不同亚群肝脏巨噬细胞在猪血清诱导大鼠免疫性肝损伤中作用

目的:我们前期研究发现在正常大鼠肝脏中存在2个巨噬细胞亚群,本实验将探讨猪血清诱导免疫性肝损伤大鼠肝脏巨噬细胞的构成及作用。方法:(1)制作大鼠免疫性肝损伤模型。(2)肝脏病理组织学及免疫荧光研究。(3)流式细胞技术研究并分离肝脏巨噬细胞。(4)实时定量PCR检测肝脏巨噬细胞、炎症及纤维化相关细胞因子及Ki67(细胞增殖抗原,MKI67)的表达。结果:(1)猪血清腹腔注射组(IPS组)大鼠肝内大量纤维间隔形成,α平滑肌肌动蛋白(α-SMA)阳性的肌成纤维细胞增生,ED2(CD163,表面抗原分化簇163)阳性细胞增多。(2)免疫荧光及印度墨注射后显示IPS组肝脏中巨噬细胞大小不一,自身荧光强弱不等,在肝内分布不同;GdCl3仅可清除体积大、自身荧光强的细胞。(3)自大鼠肝脏分离出2个巨噬细胞亚群,即ED2染色强阳性、自身荧光强的细胞(ED2high/AFhigh)和ED2染色弱阳性、自身荧光弱的细胞(ED2dim/AFdim),与对照组相比IPS组2个亚群细胞数量均显著增多。(4)ED2high/AFhigh细胞中主要表达与炎症相关的细胞因子,ED2dim/AFdim细胞则主要表达与纤维化相关的细胞因子。(5)ED2dim/AFdim细胞Ki67表达显著高于ED2high/AFhigh细胞,IPS组巨噬细胞Ki67表达显著高于对照组。结论:猪血清诱导免疫性肝脏损伤大鼠肝脏中亦存在2个巨噬细胞亚群,它们在肝脏损伤发病中发挥不同的作用,ED2dim/AFdim细胞增殖活跃,主要表达与纤维化相关的基因,可能在猪血清诱导的肝纤维化形成中起着重要作用。     

BALB/c小鼠胸腺巨噬细胞不同亚群特性研究

目的研究小鼠胸腺巨噬细胞组成、分布、形态、功能。方法糖皮质激素诱导小鼠胸腺细胞凋亡,采用免疫荧光染色法,免疫组织化学和酸性磷酸酶(ACP)双重染色方法,发现小鼠胸腺巨噬细胞的不同表型。结果发现小鼠胸腺巨噬细胞存在四种表型:树状突起型细胞:可被Mac-2,F4/80单克隆抗体标记,数量最多,分布于整个胸腺实质且有吞噬作用;扁平型细胞:可被F4/80单克隆抗体标记,分布于皮质被膜下方;小卵圆型细胞:可被Mac-2单克隆抗体标记,分布于胸腺髓质和皮髓交界(CMR),此类细胞未见吞噬现象。ED1~+细胞:细胞形态不规则,主要分布于皮髓质交界区。以上不同的巨噬细胞亚群均可显示酸性磷酸酶活性。结论小鼠胸腺巨噬细胞存在不同亚群,表现在其组成、形态、分布、特点及功能差异等方面。     

巨噬细胞异质性研究的进展

近几年来,在研究巨噬细胞的免疫生物学时发现不同组织中的巨噬细胞在功能、形态、生物特性等方面都有明显的差异,而且,在同一组织中的巨噬细胞也存在明显的功能异质性。此外,由于抑制性巨噬细胞的出现,人们更加注意巨噬细胞的亚群问题,从而促进了巨噬细胞异质性的研究。     

巨噬细胞的细胞毒功能异质性及硒的影响

实验证明,巨噬细胞(Mφ)是由形态、功能及结构上各异的细胞亚群所组成,即所谓Mφ异质性。体内不同解剖部位的Mφ是异质的,M.S.Walker将其称为异源亚群(Interpopulations)。体内同一解剖部位的Mφ亦可以是异质的,Walkes称之为同源亚群(Intrapopulations)。研究mφ亚群可采用密度梯度离心法,是根据细胞的物理性状(密度)将其分成不同群体。它既     

超抗原SEB 活化的NKT 细胞亚群形态和分化途径的研究

目的:以NKT细胞形态和分化途径为焦点, 研究超抗原SEB活化的NKT 细胞亚群的特征.方法:C57BL/J小鼠脾细胞分别经SEB或者ConA诱导, 收集体外扩增10 d 和5 d的淋巴细胞以及获取正常黏附性巨噬细胞.用荧光抗体染色, 流式细胞仪测定淋巴细胞和大淋巴细胞群表面CD69的表达、 NKT细胞亚群百分数和不同NKT 细胞亚群的分化途径.倒置显微镜400倍下比较大淋巴细胞的形态.结果:SEB活化的淋巴细胞和大淋巴细胞表面CD69分子表达由正常值的0.11%分别提高到55.00%和68.95%.大细胞群中CD8+ 和TCRVB8+NKT 细胞亚群的百分数由原始的0.36%和0.81%分别提高到30.29%和31.48%.这些细胞位于流式细胞图的上部, 是大体积细胞群.显微镜下显示SEB活化的大淋巴细胞体积不仅大于ConA活化的T淋巴细胞, 而且比正常巨噬细胞大5倍以上, 是非黏附性细胞.胞内质粒松散、胞质与核之比＞1.SEB活化的CD8+ 和TCRVβ8+NKT细胞亚群均由T细胞直接分化而来.结论:SEB活化10 d的NKT细胞亚群主要是CD8+ NK1.1+和TCRVβ8+ NK1.1+的NKT细胞.这些细胞是大体积淋巴细胞, 应属于T细胞的亚群.     

PGE_2及IL_1调节胸腺细胞增殖反应

巨噬细胞(Mφ)受不同物质刺激后能产生具有拮抗作用的各种因子,一种是增强胸腺细胞增殖的IL-1,另一种是抑制胸腺细胞增殖的PGE_2,这两种因子是Mφ受同一种物质刺激而产生的.本文着重研究这两种因子对胸腺细胞增殖作用的相反效应,并探讨它们是否由巨噬细胞不同亚群所产生.     

microRNA调控树突状细胞分化与成熟的机制

树突状细胞(DC)含有不同的异质性亚群,在获得性免疫的启动、定向激活及调节中发挥重要作用.DC的自身发育分化及功能性成熟受细胞因子及转录因子构成的复杂网络调控.近期研究发现,microRNA (miRNA)通过抑制蛋白翻译或降解mRNA转录本来调控基因表达,调节包括免疫系统在内的多种生物学过程.许多miRNA在B、T淋巴细胞、DC、巨噬细胞及其他类型免疫细胞的发育、分化、存活及功能成熟起到重要作用,部分DC相关的miRNA如miR-155和miR-146a同时参与其他免疫细胞的调节.本文综述了DC亚群的功能,靶向不同DC亚群的免疫后果及细胞表面受体的种类;同时也总结了miRNA在DC由髓系前体细胞发育并分化为特异性亚群过程以及其在DC特异性功能中所发挥的重要作用.     

头颈部鳞状细胞癌巨噬细胞关键基因的筛选分析

目的 应用生物信息学的方法分析筛选头颈部鳞状细胞癌(HNSCC)巨噬细胞中的潜在关键基因,为HNSCC的预后提供靶点。方法 基于在线数据库,利用一致流形近似与投影(UMAP)降维,捕获巨噬细胞群;进一步通过t-分布随机近邻嵌入(tSNE)聚类降维分析肿瘤组织与正常组织细胞群分布的变化并筛选差异基因的表达;运用Monocle包对关键风险基因在不同发育阶段细胞中的表达情况进行分析;利用Kaplan-Meier Plotter在线数据平台分析生存曲线;运用空间转录组技术验证关键基因在组织中的表达映射;多色荧光免疫组化进行临床样本的验证。结果 捕获得到7个巨噬细胞亚群,其中第1亚群仅存在于肿瘤组织中且分泌型磷蛋白1(SPP1)基因高富集。SPP1高表达趋向巨噬细胞M2型极化并处于细胞分化的终末阶段。SPP1+巨噬细胞糖酵解、缺氧、上皮间质化、血管生成等功能活跃,与HNSCC患者的预后呈负相关。结论 SPP1可能成为HNSCC中有价值的预后生物标志物。     

巨噬细胞及组蛋白乙酰化对其极化的调控

巨噬细胞是极具异质性及多能性的免疫细胞,可响应不同环境信号而极化为对组织稳态或宿主防御具有特定功能的表型。经典活化的巨噬细胞和替代活化的巨噬细胞是巨噬细胞极化后的两类主要亚群。表观遗传修饰可通过调控基因表达对巨噬细胞极化发挥调节作用,组蛋白乙酰化是其修饰机制中最常见的类型之一,本文就组蛋白乙酰化调控巨噬细胞极化的研究进展作一综述。     

人类单核细胞的异质性

单核细胞能辅助并调节体液免疫及细胞免疫,它们的不同功能与异质性有关,与T细胞亚群的各种功能相似。单核/巨噬细胞     

Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18)

A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno- and enzyme-histochemistry on cryostat sections. The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of Mr 160,000 and 95,000.     

Rat bone marrow and monocyte cultures: influence of culture time and lymphokines on the expression of macrophage differentiation antigens

A set of seven monoclonal antibodies (moabs) has been shown to discriminate in situ between distinct subpopulations of macrophages in the rat. It is still controversial if this heterogeneity is caused by the existence of different lineages or by differentiation of a common precursor. In both cases, the differentiation process might be regulated by microenvironmental factors. The present study examines the expression of the macrophage markers recognized by the seven ED-moabs in bone marrow and monocyte cultures. Furthermore, the impact of culture time and stimulating factors on the antigen expression in these cultures was tested. The expression of the ED3 antigen is highly inducible in bone marrow cultures. Factors that might be responsible for the increased ED3 expression are investigated. This strong ED3 expression by bone marrow-derived macrophages is nearly absent by monocyte-derived macrophages. This implies that the ability to express ED3 is blocked before the macrophage precursor cells enter the circulation to become monocytes. The ED2 expression cannot be induced under the tested circumstances bone marrow macrophages in vivo do not express these antigens. In culture, these macrophages stain positive for these markers already after the first day of culturing. The other three antigens are expressed on all macrophages under all tested circumstances.     

Ontogenetic development, differentiation, and phenotypic expression of macrophages in fetal rat lungs.

Development, differentiation, and distribution of macrophages in fetal rat lungs were investigated immunohistochemically using anti-rat macrophage monoclonal antibodies. In the lung buds, RM-1+ macrophages were first detected on fetal day 13, and some showed reactivity for TRPM-2. They populated in the peribronchial mesenchyme of the lung buds, proliferated in loco, and showed no peroxidase activity in any intracellular organelles. Their immunophenotypic and ultrastructural features were consistent with those of primitive/fetal macrophages. By fetal day 16, some of them expressed ED1, but ED1+ cells were a minor subpopulation throughout the fetal period. On fetal day 18, ED2+ macrophages developed; some also were positive for RM-1, but the others were negative. Both the RM-1+ and ED2+ macrophages were major macrophage subpopulations and expressed Ki-M2R and/or TRPM-3; ED2+ and/or Ki-M2R+ cells are regarded as pulmonary interstitial resident macrophages. In organ culture, a similar expression of differentiation antigens by macrophages was confirmed. None of these macrophages cytochemically showed any peroxidase activity in vivo or in vitro. In the fetal stage, both RM-1+ and ED2+ macrophage subpopulations showed proliferative potential, suggesting their ability to proliferate and survive in vivo.     

Characterization of pulmonary macrophages and bronchus-associated lymphoid tissue (BALT) macrophages in the rat. An enzyme-cytochemical and immunocytochemical study

Three subpopulations of macrophages present in the lung, viz. pulmonary alveolar macrophages (PAM), pulmonary tissue macrophages (PTM), and macrophages in the bronchus-associated lymphoid tissue (BALT), were compared using enzyme-cytochemical and immunocytochemical methods. BALT macrophages can be distinguished from PAM and PTM on the basis of their enzyme reaction products. With three monoclonal antibodies, ED1, ED2, and ED3, that recognize different antigen determinants of rat macrophages, the three subpopulations could be clearly distinguished. PAM are ED1-positive and PTM are ED2-positive. The macrophage subpopulation scattered throughout BALT is ED1-positive; macrophages situated at the peripheral site of BALT, near artery and bronchus, are ED2-positive. No ED3-positive macrophages were observed either in the lung or in BALT. These results are discussed with respect to the possible relationship between the subpopulations of pulmonary macrophages.     

Ontogenic development of macrophage subpopulations and Ia-positive dendritic cells in fetal and neonatal rat spleen.

Development, differentiation, and distribution of macrophage subpopulations and Ia+ dendritic cells in the fetal and neonatal rat spleen were investigated by means of double immunohistochemical staining and immunoelectron microscopy. To characterize these cell populations, a panel of anti-rat macrophage monoclonal antibodies (RM-1, ED2, ED3, TRPM-3, Ki-M2R) and an anti-rat Ia antibody (OX6) were used. In the fetal rat spleen, macrophages were first detected by RM-1 at fetal day 15. ED2+ and/or Ki-M2R+ macrophages appeared at fetal day 16. TRPM-3+ and/or ED3+ macrophages appeared a day later. During the fetal and neonatal development, ED2+ and TRPM-3+ macrophages differentiated independently, maturing into red pulp macrophages and marginal metallophilic and marginal zone macrophages respectively. Intimate topographical relations were observed between ED2+ macrophages and hematopoietic cells and between TRPM-3+ macrophages and marginal zone lymphocytes. Ia+ cells were first observed around arterioles at fetal day 15. In the fetal and neonatal period, the number of Ia+ cells gradually increased, their shape became dendritic, and they matured into interdigitating cells in the inner periarteriolar lymphatic sheath. In ontogeny, Ia+ dendritic cells were not stained with ED2 or TRPM-3. These results suggest that ED2+ macrophages, TRPM-3+ macrophages, and Ia+ dendritic cells are distinct cell lines that pursue independent developmental process in spleen ontogeny.     

Primary culture of rat thymic non-lymphoid cells: influence of culture time on the expression of macrophage differentiation antigens defined by monoclonal antibodies.

A panel monoclonal antibodies (mAbs) raised to rat thymic non-lymphoid cells has been shown to discriminate between distinct subpopulations of macrophages depending on their anatomic localization in the thymus. These reagents were used in this study to examine the expression of macrophage-associated antigens in primary culture of rat thymic stromal cells. The phenotype of both adherent macrophage (AM) monolayers and non-adherent cells (NAC) released in culture medium was studied at different time points after cultivation. More than 95% AM expressed ED1 and R-MC 38 antigens (pan-macrophage markers), class I MHC antigens (OX-18) and iC3b receptor recognized by OX-42 mAb. Most of them (70-85%) were reactive with ED2, R-MC 40, 41 and 42 mAbs specific for cortical and cortico-medullary zone (CMZ) macrophages. A much smaller percentage was positive with R-MC 43/44 and R-MC 46/47 mAbs staining CMZ/medullary macrophages and a subset of cortical macrophages, respectively. A minor subset of AM expressed class II MHC molecules which progressively decreased during cultivation. NAC were phenotypically heterogeneous. In comparison with adherent cells they contained a lower percentage of cortical/CMZ phenotype macrophages. In addition, NAC were slightly enriched in R-MC 43+ cells and more significantly expressed IA/E antigens (85-95%). ED3, R-MC 39 and 45 mAbs reactive with thymic macrophages in situ were mostly non-reactive with AM and NAC in culture.     

Embryonic and fetal rat myoblasts form different muscle fiber types in an ectopic in vivo environment.

Limb muscle development is characterized by the migration of muscle precursor cells from the somite followed by myoblast differentiation and the maturation of myotubes into distinct muscle fiber types. Previous in vitro experiments have suggested that rat limb myoblasts are composed of at least two distinct myoblast subpopulations that appear in the developing hindlimb at different developmental stages. These embryonic and fetal myoblast subpopulations are believed to generate primary and secondary myotubes, respectively. To test this hypothesis, cells obtained from embryonic day 14 (ED 14) and ED 20 rat hindlimbs were analyzed for myosin heavy chain expression after long-term differentiation in adult rat brains. Fetal myoblasts from ED 20 hindlimbs produced muscle fibers with a phenotype similar to that seen in tissue culture--predominantly fast myosin with a small proportion also coexpressing slow myosin. However, injection sites populated by embryonic myoblasts from ED 14 hindlimbs produced a different phenotype from that previously reported in culture, with fibers expressing an entire array of myosin isoforms. In addition, a subpopulation of fibers expressing exclusively slow myosin was found only in the embryonic injection sites. Our results support the existence of at least three myogenic subpopulations in early rat limb buds with only one exhibiting the capability to differentiate in vitro. These findings are consistent with a model of muscle fiber type development in which the fiber type potential of myoblast populations is established before differentiation into myotubes. This process establishes myogenic subpopulations that have restricted adaptive ranges regulated by both intrinsic and extrinsic factors.     

Rat thymus macrophages: an immunohistochemical study on fetal, neonatal and adult thymus

The pre- and postnatal development of the macrophage population of rat thymus is investigated applying enzyme-histochemical and immunohistochemical techniques, both on tissue sections and cell suspensions. A set of three monoclonal antibodies (ED1, ED2 and ED3), each of which recognizes cells of the monocyte-macrophage lineage in the rat, enabled us to distinguish between macrophages in the various compartments of the thymus. The medulla is characterized by ED1-positive dendritic cells, the corticomedullary region comprises numerous monocyte-like ED1-positive macrophages and the cortex contains a particular subpopulation of branched ED2-positive macrophages. Both the medullary dendritic cells and the cortical branched cells show Ia-membrane staining. ED3-positive cells are only occasionally present. During fetal life ED1-positive monocyte-like macrophages and dendritic cells are present. Just after birth ED2-positive cortical macrophages start to develop. Their number increases strongly during the first week after birth. The role of the various subpopulations of thymic macrophages is discussed.     

Tumor-derived monocyte chemoattractant protein-1 induces intratumoral infiltration of monocyte-derived macrophage subpopulation in transplanted rat tumors.

By immunohistochemistry using anti-rat macrophage monoclonal antibodies RM-1, ED1, ED2, ED3, TRPM-3, and Ki-M2R, we studied transplanted rat tumors of 9L (rat gliosarcoma), Ad-2 (rat mammary carcinoma), and MT-P (rat malignant fibrous histiocytoma) cell lines to examine the distribution pattern of macrophages within and around the tumors. Most tumor-associated macrophages expressed RM-1, ED1, and Ia antigens, indicating activated macrophages. Based on differences in their immunophenotypical expression, these macrophages were distinguished into two major subpopulations. One expressed TRPM-3 and/or ED3, and the other was positive for ED2 and Ki-M2R. The former was considered to be monocyte-derived macrophages, whereas the latter showed the immunophenotype of tissue-fixed, resident macrophages. Infiltration and distribution patterns in the two macrophage subpopulations differed in the three different tumors. Monocyte-derived, activated macrophages infiltrated into 9L- and Ad-2-transplanted tumors, which markedly produced monocyte chemoattractant protein-1 (MCP-1). Additionally, numerous ED2- and Ki-M2R-positive macrophages were observed within the Ad-2-transplanted tumors, and some of them expressed TRPM-3. However, there were few macrophages in the MT-P-transplanted tumors that showed no MCP-1 production. In transplanted tumors of four MT-P/MCP-1 cell lines established by transfecting a rat MCP-1 gene expression vector (pCEP4/MCP-1) into the MT-P cell line, different levels of MCP-1 production were detected, which correlated well with the numbers of intratumorally infiltrated TRPM-3-positive macrophages. In contrast, ED2- and Ki-M2R-positive macrophages were not detected in any MT-P/MCP-1-transplanted tumors. MT-P/MCP-1-transplanted tumors exhibited lower growth rate than parental MT-P-transplanted tumors. These results indicate that tumor-derived MCP-1 induces intratumoral infiltration of monocyte-derived macrophages, but not macrophages with the immunophenotype of tissue-fixed, resident type. The former population of macrophages seems to have a suppressive effect on the growth of tumors.     

Macrophages and Dendritic Cells during the Early Stages of Antigen-Induced Arthritis in Rats: Immunohistochemical Analysis of Cryostat Sections of the Whole Knee Joint

The appearance of different macrophage subpopulations, Ia-positive antigen-presenting dendritic cells and of T and B lymphocytes was studied in early phases of antigen-induced arthritis in rat knee joints. Cryostat sections of whole knee joints were analysed with immunohistochemical techniques using monoclonal antibodies against rat macrophages, Ia-antigen, and lymphocyte subpopulations. The results showed that in the early phases of the development of arthritis, the synovium was already infiltrated by many monocytes, young macrophages, granulocytes, perivascular Ia-positive non-lymphoid cells, some mature tissue macrophages, and only few T lymphocytes. In later phases not only monocytes, young macrophages and Ia-positive cells became more prominent but also the more mature ED2 positive macrophages and the ED3 positive macrophages that are normally confined to lymphoid organs became increasingly important. The T-cell population increased to some extent in later phases of arthritis induction, possibly induced by clustering with the Ia-positive cells.