究显示慢性乙型肝炎患者肝星状细胞大量活化增生,用复方861治疗6个月后有肝细胞再生,同时活化的星状细胞数量显著减少,可观察到其凋亡。结论：正常肝细胞可以促进星状细胞凋亡,复方861对星状细胞有直接和间接的促凋亡作用。     

白藜芦醇和吉西他滨联合用药对人胰腺癌细胞作用的研究

背景：吉西他滨是治疗进展期胰腺癌的一线化疗药物,单独用药临床效果欠佳。白藜芦醇为脱嘌呤脱嘧啶核酸内切酶1／氧化还原因子－1（APEl／Ref-1）的氧化还原功能抑制剂,具有抑制恶性肿瘤生长的特性．可能在胰腺癌的预防和治疗中发挥重要作用。目的：检测白藜芦醇和吉西他滨联合用药对人胰腺癌细胞株生长和凋亡的影响．并进一步探讨发挥该作用的分子机制。方法：将胰腺癌细胞株SWl990和BxPc-3分为4组：溶剂对照组、吉西他滨组、白藜芦醇组和联合用药组。采用CCK-8法和流式细胞术分别检测细胞增殖和凋亡．以蛋白质印迹法检测APE1／Ref-1蛋白的表达。结果：作用于SWl990和BxPc-3细胞24h、48h和72h后．与溶剂对照组相比,三组用药组的细胞存活率均明显降低;作用于SWl990和BxPc-3细胞48h后,与溶剂对照组相比,三组用药组的细胞凋亡率均明显增加：联合用药组对细胞增殖和凋亡的影响均强于两组单独用药组。与空白对照组相比．吉西他滨组SWl990和BxPc－3细胞APEl／Ref-1蛋白表达均明显增加。结论：白藜芦醇和吉西他滨联合用药可明显加强对胰腺癌细胞增殖和凋亡的影响．白藜芦醇可能通1寸抑制APEl,Ref-1蛋白的氧化还原功能而增加胰腺痛细胞对吉西他滨的敏感十牛．     

氧化型脂蛋白(a)对兔主动脉平滑肌细胞增殖的影响

为探讨脂蛋白（a）高度致动脉粥样硬化作用的可能机制，本文通过体外CU2 氧化法进行了低密度脂蛋白和脂蛋白（a）的氧化，并利用细胞计数及氚标胸腺密啶脱氧核苷掺入法，比较观察了天然和氧化的脂蛋白（a）及低密度脂蛋白对培养的兔主动脉平滑肌细胞增殖的影响。结果发现，在相同氧化时间内脂蛋白（a）对Cu2 的氧化敏感性较低密度脂蛋白低；氧化后的脂蛋白（a）和低密度脂蛋白均可明显促进平滑肌细胞增殖使DNA合成增加（P＜0．01），且其作用较相应的天然脂蛋白大（P＜0．05．P＜0．01）、此结果提示：脂蛋白（a）经氧化后促进平滑肌细胞增殖可能是其致动脉粥样硬化的机制之一。