抗Vn、抗CD44单克隆抗体抑制内皮—单个核细胞粘附的研究

<正>单个核细胞粘附于动脉内膜表面可见于机体的许多病理过程和正常的生命活动,如炎症、免疫反应、动脉粥样硬化等.但有关粘附的机制尚不清楚,为此,我们对粘附相关蛋白介导粘附的作用进行了系列研究.本实验采用单克隆抗体阻断法,观察了Vn和CD44在内皮一单个核细胞粘附过程中的作用和作用特点.实验采用培养的血管内皮细胞,传至第二至三代,分离纯化外周血单个核细胞,将单个核细胞用单克隆抗体处理后,     

TLR4激动对内皮细胞粘附功能的影响及阿托伐他汀的干预研究

目的：观察Toll样受体4（TLR4）激动对脐静脉内皮细胞（HUVECs）氧化低密度脂蛋白受体LOX-1的调节和对单核细胞与HUVECs粘附率的影响，以及LOX-1在内皮细胞粘附功能中的作用，并观察阿托伐他汀的干预作用方法：RT-PCR方法检测TLR4、LOX-1 mRNA表达水平，流式细胞术检测TLR4、LOX-1蛋白表达水平，细胞计数法计算单核细胞与HUVECs粘附率。结果：脂多糖(1 mg/L) 孵育24 h上调HUVECs TLR4、LOX-1 mRNA和蛋白的表达，增加单核细胞与HUVECs粘附率,抗LOX-1抗体部分抑制LPS介导的单核细胞与HUVECs粘附率的增加，阿托伐他汀（10 μmol/L）抑制脂多糖介导的上述效应。结论：TLR4激动上调LOX-1表达及增加内皮细胞粘附功能，LOX-1在LPS介导的单核内皮细胞粘附功能中起部分作用，阿托伐他汀可能通过抑制TLR4表达及TLR4 介导的LOX-1表达而发挥其内皮细胞保护作用。     

肺炎链球菌粘附肺Ⅱ型上皮细胞触发宿主细胞酪氨酸蛋白磷酸化

目的 探讨细胞内信号传导与肺炎球菌侵袭、致病的关系,在体外研究Ⅱ型肺炎链球菌粘附肺Ⅱ型上皮细胞（A549）是否能触发细胞内酪氨酸蛋白激酶（TPK）信号传导途径 ,以及触发该信号传导可能的细菌亚组分。方法 用FTTC荧光标记肺炎链球菌,在体外观察肺炎链球菌粘附肺Ⅱ型上皮细胞的粘附动力学特征;用免疫组织化学和ELISA方法观察完整细菌触发的细胞内酪氨酸蛋白磷酸化,用各种因素预处理肺炎链球菌后,观察触发细胞内酪氨酸蛋白磷酸化可能的细菌亚组分。结果 证实了上述粘附过程存在剂量依赖和时间的依赖关系,而且是特异的过程;细菌粘附使细胞内酪氨酸磷酸化由细菌表面蛋白质介导。结论 肺炎链球菌粘附肺Ⅱ型上皮细胞能触发细胞内信号传导,且细菌表面蛋白质在触发胞内酪氨酸蛋白磷酸化传导中起着重要作用。     

小鼠脾细胞CD4~+ CD25~+调节性T细胞的表型特征

观察CD4+CD25+T和CD4+CD25-T细胞的表型和细胞因子的表达。自小鼠脾脏制备单个细胞悬液,分离CD4+T细胞、CD4+CD25+和CD4+CD25-T细胞,进行细胞表面标记,激活后进行细胞内细胞因子染色,利用流式细胞仪在单个细胞水平上分析细胞表面分子、转录因子和细胞因子表达之间的关系。结果:在CD4+T细胞中,约有7.8%的细胞同时表达CD25分子。与CD4+CD25-T细胞相比,CD4+CD25+T细胞CD44的表达略有增加,CD45RB的表达明显下降,CTLA-4和Foxp3明显增加。以同时表达CTLA-4和Foxp3的细胞为主,其次为单独表达Foxp3的细胞。细胞因子的研究结果表明,与CD4+CD25-T细胞相比,CD4+CD25+T细胞IL-2、IFN-γ明显减少,而只产生IL-10的细胞略有增加。CD4+CD25+调节性T细胞无论在表型、转录因子的表达以及细胞因子表达方面均于非调节性T细胞不同。     

透明质酸及其受体对细胞运动的作用

透明质酸 (HA)是细胞外基质的主要成分之一。细胞外基质中的HA通过对基质装配产生影响 ,形成多孔性疏松的基质 ,从而促进细胞运动。细胞表面的HA不仅能够介导细胞与基质的初黏附 ,而且在细胞运动时可促进细胞与基质的部分去黏附。细胞内的HA通过与细胞内HA结合蛋白以及信号转导相关的酶相结合 ,作用于细胞骨架 ,调节细胞运动。HA与其受体CD4 4 ,RHAMM或Layilin结合 ,转导信号入细胞内 ,使细胞骨架的构型发生改变 ,并调节与细胞运动有关的蛋白水解酶的合成和释放 ,由此促进细胞的黏附和运动。     

β-1,4-半乳糖基转移酶-Ⅰ在树突状细胞中的表达及作用

目的：观察β-1,4-半乳糖基转移酶-Ⅰ（β-1,4-galactosyltransferase-Ⅰ,β-1,4-GalT-Ⅰ）在人外周血来源的树突状细胞（Dendritic cells,DCs）中的表达与定位,研究其对DCs免疫功能的影响。方法：人外周血单核细胞经GM-CSF＋IL-4＋TNF-α诱导培养分化为DCs,采用RT-PCR、流式细胞术和激光共聚焦技术观察DCs中β-1,4-GalT-Ⅰ的表达与定位;通过细胞粘附试验分析β-1,4-GalT-Ⅰ表达与细胞粘附能力的关系。用α-乳清蛋白（α-lactalbumin,LA）作为细胞表面β-1,4-GalT-Ⅰ抑制剂,研究β-1,4-GalT-Ⅰ在DCs细胞粘附以及与CD4＋Th细胞形成免疫突触过程中的作用。结果：在人外周血来源DCs细胞表面和细胞浆内可表达长型和短型β-1,4-GalT-Ⅰ;长型β-1,4-GalT-Ⅰ或细胞表面β-1,4-GalT-Ⅰ的表达水平与细胞粘附能力呈正相关;α-LA既可抑制DCs对层粘连蛋白的粘附,又可抑制DCs与CD4＋Th形成免疫突触。结论：β-1,4-GalT-Ⅰ在DCs中表达,他们作为粘附分子参与细胞粘附和免疫突触形成。     

血管内皮-钙粘附素/连环蛋白复合体在炎症微血管通透性方面的作用

血管内皮 -钙粘附素 (ｖａｓｏｅｎｄｏｔｈｅｌｉａｌ -ｃａｄｈｅｒｉｎ ,ＶＥ -ｃａｄｈｅｒｉｎ)属于钙粘附素家族成员 ,它存在于不同类型血管的内皮细胞 ,可与细胞内介导蛋白相互作用而形成血管内皮 -钙粘附素 /连环蛋白复合体。血管内皮 -钙粘附素 /连环蛋白复合体是粘附连接(ａｄｈｅｒｅｎｓｊｕｎｃｔｉｏｎｓ ,ＡＪ)的重要组成部分 ,它在调节微血管内皮通透性方面扮演了重要角色。1　血管内皮 -钙粘附素的表达血管内皮 -钙粘附素是一种内皮特异性钙粘附素。在胚胎期血管发生的最初时期 ,血管内皮 -钙粘附素表达于卵黄囊间质的中胚…     

儿童癫痫患者T淋巴细胞活化并产生促炎因子

目的检测儿童癫痫患者外周T淋巴细胞活化状态以及细胞因子水平变化。方法选取20名儿童癫痫患者和19名健康对照儿童,采集外周血,分离外周血单个核细胞(PBMC),用细胞膜表面标记抗体流式细胞术检测T淋巴细胞表面共刺激分子CD69、CD25和细胞毒性T淋巴细胞相关蛋白4(CTLA4)的表达,用细胞内因子染色结合流式细胞术检测T细胞细胞因子γ干扰素(IFN-γ)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和IL-17A的表达,利用细胞内因子染色结合流式细胞术检测调节性T淋巴细胞(Treg)表达IL-10的情况。结果与对照组相比,儿童癫痫患者外周血中T淋巴细胞表面高表达共刺激分子CD69、CD25和CTLA4,活化的CD4+T细胞高表达IFN-γ、TNF-α、IL-6和IL-17 A。在儿童癫痫患者高表达IL-10的Treg数目增加。结论儿童癫痫患者外周T淋巴细胞活化并产生细胞因子。     

PPD对人内皮细胞与外周血单个核细胞粘附的影响

为探讨热休克蛋白 6 0kD在动脉粥样硬化早期形成机制中的作用 ,本实验采用病原微生物同源性蛋白即结核杆菌纯化蛋白衍生物 (PPD ) ,观察其在人脐静脉内皮细胞 (EC )和外周血单个核细胞 (PBMC )粘附过程中的作用。利用培养的人脐静脉内皮细胞株 ,测定人PBMC和EC的粘附率 ;采用McAbCD5 4和McAbIL 1β抑制试验 ,检测PPD对EC表达粘附分子的作用。结果显示 ,经PPD处理的EC对PBMC的粘附率增加 ,并有一定的时相关系 ,粘附分子ICAM 1在EC表面表达增加 ;McAbCD5 4可以降低PPD的EC和PBMC的粘附率 ,而McAbIL 1β对粘附率没有影响。PPD可以直接刺激EC表达粘附分子CD5 4,进一步参与介导白细胞与内皮细胞的粘附。     

CD137研究新进展

CD137是近年新发现的肿瘤坏死因子受体超家族的新成员 ,是一种新的共刺激信号分子。它参与T细胞的活化、分化、增殖、凋亡、信号传导及细胞外粘附 ;活化的CD137参与调节细胞因子对粒细胞的抗凋亡作用 ;CD137作为一个独特的潜在的单核细胞存活因子 ,可诱导单核细胞存活、增殖与粘附 ;CD137作为一种重要的受体亦参与调节树突状细胞 (DC)的功能 ;CD137的配体 (CD137L)表达于癌细胞表面 ,在肿瘤免疫及治疗中起作用 ;自身免疫病中慢性活化的自身反应T细胞表达CD137,CD137的异常表达与免疫疾病的发生存在某种关系 ;CD137在改善骨髓移植后异源T细胞的移植物抗宿主病 (GVHD)和移植物抗白血病 (GVL)等异源免疫反应中亦起重要作用。     

Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells

Upon inflammation, stimulated, but not resting T lymphocytes cross the blood-brain barrier and migrate into the central nervous system. This study shows that direct contact between stimulated T lymphocytes and human brain microvascular endothelial cells (HB-MVEC) induces phenotypic and functional changes on the latter cells. Plasma membranes isolated from stimulated T lymphocytes (S-PM) up-regulated the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on isolated HB-MVEC. In addition, HB-MVEC activated by S-PM secreted interleukin (IL)-6 and IL-8. The levels of ICAM-1, E-selectin, IL-6, and IL-8 expressed in S-PM-activated HB-MVEC were similar to those observed with 1000 U/ml tumor necrosis factor (TNF). In contrast, VCAM-1 expression was 15% of that induced by TNF. Inhibitors of TNF diminished (≤ 45 %), but did not abolish the expression of cell adhesion molecules and IL-6 induced by S-PM, IL-8 production being insignificantly affected (≤ 10 %). This suggests that membrane-associated TNF was partially involved in HB-MVEC activation. The present study demonstrates that stimulated T lymphocytes are able to activate HB-MVEC upon direct cell contact. This novel mechanism of inducing the expression of cell adhesion molecules may prompt the initial adhesion of stimulated T lymphocytes to brain endothelium.     

Adhesive and invasive features in gliomas

This study aims at the in situ identification of factors mediating glioma cell invasion requiring adhesion, extracellular matrix degradation, and migration. Forty-five gliomas (astrocytomas, glioblastomas, oligodendrogliomas, and mixed gliomas) were investigated for the immunohistochemical expression of the membrane protein CD44s, the basal lamina proteins laminin, collagen IV, and fibronectin, the lectin galectin-3 recognizing tenascin and N-CAM, as well as for the matrix-degrading enzymes metalloproteinases MMP-2, MMP-9, and cathepsin D. Besides vessels expressing basal lamina proteins, tenascin, MMP-2, MMP-9, and galectin-3, tumor cells revealed strong immunoreactivity for CD44s, tenascin, galectin-3, and N-CAM, which was restricted to solid tumor masses. Single invading cells displayed distinct expression of MMP-2 and MMP-9, also found in solid tumor areas, as well as of cathepsin D. Restricted expression of CD44s, galectin-3, tenascin, and N-CAM in solid tumor masses seems to contribute to homotypical tumor cell adhesion. However, switching to an invasive phenotype, single tumor cells lack this expression pattern and acquire degrading and phagocytic activities by expressing cathepsin D, MMP-2, and MMP-9, which are also expressed by solid tumor masses facilitating the loosening and invasion of single neoplastic cells. The blocking of these factors may be of potential benefit in anti-invasive therapy.     

Human interleukin 6 and tumor necrosis factor alpha production studied at a single-cell level

Individual peripheral blood mononuclear cells, which produced interleukin 6 (IL 6) or tumor necrosis factor alpha, (TNF alpha), were studied by cytokine-specific polyclonal or monoclonal antibodies (mAb) and immunofluorescence technique with UV microscopy. Lipopolysaccharide (LPS) induced IL 6 as well as TNF alpha production in the majority of the monocytes, but not at all in lymphocytes. Approximately every second monocyte made TNF alpha in response to LPS within 0.5 h from start of the cultures, when no IL 6 or TNF alpha production occurred. The maximal number of TNF alpha-synthesizing monocytes was observed 1.5 h later and then rapidly declined. LPS stimulation led to optimal IL 6 production 3 h after initiation of the cultures, with 90% of the monocytes expressing intracellular IL 6. LPS-induced IL 6 synthesis started about 1 h after that of TNF alpha. Polyclonal T cell activation with staphylococcal enterotoxin A or anti-CD3 mAb induced a biphasic production pattern of IL 6 as well as TNF alpha. Early IL 6 synthesis, which peaked 6-8 h from start of the cultures, occurred exclusively in monocytes, while late IL 6 production at 48 h was restricted to a small fraction of lymphoid cells. T cell mitogen induced early TNF alpha production, which peaked at 6 h, took mainly place in monocytes and to a minor degree in CD4+ as well as CD8+ T cells. The majority of the TNF alpha-producing mononuclear cells at 24 h were of the CD4+ T cell lineage in the staphylococcal enterotoxin A- or anti-CD3 mAb-activated cultures. IL 6 as well as TNF alpha accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.     

Differential effects of interleukin-1 and formylmethionylleucylphenylalanine on chemotaxis and human endothelium adhesivity for A549 tumor cells

We investigated the effects of formylmethionylleucylphenylalanine (FMLP), interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) on tumor cell chemotaxis and tumor cell/endothelial cell adhesion. Chemotaxis of A549 human lung carcinoma cells was measured as the number of tumor cells which migrated across a nitrocellulose filter in a Boyden chamber. Tumor cell/endothelial cell adhesion was measured as the number of 125IUdR tumor cells adherent to monolayers of endothelial cells. Confluent monolayers of human umbilical endothelial cells were incubated from 10 to 240 minutes with FMLP, monocyte-derived interleukin-1, or recombinant IL1 alpha or IL1 beta. The endothelial cells were washed and then incubated with 125IUdR-tumor cells. Thirty minutes later the number of adherent tumor cells was assessed isotopically. Our results demonstrate that (a) interleukin-1 but not FMLP, has chemotactic activity for tumor cells, and (b) both FMLP and interleukin-1 enhance tumor cell adhesion to the endothelium independent of any chemotactic activity. Furthermore, we demonstrate that IL1 alpha and IL1 beta have different effects on tumor cell/endothelial cell adhesion, and raise the possibility that IL1 alpha but not IL1 beta is continuously synthesized and stored within the endothelium. We postulate that IL1 alpha and IL1 beta influence tumor cell/endothelial cell adhesion independent of chemotaxis through the expression of adhesive receptors on the endothelial cell surface.     

Effects of hyaluronan on the invasive properties of human breast cancer cells in vitro

Hyaluronan (HA) is a high molecular weight glycosaminoglycan present mostly in the extracellular matrix (ECM). HA binds to specific receptors such as CD44. Its production is increased at the tumour-stroma interface, including those in breast cancer tumours. It has been suggested that it facilitates invasion of tumour cells into the ECM by a hydrodynamic effect, or by altering tumour cell behaviour. Using in vitro tests we studied the effect of immobilized (iHA) and soluble (sHA) HA on the invasive properties of four human breast cancer cell lines with different levels of CD44 expression. Our results show that iHA acts as an adhesive, haptotactic, and motility stimulating factor for the CD44 positive Hs578T cells and induces the expression of membrane CD44. sHA also changes the motility properties of the Hs578T and MDA-231 cells and increases their CD44 expression. sHA or iHA have no measurable effect on the adhesion, motility or CD44 expression of the ZR-75-1 and MCF-7 breast cancer cells. Our results establish that in high CD44 expressing breast cancer cells HA modulates tumour cell adhesion and motility and also increases the expression of its own receptor, CD44.     

Confirmation by peptide sequence and co-expression on various cell types of the identity of CD44 and P85 glycoprotein

The p85 glycoprotein expressed on a variety of human cell types including astrocytes and lymphocytes has not been associated with the CD44 cluster. The recent demonstration that Hermes, a glycoprotein implicated in the adhesion of lymphocytes to endothelium, belongs to the CD44 cluster raises interesting questions concerning the role of this molecule on astrocytes and on non-lymphoid cells. To obtain confirmation of the identity of p85 glycoprotein and CD44, p85 glycoprotein was purified from B-chronic lymphocytic leukemia cells by affinity to monolonal 50B4-IgG and electrophoretic elution, digested with trypsin or CNBr and fractionated by reversed-phase HPLC. The sequences of three peptides were obtained which could be aligned with the amino acid sequence deduced from the CD44 cDNA at residues 49-54, 59-66 and 309-323. These constitute the first reported peptide sequences for antigens of the CD44 cluster and confirm that p85 glycoprotein is indeed the product of the CD44 gene. Since two different cDNA clones encoding molecules with cytoplasmic tails of 72 and 5 amino acids have been isolated, the isolation of peptide 309-323 confirms the existence of a processed protein with the longer cytoplasmic domain. Using a cDNA probe, we have characterized the expression of CD44 in several normal and malignant cell types. The level of CD44 mRNA was correlated with the surface expression of CD44 antigens (50B4) in several leukemic cell lines, in astrocytoma lines and in normal granulocytes. Negative cells included the Y79 retinoblastoma line, the NALM-6 leukemic line and endothelial cells. Identical mRNA species of 5.0, 2.3 and 1.7 kb were present in all CD44-positive samples, including normal granulocytes, astrocytoma, melanoma and leukemia cell lines and leukemic cells from patients. The highest level of expression of CD44 was observed on astrocytoma lines and on acute lymphoblastic leukemia cells of immature phenotype. The presence of high levels of CD44 on malignant cells could increase the ability of these cells to adhere to matrix proteins and/or to interact with endothelium, thus potentially altering their capacity for invasiveness and metastasis.     

Accessory factors involved in murine T cell activation. Distinct roles of interleukin 6, interleukin 1 and tumor necrosis factor

Interleukin (IL) 6 was compared to other macrophage-derived products for its capacity to support the proliferation of accessory cell-depleted T cells. Monoclonal anti-IL6 antibodies were found to inhibit completely the "accessory activity" of macrophage supernatants, thus demonstrating the central role played by IL6 in T cell activation. IL6 was apparently more critical for initiating than in maintaining T cell proliferation because anti-IL6 antibodies lost all inhibitory activity when added late to the culture. Moreover, IL6 was not the only accessory factor required for optimal T cell proliferation. Using low-density cultures to minimize the number of contaminating accessory cells, we found that significant proliferation of CD4 cells was obtained only in the presence of both IL6 and IL1. In contrast, with CD8 cells substantial proliferation was obtained with IL6 alone. This response could, however, be enhanced by IL1. Tumor necrosis factor (TNF) and granulocyte/macrophage colony-stimulating factor showed no activity in these assays. The concentrations of IL1 and of IL6 required to support optimal proliferation were 10 pg/ml and 1 ng/ml, respectively. Analysis of the mechanisms underlying T cell activation by IL1 and IL6 indicated that both cytokines were required for optimal production of IL2 but that IL6 alone was sufficient to confer IL2 responsiveness. For CD8 cells, this effect was observed with doses of IL6 about 100 times lower than those required for the induction of IL2 secretion (0.001 vs. 0.1 ng/ml). TNF, which was not capable of inducing IL2 secretion, was also found to induce IL2 responsiveness but only at a concentration approximately equal to 1000 times higher than that of IL6. In accordance with these observations, IL6 and to a lesser extent TNF were found to enhance IL2R expression by CD8 cells. Interestingly, this enhancing effect was totally dependent on the presence of IL2.     

ICA和PJA对高转移性人肺癌细胞体外侵袭转移能力抑制的研究

目的：将新型抗癌药物淫羊藿甙（ＩＣＡ）和济南假单胞菌制剂（ＰＪＡ）作用于ＰＧ细胞,从肿瘤转移抑制的多个方面和环节探讨ＰＪＡ、ＩＣＡ抗转移作用的机制。方法：采用粘附实验、运动侵袭实验、逆转录ＰＣＲ（ＲＴ－ＰＣＲ）、细胞免疫组化等多种方法进行了检测。结果：ＰＪＡ、ＩＣＡ可降低ＰＧ细胞对胞外基质的粘附性及侵袭、运动能力,减少ＰＧ细胞表面粘附分子ＣＤ４４Ｖ６、ＬＮ－Ｒ及胞浆内ＣＫ１８的表达,同时细胞内ｃ－ｍｙｃ、Ｔｉａｍ－１基因ｍＲＡＮ水平均有不同程度的降低,而Ｎｍ２３－Ｈ１ ｍＲＮＡ水平有不同程度的升高,且两药有明显的协同作用。结论：ＰＪＡ、ＩＣＡ通过对肿瘤转移多个步骤的抑制而发挥抗转移作用。