乳杆菌肽聚糖调节小鼠免疫细胞基因表达的通路分析

目的:探索乳杆菌肽聚糖免疫调节作用的机制.方法:BALB/c小鼠腹腔注射乳杆菌肽聚糖,从腹腔巨噬细胞和脾淋巴细胞提取RNA,基因芯片分析基因表达情况,基于已知的基因网络数据库利用PathwayExplorer和GeneMAPP分析乳杆菌肽聚糖刺激特异性的基因网络.结果:PathwayEx-plorer分析得到的最显著变化的基因网络是"细胞因子-细胞因子受体相互作用"和"辅助性T细胞表面分子".GeneMAPP分析得到最显著基因网络是核糖体蛋白、炎性反应基因网络和血红素合成基因网络.结论:乳杆菌肽聚糖刺激引起大量蛋白表达,引起保护性炎性反应,激活Th细胞,可能引起Th1免疫反应.     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞线粒体膜电位的影响

为探讨双歧杆菌的完整肽聚糖(WPG)对小鼠腹腔巨噬细胞线粒体膜电位的影响,首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞,用四甲基罗丹明乙酯(TMRE)标记线粒体膜电位,最后用激光共聚焦显微镜检测巨噬细胞内荧光强度的变化。和对照组相比,WPG刺激巨噬细胞后,巨噬细胞内反映线粒体膜电位的荧光强度明显增强。双歧杆菌的WPG在激活巨噬细胞的过程中可提高其线粒体膜电位。     

prtP基因对副干酪乳杆菌调节小鼠肠道免疫作用的影响北大核心CSCD

目的研究副干酪乳杆菌丝氨酸蛋白酶编码基因prtP对小鼠肠道免疫调节作用的影响。方法BALB/c雌性小鼠随机分7组：PBS对照组、副干酪乳杆菌亲本株（Lactobacillus paracasei, Lp）、prtP缺失突变株（Lp△prtP）及回复株ReLp△prtP对应的高、中剂量组（1×109 CFU/ml、5×108 CFU/ml）连续灌胃0～21 d后对免疫器官指数、脾脏淋巴细胞转化试验、巨噬细胞能量代谢水平、腹腔巨噬细胞吞噬能力、细胞因子、脾脏CD11c＋、CD80＋有核细胞数进行检测。 结果与prtP基因缺失突变株相比,副干酪乳杆菌亲本株能显著提高小鼠脾脏淋巴细胞转化率、腹腔巨噬细胞的能量代谢水平以及腹腔巨噬细胞吞噬能力,能显著提高血清中IFN-α、IL-10、IFN-γ的表达,显著提高CD11c＋、CD80＋双阳性细胞数量。结论副干酪乳杆菌对肠道免疫功能的促进作用与prtP基因有关。     

双歧杆菌的完整肽聚糖对LPS激活裸鼠腹腔巨噬细胞的影响

目的 了解被ＬＰＳ激活的裸鼠腹腔巨噬细胞在用双歧双歧杆菌的完整聚糖刺激后产生的ＮＯ、ｉＮＯＳ及ｃＧＭＰ的水平。方法 以Ｇｒｉｅｓｓ试剂、激光截聚焦显微镜以及入免法分别测定裸鼠腹腔巨噬细胞产生的ＮＯ、ｉＮＯＳ及ｃＧＭＰ的水平。结果 完整肽聚糖注射组裸鼠腹腔巨噬细胞在ＬＰＳ诱导下产生ＮＯ、ｉＮＯＳ及ｃＧＭＰ含量均显著高于对照组（Ｐ〈０．０１）。结论 双歧双歧杆菌的完整肽聚糖能协同ＬＰＳ激活巨噬细胞,使     

双歧杆菌的全肽聚糖对裸鼠腹腔巨噬细胞产生IL-6、IL-12及一氧化氮的影响

目的 :探讨分叉双歧杆菌 (B bifidum)的全肽聚糖对巨噬细胞功能的调节作用。方法 :首先用全肽聚糖注射于裸鼠腹腔 ,以ELISA法测定裸鼠腹腔巨噬细胞产生的IL - 6和IL - 12含量 ,同时用Griess试剂检测一氧化氮 (NO)的水平。结果 :全肽聚糖注射组裸鼠腹腔巨噬细胞产生的IL - 6、IL - 12及NO含量分别为732 5 4± 190 30 (pg/mL)、816 37± 96 40 (pg/mL)和 48 90± 6 5 1(μmol/L) ;对照组分别为 30 3 78± 171 75 (pg/mL)、5 10 2 7± 12 3 46 (pg/mL)以及 30 6 7± 12 83(μmol/L) ,三者比较均具有显著差异 (P <0 0 1)。 结论 :分叉双歧杆菌的全肽聚糖能激活巨噬细胞 ,使之分泌多量的IL - 6、IL - 12及NO ,这些细胞毒性效应分子介导了全肽聚糖的多种重要生理功能。     

β-casomorphin-7对小鼠的免疫调节作用

目的：通过体外和体内方法研究β-casomorphin-7对小鼠脾脏淋巴细胞和腹腔巨噬细胞的作用.方法：利用脾细胞增殖试验和腹腔巨噬细胞中NO浓度的变化来研究体外不同的β-casomorphin-7浓度对脾脏淋巴细胞的增殖和腹腔巨噬细胞中NO浓度变化的影响,以及腹腔注射β-casomorphin-7和饮用β-casomorphin-7溶液对上述两个指标的影响.结果：体外试验表明,β-casomorphin-7在不同浓度对脾脏淋巴细胞的增殖显示了刺激和抑制的双向作用,而对NO的产生显示了明显的抑制作用（P＜0.01）.体内试验表明,β-casomorphin-7通过腹腔注射和饮用两种给药方式对脾淋巴细胞和腹腔巨噬细胞的作用是一致的.β-casomorphin-7显著地增强了脾淋巴细胞的增殖反应（P＜0.01）,且抑制了腹腔巨噬细胞NO的产生.结论：当前的试验表明,β-casomorphin-7具有免疫调节作用,且小鼠在2～3周龄时,可吸收入血发挥免疫调节作用.     

MicroRNA-126基因敲减小鼠腹腔巨噬细胞功能的变化及意义

研究microRNA-126(miR-126)基因敲减小鼠腹腔巨噬细胞的功能变化,初步探讨其意义。采用real-time PCR检测LPS刺激前后野生型(wild type,WT)小鼠腹腔巨噬细胞miR-126表达变化,CCK8法检测其增殖情况;观察miR-126基因敲减(knock down,KD)小鼠腹腔巨噬细胞的形态变化,并用real-time PCR检测miR-126表达变化;CCK8法检测LPS刺激下miR-126KD小鼠腹腔巨噬细胞增殖变化;FACS检测巨噬细胞表面MHCⅡ类分子和CD86分子的表达变化;最后,real-time PCR检测巨噬细胞表达炎症因子IL-6、TGF-β和Ⅰ型精氨酸酶(arginase 1,Arg-1)等的变化情况。结果显示,WT小鼠腹腔巨噬细胞在LPS刺激后miR-126表达水平下调,显著低于未刺激组(P0.05),而增殖能力明显增强(P0.05);与WT小鼠相比,miR-126KD小鼠腹腔巨噬细胞的miR-126表达量明显下调(P0.05);形态上,WT小鼠腹腔巨噬细胞有较长伪足,多呈梭形,而miR-126KD小鼠腹腔巨噬细胞多呈圆形,细胞边缘光滑较少见伪足;LPS作用48h后,miR-126KD小鼠腹腔巨噬细胞的增殖能力明显强于WT小鼠(P0.05);且其膜MHCⅡ类分子和CD86分子表达也较WT小鼠显著上调(P0.05);LPS刺激下,miR-126KD小鼠腹腔巨噬细胞CCL-1表达显著增加,而IL-6、TGF-β和Arg-1的表达水平显著减少(P0.05)。这些结果提示,miR-126基因敲减可显著影响小鼠腹腔巨噬细胞的增殖能力和功能相关分子的表达,提示miR-126对小鼠腹腔巨噬细胞的功能具有重要的调控作用。     

TLR2和TLR7信号协同作用导致小鼠炎性反应增强和胚胎丢失率增高的机制研究

Toll样受体(Toll-like receptors,TLR)识别致病原并激活宿主固有免疫反应。其中TLR2可识别革兰氏阳性细菌的细胞壁成分,而TLR7可识别病毒所产生的单链RNA。本研究采用小鼠巨噬细胞株(RAW264.7)和新鲜获得的小鼠腹腔巨噬细胞体外培养体系,用TLR2配体肽聚糖(peptidoglycan,PGN)或TLR7配体R837单独或联合刺激,观察其刺激效应。此外,采用上述TLR配体诱导建立小鼠胚胎吸收模型,观察体内条件下TLR2和TLR7信息转导对妊娠结局的影响。结果显示,两种配体同时刺激可呈现协同效应,导致巨噬细胞表达炎性细胞因子IL-1β、CCL5和TNF-α的水平显著增高。同时注射PGN和R837可使孕鼠胚胎丢失率显著增高。而预先注射中和抗体可显著降低PGN和R837所引起的胚胎丢失率增高。这些结果提示,细菌和病毒同时感染可发挥协同效应导致流产的发生,而IL-1β、CCL5和TNF-α等可能参与这一过程。     

双歧杆菌DNA对小鼠巨噬细胞中PKC和NF-κB的影响

目的：探讨青春型双歧杆菌的DNA对巨噬细胞中6种PKC和NF-κB的影响。方法：通过激光共聚焦显微镜观察小鼠腹腔巨噬细胞中PKCα、PKCβI、PKCβII、PKCγ、PKCε和PKCζ的荧光强度,以免疫细胞化学染色法检测巨噬细胞中NF-κB^＋细胞的密度。结果：双歧杆菌DNA注射组小鼠腹腔巨噬细胞中,PKCα和PKCβII的平均荧光强度明显高于对照组（P〈0.01）;而PKCβI、PKCγ、PKCε和PKCζ的平均荧光强度在两组间则无统计学意义（P〉0.05）。双歧杆菌DNA注射组巨噬细胞中,NF-κB^＋细胞的密度显著高于对照组（P〈0.01）。结论：青春型双歧杆菌的DNA可通过活化PKCα、PKCβII和NF-κB来激活巨噬细胞。     

淋巴细胞缺陷对内毒素血症小鼠腹腔巨噬细胞活化的影响

目的观察淋巴细胞缺陷对内毒素血症小鼠腹腔巨噬细胞活化的影响。方法采用腹腔注射脂多糖(LPS)建立Balb/c小鼠和T、B细胞缺陷的重症联合免疫缺陷(SCID)小鼠内毒素血症模型,ELISA检测2种小鼠腹腔灌洗液TNF-α和IL-10水平,实时荧光定量PCR检测腹腔巨噬细胞(peritoneal macrophage,PMa)TNF-α、IL-10及丝裂原蛋白激酶磷酸酶-1(mito-gen-activated protein kinase phosphatase-1,MKP-1)mRNA表达;ELISA检测Balb/c及SCID小鼠PMa体外刺激后细胞因子分泌情况。结果 LPS注射后1 h,SCID小鼠腹腔灌洗液TNF-α水平高于Balb/c小鼠,注射后3 h,IL-10水平低于Balb/c小鼠;LPS注射前及注射后,SCID小鼠PMa TNF-αmRNA表达高于Balb/c小鼠PMa,IL-10 mRNA表达低于Balb/c小鼠PMa;体外实验LPS刺激下,SCID小鼠PMa较Balb/c小鼠PMa分泌更多的TNF-α,IL-10的分泌却偏少。LPS注射前及注射后,Balb/c小鼠PMa MKP-1 mRNA表达均明显高于SCID小鼠。结论淋巴细胞缺陷导致内毒素血症小鼠腹腔巨噬细胞活性的增加,淋巴细胞抑制巨噬细胞的活化并可能调控其发育及成熟;MKP-1表达的减少可能是淋巴细胞缺陷导致腹腔巨噬细胞活性增加的分子机制之一。     

大鼠免疫细胞的促肾上腺皮质激素受体的研究

大鼠免疫细胞都有高、低亲和力两类ＡＣＴＨ受体。脾脏细胞高亲和力促肾上腺皮质激素（ＡＣＴＨ）受体的解离常数（ＫＤ）为０．１１ｎｍｏｌ／Ｌ，最大结合量（Ｂｍａｘ）为１．２ｆｍｏｌ／２×１０￣６细胞，３６６位点／细胞，低亲和力受体ＫＤ为５．７ｎｍｏｌ／Ｌ，Ｂｍａｘ为１４７ｆｍｏｌ／２×１０￣６细胞，４２０００位点／细胞。外周血淋巴细胞（ＰＢＬ）高亲和力ＡＣＴＨ受体的ＫＤ值为０．１５ｎｍｏｌ／Ｌ，Ｂｍａｘ为１．２ｆｍｏｌ／１×１０￣６细胞，１５８０位点／细胞，低亲和力受体ＫＤ值为６．１ｎｍｏｌ／Ｌ，Ｂｍａｘ为３７．５ｆｍｏｌ／１×１０￣６细胞，３２０００位点／细胞。胸腺细胞、Ｔ、Ｂ淋巴细胞及腹腔巨噬细胞（Ｍφ）亦具有高、低亲和力不同的ＡＣＴＨ受体。     

Role of immune cells in pregnancy

The human uterus is generally considered to be an immunologically privilege site that isolates the implanted allogeneic embryo from an aggressive maternal immune response. Maternal tolerance of the fetal allograft could be the result of the integration of numerous mechanisms promoted by decidual natural killer (NK) cells, macrophages and T cells. In this review, we outline the possible role of all these immune cells on the maintenance of pregnancy, focusing on the role of the T cells.     

Nox enzymes in immune cells

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes is a multi-component electron transferase that uses cytoplasmic NADPH to convert molecular oxygen to superoxide anion, consequently delivering reactive oxygen species to the site of invading microorganisms. Together with soluble factors and other phagocyte-derived agents, the resultant toxic species kill and degrade the ingested microbe. Flavocytochrome b (558), a heterodimeric protein composed of gp91 phox and p22 phox, is the membrane component of the NADPH oxidase and was previously thought to be uniquely expressed in phagocytes. Based on structural homology with gp91 phox, recent studies have defined a family of NADPH oxidase proteins (Nox) that is widely distributed throughout the plant and animal kingdoms and in many tissues in multicellular organisms. The goals of this review are to review features of the phagocyte NADPH oxidase that serve as a paradigm for exploiting oxidants for host defense, and to discuss contributions of other Nox proteins to innate immunity.     

Aging and innate immune cells

The innate immune system serves an important role in preventing microbial invasion. However, it experiences significant changes with advancing age. Among the age-associated changes are: Aged macrophages and neutrophils have impaired respiratory burst and reactive nitrogen intermediates as a result of altered intracellular signaling, rendering them less able to destroy bacteria. Aged neutrophils are also less able to respond to rescue from apoptosis. Aged dendritic cells (DC) are less able to stimulate T and B cells. The altered T cell stimulation is a result of changes in human leukocyte antigen expression and cytokine production, and lower B cell stimulation is a result of changes in DC immune complex binding. Natural killer (NK) cells from the elderly are less capable of destroying tumor cells. NK T cells increase in number and have greater interleukin-4 production with age. Levels of various complement components are also altered with advancing age.     

Lysis of herpesvirus-infected cells by immune spleen cells.

Spleen cells from herpes simplex-infected mice have been shown to lyse 51Cr-labeled virus-infected target cells. The cell-mediated lysis was shown to be antibody dependent but not involving adherent cells. Lysis of infected cells by this mechanism may be one form of host defense in infection by some viruses.     

Mechanism of immune interferon production in vitro: interaction between immune interferon-producing cells and antigenic cells.

Various processes of in vitro immune interferon production by sensitized spleen cells stimulated with allogeneic cells were investigated. When L cells, an interferon-inducing antigen, were fixed with methyl alcohol or paraformaldehyde, the ability to induce immune interferon disappeared. In this immune interferon production system, the majority of sensitized spleen cells adhered to target cells within 1 h of cocultivation. Adherence of immune interferon-producing cells to target cells was observed only when L cell-sensitized spleen cells were cocultured with L cells or with mouse embryo cells derived from C3H mice. Fixation of antigenic cells with methyl alcohol or paraformaldehyde significantly reduced cell adherence. When L cells alone or sensitized spleen cells alone were pretreated separately with cytochalasin D, neither cell type could bind to partner cells. Specific adherence did not take place at 4 degrees C, nor in the presence of dinitrophenol or sodium azide. Continuous protein synthesis in both cells was not required for immune cell adherence. Divalent cations, Ca2+ or Mg2+, were required for this immune specific adherence to take place. However, once stable adherence was established, treatment with cytochalasin D, ethylenediaminetetraacetic acid, or sodium azide, or simple reduction of temperature, did not disrupt the binding. Interaction between immune interferon-producing cells and antigenic cells can be subdivided into two phases according to the requirement for divalent cations: (i) lymphocytes and antigenic cells interact transiently, and divalent cations are required to maintain the binding; (ii) lymphocytes and antigenic cells form a stable interaction, and deprivation of divalent cations does not disrupt the binding. Colchicine showed an inhibitory effect in the period after cell-to-cell adherence. Colchicine did not inhibit the release of interferon. On the other hand, vinblastine, another antimicrotubule agent inhibited the secretion of immune interferon. Since interferon synthesis was not stopped immediately after addition of cycloheximide, continued protein synthesis of sensitized spleen cells was not required for interferon secretion. The present study showed that adherence of immune interferon-producing cells to antigenic cells was a complex phenomenon involving a series of successive events.