NOD/SCID雌性小鼠动情周期的观察

目的观察NOD/SCID小鼠动情周期及卵巢切除术后阴道涂片的变化。方法连续观察9 d NOD/SCID鼠,每日进行两次阴道涂片,计算动情周期时间及发生率。卵巢切除后,阴道涂片并观察阴道涂片变化。结果NOD/SCID小鼠动情周期为4~6 d。有规律动情的小鼠占80%。阴道口状态与动情周期无明显相关。卵巢切除术后,阴道涂片呈动情后期或动情间期改变。结论采用阴道脱落细胞涂片法可判断NOD/SCID雌性小鼠的动情周期及特点,NOD/SCID雌性小鼠卵巢切除手术有效。     

NOD/SCID小鼠模型在实验血液学研究中的应用

NOD/SCID（非肥胖糖尿病/重症联合免疫缺陷）小鼠是在SCID（重症联合免疫缺陷）小鼠的基础上与非肥胖性糖尿病小鼠（NOD/Lt）品系回交的免疫缺陷鼠。NOD/SCID小鼠既有先天免疫缺陷,又有T和B淋巴细胞缺乏,各种肿瘤细胞可以植入,且较少发生排斥反应及移植物抗宿主病（GVHD）,所以NOD/SCID小鼠逐渐成为血液学实验研究的有用工具。本文从NOD/SCID小鼠的生物学特性、建立人类白血病模型、干细胞移植、药物研究及NOD/SCID小鼠应用中存在的不足和改良等方面综合述评。     

NOD1和NOD2基因在牦牛雄性生殖器官中的表达检测

为了解NOD1和NOD2基因在牦牛生殖系统中的表达情况,从脾脏组织总RNA中RT-PCR扩增牦牛NOD1和NOD2基因,半定量RT-PCR分析NOD1和NOD2 mRNA在牦牛雄性生殖组织的表达水平。结果显示,NOD1在脾脏、睾丸、附睾头、附睾体、附睾尾、输精管和阴茎中均相对低表达,而NOD2在脾脏、睾丸、附睾头、附睾体、附睾尾、输精管和阴茎中均相对高表达。结果表明,NOD1和NOD2在牦牛雄性生殖组织中广泛表达,提示其在牦牛生殖过程中可能发挥一定的作用。     

NOD1和NOD2：介导天然免疫的两种胞浆蛋白

NOD1和NOD2是新发现的一类参与天然免疫的胞浆蛋白质家族—核苷酸结合寡聚域样受体（the nucleotide bindingolig omerization domain-like receptor,NLRs）中的两个重要蛋白受体,它们通过识别外源病原菌的模式抗原分子而激活NF-κB等核转录因子,启动相关细胞因子的基因表达,释放炎性因子和抗菌肽等,其介导的信号通路在宿主抵御病原体感染的天然免疫中发挥着重要作用。     

Tle1 attenuates hepatic ischemia/reperfusion injury by suppressing NOD2/NF-κB signaling

ABSTRACT

Liver damage induced by ischemia/reperfusion (I/R) remains a primary issue in multiple hepatic surgeries. Innate immune-mediated inflammatory responses during the reperfusion stage aggravate the injury. Nevertheless, the detailed mechanism of hepatic I/R has not been fully clarified yet. Our research focuses on the role of Transducin-like enhancer of split-1 (Tle1) in the liver I/R injury and the relation between Tle1 and Nucleotide-binding oligomerization domain 2 (NOD2). To answer these questions, we constructed mouse models of I/R and cell models of hypoxia/reoxygenation (H/R). We found decreased Tle1 accompanied by increased NOD2 during reperfusion. Mice pro-injected with Tle1-siRNA emerged aggravated liver dysfunction. Repression of Tle1 had a significant impact on NOD2 and downstream NF-κB signaling in vitro. However, alteration of NOD2 failed to affect the expression of Tle1. To conclude, our study demonstrates that Tle1 shelters the liver from I/R injury through suppression of NOD2-dependent NF-κB activation and subsequent inflammatory responses.     

In vivo assay of human NK-dependent ADCC using NOD/SCID/γc (NOG) mice

Monoclonal antibodies are essential to the success of molecularly targeted therapies. Recently, numerous therapeutic antibodies have been developed for various diseases, including cancer and autoimmune diseases. Experimental systems to effectively evaluate these candidate antibodies are urgently needed. One of the mechanisms used by antibodies to kill tumor cells is antibody-dependent cellular cytotoxicity (ADCC), in which natural killer cells (NK) are the main mediator. The capacity to induce ADCC has conventionally been assessed in the human-mouse xeno-graft model, in which human peripheral blood mononuclear cells (PBMC), containing NK cells along with antibodies, are administered to tumor-bearing immunodeficient mice. However, contamination from other cellular populations often affects tumor growth, making it difficult to evaluate the antibody’s effect. In this study, we established a new NK-dependent ADCC assay model using a supra-immunodeficient strain of mice, NOD/SCID/γc^null (NOG). Our model system simply consisted of three elements: isolated human NK cells, a Burkitt’s lymphoma cell line (Daudi), and an anti-CD20 antibody (Rituximab). In this experimental setting, human NK cells from healthy donors retained their killing activity and suppressed the growth of Daudi cells in NOG mice when they were administered along with Rituximab. This system, therefore, is useful for evaluating the in vivo function of human NK cells.     

干燥综合征NOD小鼠模型研究进展

回顾国外NOD小鼠模型研究干燥综合征病理所取得的成果.查阅PUBMED上自2000年以来的相关文献,发现研究成果为疾病早期的细胞凋亡和疾病期的自身免疫反应提供了有利的证据.说明NOD小鼠模型在干燥综合征的病理机制研究中具有重要的作用,同时新抗原受体的发现为今后的研究提供了方向.     

Assessment of Benzene-Induced Hematotoxicity Using a Human-Like Hematopoietic Lineage in NOD/Shi-scid/IL-2R��null Mice

Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγ^null (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.     

细胞内模式识别受体NOD2信号转导及其调节

近年新发现的NOD2被证实是一细胞内模式识别受体,广泛参与宿主对病原体的多种免疫和炎症应答。与Toll样受体一样,它在调节天然免疫和获得性免疫方面具有重要意义,是联系天然免疫与特异性免疫的重要桥梁。该文着重介绍NOD2识别的配体及其方式、NOD2信号转导通路及其调节机制方面的有关进展。     

根瘤菌NOD因子的感知与信号传导

根瘤菌是一类引起豆科植物结瘤固氮的土壤细菌。根瘤中的类菌体固定空气中的氮气为宿主植物提供充足的氮源。共生体系的建立始于细菌与宿主植物间复杂的信号交换过程。植物产生类黄酮诱导相应的根瘤菌合成分泌结瘤因子 ,后者进而诱导宿主植物根系形态变化以及早期根瘤素基因表达。以下将就宿主植物结瘤因子的特异识别和早期信号传导进行讨论。     

NOD:一类新的固有免疫模式识别受体

哺乳动物主要通过Toll样受体(TLR)识别微生物。最近,发现一个新的蛋白质家族,核苷酸结合寡聚化结构域(NOD),参与胞内微生物的模式识别。NOD是一类位于胞质有典型的LRR-NBS结构的蛋白质家族,可以识别细菌细胞壁成分——细菌肽聚糖(peptidoglycan,PGN),活化NF-κB,参与固有免疫应答并诱导炎症反应和细胞凋亡。其中最有代表性的是NOD1和NOD2。最近的研究发现,NOD1和NOD2能识别细菌特殊结构。对该家族的研究将有助于防治胞内病原体感染、探索炎症性疾病的发病机制和治疗方案。     

CD34+和CD34-细胞在NOD/SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型, 比较了新鲜及培养后的CD34+和CD34-细胞在体内植入及重建造血能力。从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34+和CD34-细胞, 经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内, 6周后处死存活的小鼠, 取其骨髓、脾脏和外周血细胞, 分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测。经检测, 输注CD34+细胞和混合细胞的小鼠, 其体内CD45+细胞及人源各系血细胞的含量相近, 两者均远远高于输注CD34-细胞的小鼠。输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34+细胞的小鼠存活率约为66.7%, 而输注培养后混合细胞的小鼠全部存活, 且在两组存活的小鼠体内均能检测到CD45+细胞及人源各系血细胞。结果表明: 无论是新鲜还是培养后的CD34+细胞均具有在NOD/SCID小鼠体内植入和重建造血能力, 而CD34-细胞不具有该能力, 但CD34-细胞与CD34+细胞同时输注有助于提高小鼠的存活率, 说明其对CD34+细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用。     

Fracture Healing Is Delayed in Immunodeficient NOD/scid??IL2Rγ?cnull Mice

Following bone fracture, the repair process starts with an inflammatory reaction at the fracture site. Fracture healing is disturbed when the initial inflammation is increased or prolonged, whereby, a balanced inflammatory response is anticipated to be crucial for fracture healing, because it may induce down-stream responses leading to tissue repair. However, the impact of the immune response on fracture healing remains poorly understood. Here, we investigated bone healing in NOD/scid-IL2Rγ_c^null mice, which exhibit severe defects in innate and adaptive immunity, by biomechanical testing, histomorphometry and micro-computed tomography. We demonstrated that NOD/scid-IL2Rγ_c^null mice exhibited normal skeletal anatomy and a mild bone phenotype with a slightly reduced bone mass in the trabecular compartment in comparison to immunocompetent Balb/c mice. Fracture healing was impaired in immunodeficient NOD/scid-IL2Rγ_c^null mice. Callus bone content was unaffected during the early healing stage, whereas it was significantly reduced during the later healing period. Concomitantly, the amount of cartilage was significantly increased, indicating delayed endochondral ossification, most likely due to the decreased osteoclast activity observed in cells isolated from NOD/scid-IL2Rγ_c^null mice. Our results suggest that—under aseptic, uncomplicated conditions—the immediate immune response after fracture is non-essential for the initiation of bone formation. However, an intact immune system in general is important for successful bone healing, because endochondral ossification is delayed in immunodeficient NOD/scid-IL2Rγ_c^null mice.     

Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rγKO mice

Although physiological development of human lymphoid subsets has become well documented in humanized mice, in vivo development of human myeloid subsets in a xenotransplantation setting has remained unevaluated. Therefore, we investigated in vivo differentiation and function of human myeloid subsets in NOD/SCID/IL2rγ(null) (NSG) mouse recipients transplanted with purified lineage(-)CD34(+)CD38(-) cord blood hematopoietic stem cells. At 4-6 mo posttransplantation, we identified the development of human neutrophils, basophils, mast cells, monocytes, and conventional and plasmacytoid dendritic cells in the recipient hematopoietic organs. The tissue distribution and morphology of these human myeloid cells were similar to those identified in humans. After cytokine stimulation in vitro, phosphorylation of STAT molecules was observed in neutrophils and monocytes. In vivo administration of human G-CSF resulted in the recruitment of human myeloid cells into the recipient circulation. Flow cytometry and confocal imaging demonstrated that human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic function. Human bone marrow-derived monocytes/macrophages were further confirmed to exhibit phagocytosis and killing of Salmonella typhimurium upon IFN-γ stimulation. These findings demonstrate the development of mature and functionally intact human myeloid subsets in vivo in the NSG recipients. In vivo human myelopoiesis established in the NSG humanized mouse system may facilitate the investigation of human myeloid cell biology including in vivo analyses of infectious diseases and therapeutic interventions.     

How NOD2 mutations predispose to Crohn's disease?

NOD2 mutations are associated with the development of granulomatous inflammatory diseases, such as early-onset sarcoidosis (EOS), Blau syndrome (BS) and Crohn's disease (CD). As a pathogen-recognition molecule for muramyl dipeptide (MDP), NOD2 controls both innate and adaptive immune responses, through the regulation of cytokines, chemokines and antimicrobial peptides production. Notably, Nod2-deficient mice experienced increased susceptibility to enteric infection and to antigen-specific colitis. Furthermore, mutant mice bearing the orthologue of the major CD-associated NOD2^3020ins allele showed increased susceptibility to DSS-induced colitis. However, many questions remain open. (i) Is antimicrobial function deficiency sufficient to initiate the development of CD? (ii) How impaired and mutant NOD2 might lead to increased adaptive immune response? (iii) How do the other disease-associated NOD2 mutations contribute to the development of chronic intestinal inflammation? Whatever the relevant mechanism(s), it provides a casual link between abnormal bacterial sensing and development of inflammatory disorders. Further work should now focus on restoring abnormal NOD2 function by modulating antimicrobial function and regulatory mechanisms of the adaptive immune system.     

NOD1真核表达质粒的构建及表达

目的:构建天然免疫胞内识别受体核苷酸寡聚域1(NOD1)真核表达质粒。方法:NOD1基因片段经PCR扩增获得,经酶切后连接到真核表达载体pcDNA3/flag中,对挑选出的阳性克隆测序,将序列正确的重组质粒pflag-NOD1转染293T细胞,用Western印迹检测目的蛋白的表达,同时用NF-κB的萤光素酶报告基因检测NOD1蛋白的活性。结果:pflag-NOD1可以在真核细胞293T中表达,并可以增强NF-κB报告基因的转录活性。结论:构建了重组质粒pflag-NOD1,在细胞中表达NOD1后能够提高NF-κB转录的生物活性,为进一步研究NOD1的功能奠定了基础。     

NOD样受体在炎症反应中的调控作用

天然免疫(innate immunity)是机体免疫系统直接抵御病原体入侵的最初阶段,通过机体自身的特异性模式识别受体(pattern-recognition receptors,PRRs)来识别病原体特有的保守结构病原相关分子模式(pathogen-associated molecular patterns,PAMPs)。细胞内NOD样受体(NLRs)是胞浆型PRRs中的一个重要家族,病原体侵袭细胞可上调其表达,启动机体的免疫应答和炎症反应,在机体天然免疫应答中发挥独特的功能。最近有研究证明,NLRs的突变与一些人类免疫性疾病相关,并且在细菌感染和炎症反应的控制中起重要作用。该文将讨论NLRs在炎症疾病中的调控作用。     

Successful establishment of an orthotopic hepatoblastoma in vivo model in NOD/LtSz-scid IL2Rγnull mice

Investigation of hepatoblastoma in experimental conditions contributes relevantly to a detailed understanding of tumor biology and the investigation of new treatment approaches. Most systematical analyses currently use subcutaneous xenografts. We established a reproducible intrahepatic model with the hepatoblastoma-cell lines HuH6 and HepT1. The cells were stably transfected with a plasmid vector encoding for Gaussia luciferase. HuH6 and HepT1 were injected intrasplenically in NOD/LtSz-scid IL2Rγnull mice. Mice were splenectomized in order to avoid intrasplenical tumor growth. Multifocal intrahepatic tumor growth was observed in 85% (11/13) of HuH6 tumors and 55% (5/9) of HepT1 tumors. Serum Alpha-fetoprotein and Gaussia luciferase increased 5 weeks after tumor-cell inoculation. Tumors were detected by MRI at this time point. Immunhistochemical analysis such as vascularity (CD31), proliferation index (Ki-67), cytokeratin 7 and distribution of β-catenin in intrahepatic tumors were different to subcutaneous tumors. We established a reproducible xenograft model for intrahepatic hepatoblastoma growth with a high tumor incidence. Monitoring of tumor cell viability was optimized by measuring GLuc. This model enables further experimental investigations of HB in a more physiological milieu as emphasized by the β-catenin distribution.     

NOD2信号传导负调控相关因子的研究进展

核苷酸结合寡聚化结构域蛋白2(nucleotide-binding oligomerization domain protein 2, NOD2)是一类参与先天性免疫应答的受体,属于核苷酸寡聚NOD样受体家族(NOD-like receptors, NLRs)。NOD2通过识别病原菌表面的模式抗原分子从而激活丝裂原活化蛋白激酶(MAPK)和核因子-κB(nuclear factor-κB,NF-κB)等信号通路诱导炎症因子以及抗菌肽等物质,抵抗病原微生物的入侵。NOD2的负调控因子(去泛素化酶以及与通路相关的负调控蛋白等)可以阻断NOD2的信号传导从而削弱由NOD2引起的炎症反应并缓解炎症性疾病。该文总结了NOD2的负调控因子的种类以及相关作用机制,为进一步研究NOD2的调控机制以及临床干预NOD2相关性疾病提供参考。     

沙眼衣原体感染活化NOD1信号通路的研究

目的:探索沙眼衣原体(Chlamydia trachomatis,Ct)持续感染状态下,NOD1、IL-6及STAT3分子的表达情况和相互关系。方法:利用HeLa229细胞或STAT3基因沉默的HeLa229细胞,分别建立沙眼衣原体的急性感染和持续感染模型;应用Western Blot及ELISA等方法检测不同感染状态下STAT3及NOD1蛋白表达水平以及细胞因子IL-6的分泌水平。结果:HeLa229细胞在Ct感染状态下,STAT3和NOD1以及IL-6表达水平均升高,且于持续感染状态下的升高较急性感染状态下的升高更明显;沉默STAT3基因后,Ct感染的细胞NOD1及IL-6的表达水平下降明显。结论:HeLa229细胞在Ct持续感染状态下,STAT3能上调NOD1及IL-6表达水平,上述分子间存在NOD1-IL-6-STAT3正反馈信号通路。