MBL与人单核细胞系THP1/CD14细胞结合及其特性研究

采用Cell-ELISA和流式细胞术,发现人单核细胞系THP1/CD14在生理条件下可结合甘露聚糖结合凝集素(MBL),这种结合具有Ca2+依赖性,能被甘露糖、N-乙酰葡糖胺、D-葡萄糖、半乳糖、蔗糖、海藻糖等及重组人MBL-CRD蛋白和MBL-CLR蛋白所部分抑制,但C1q或抗人C1qR单克隆抗体对之无影响。还发现,炎性状态下的THP1/CD14细胞与MBL的结合增强。结果表明,THP1/CD14细胞表达Ca2+依赖的、糖敏感的MBL受体或结合蛋白,包括对CLR特异和CRD特异的两种受体,均与C1q受体无关;炎性刺激可上调THP1/CD14细胞MBL受体或结合蛋白的表达。     

从噬菌体随机环七肽库筛选可模拟C1qR的C1q结合肽

为获得能结合C1q并模拟C1q受体 (C1qR )配体结合位点的短肽 ,以C1q为钓饵蛋白筛选噬菌体环七肽库 ,采用C1q结合ELISA、U937细胞配体结合抑制试验、多聚IgG (AIgG )竞争抑制试验鉴定阳性克隆 ,再进行单链DNA测序和分析。结果经 3轮筛选后 ,随机挑选 2 3个噬菌体克隆进行ELISA鉴定 ,10个克隆与C1q有较强的结合 ;利用U937细胞配体结合抑制试验 ,得到了 7个阳性克隆 ;从其展示肽DNA测序结果推导氨基酸序列 ,获得 7个短肽序列 :QTPFQLW、NPFNWTS、SPFXLTS、FLTWLDP、FSTFLYP、GPMWWSY和NPFXLIL。     

C1q结合十二肽的活性研究

目的研究5个C1q结合十二肽及其牛血清白蛋白(BSA)偶联物对C1q的结合活性和溶血活性的影响。方法人工合成十二肽，将其与BSA偶联，分别用U937细胞配体结合抑制试验、多聚IgG(AJgG)竞争抑制试验、CH50抑制试验测试其生物学活性。结果游离的线性肽对C1q的结合活性和溶血活性几乎无影响，而偶联肽对C1q与U937细胞表面受体、AJgG的结合以及C1q介导的溶血均有抑制作用。结论本实验获得5个C1q受体模拟肽，它们能抑制C1q与免疫复合物结合，从而阻断补体经典途径的激活，其对于补体异常激活有关疾病的防治具有潜在应用价值。     

聚合的Clq能增强人外周血淋巴细胞Ig的产生

鉴于B 细胞可表达C1q 受体,本文作者研究了C1q 对B 细胞的刺激作用。将B 细胞培养在含预先冷冻的C1q、T 细胞及亚适宜浓度(7ug/ml)的PWM 条件下,则B 细胞产生IgM、IgG 和IgA 的量增加;如果无T 细胞     

日本脑炎病毒受体74×103分子的分离与初步鉴定

目的 分离和初步鉴定口本脑炎病毒(JEV)易感细胞C6/36和Vere细胞上受体分子.方法 应用免疫共沉淀(Co-IP)技术,从C6/36和Vero细胞分离与JEV结合的受体分子,做质谱分析鉴定和Western blot检测.用激光共聚焦技术(LSCM)观察候选受体分子在细胞膜上的定位及与JEV结合的情况.结果 经过Co-IP反应,从C6/36、Vem细胞上分离出多个与JEV结合的分子条带.质谱分析首次鉴定出一个蛋白,即C6/36细胞上与JEV结合的相对分子质最(Mr)为74×103分子,为HSC70蛋白.进一步用抗HSC70抗体做Western blot,榆测剑从C6/36和Vem细胞膜上Co-IP分离出的74×103蛋白.LSCM观察到JEV吸附在C6/36细胞膜上时,HSCT0蛋白与JEV共定位.结论 C6/36细胞上Mr为74×103的HSC70可能是JEV的受体分子.     

C1q结合线性十二肽的改建及其活性鉴定

目的 改建以C1q为钓饵蛋白筛选随机肽库所获线性十二肽HWDPFSLSAYFP（a肽）以增强其生物学活性。方法在a肽序列两端加上噬菌体展示短肽上的支架氨基酸残基，得到线性肽GGG HWDPFSLSAYFP SHS（al肽）；在al肽内部构建环化结构，得到环肽 GGGG HWDPFSLSAYFP CSHS（a2肽）；采用U937细胞配体结合抑制试验、多聚IgG（AIgG）竞争抑制试验和CH50抑制试验鉴定其生物学活性。结果a1肽较a肽抑制活性明显提高，对C1q介导的溶血以及C1q与U937细胞表面受体、AIgG的结合均有抑制作用，而a2肽的活性不明显，对C1q的结合活性和溶血活性无影响。结论获得一个更具稳定结构和生物学活性的C1q结合短肽，它能更有效地抑制C1q与免疫复合物结合，从而阻断补体经典途径的激活。     

抑癌基因mda-7／IL-24与肿瘤治疗

mda 7/IL 2 4是在黑色素瘤细胞中发现的一个与黑色素瘤分化相关的基因 ,位于染色体 1q3 2 .2～ 1q41区域。编码蛋白MDA 7/IL 2 4通过与受体IL 2 0R1,IL 2 2R1与IL 2 0R2形成的 2种异二聚体复合物结合进行信号转导 ,对多种组织起源的肿瘤细胞均有抑制作用 ,还能在IFN β和分化诱导剂MEZ的作用下间接诱导黑素瘤细胞向终末期分化 ,但对正常细胞并无明显的毒副作用 ,在肿瘤的基因治疗上具有应用前景     

C1q受体及其生物学意义

十几年来人们逐渐发现补体第一成份的亚成份C_1q不仅在补体经典途径的活化中起重要作用,而且可以和多种细胞结合,这种结合是受体介导的。目前已经确定:血小板、淋巴细胞、单核巨噬细胞、中性粒细胞、嗜酸性细胞、内皮细胞和成纤维细胞具有C_1q受体(C_1qR)。由于正常人血清中游离C_1q浓度相当低,人们一度对诸细胞表面C_1qR是否具有生物学意义表示怀疑。后来有人发现C_1和免疫复合物(IC)结合后,一种血清调控蛋白C_1抑制剂可与活化的C_1r、C_1s共价结     

C1q结合十二肽的筛选与初步鉴定

目的　从噬菌体十二肽库中筛选结合C1q的短肽并进行初步鉴定。方法　以C1q为钓饵蛋白筛选噬菌体十二肽库 ,利用ELISA、U937细胞配体结合抑制试验、多聚IgG(AIgG)竞争抑制试验鉴定阳性克隆 ,再进行单链DNA测序和分析。结果　经 3轮筛选后 ,随机挑选 2 5个噬菌体克隆做ELISA鉴定 ,14个克隆显示与C1q有较强的结合。经过U937细胞配体结合抑制试验和AIgG竞争抑制试验鉴定后 ,将此 14个阳性克隆测序 ,从其展示肽DNA测序结果推导氨基酸序列 ,获得 9条氨基酸序列 :HWDPFSLSAYFP、WTPVRTNPFLLH、NGHLFSLSAYFP、RTQRNSPFFLCP、SPAFHPEHMGRG、SRAFHPFYRGRA、WYEGPFTLQTWP、LTQHNSPFFLLP和TSNPFFLWYPQP。结论　获得结合C1q的一些抑制性短肽 ,它们可能成为抑制补体经典途径激活的肽类先导化合物     

FcRL基因家族的研究进展

越来越多的细胞表面受体被确认具有能通过T细胞受体或B细胞受体调节免疫应答的能力.Fc受体样(Fc receptor like or homolog,FcRL/H)基因是近年发现的基因,自Georgia等[1]对骨髓瘤易位染色体(1;14)(q21;q23)断点区进行克隆发现了两个新的基因,即FcRL4(IRTA1)和FcRL5(IRTA2)后,相继有新的类似基因被发现.     

Expression and function of C1q receptors and C1q binding proteins at the cell surface

C1q is the recognition unit of the first component of complement that binds not only IgG and IgM containing immune complexes, but also recognizes foreign structures such as the lipid A of endotoxin, and molecules expressed at the surface of apoptotic cells. In this review, the plasma membrane receptors and binding proteins for C1q are discussed and new data are presented on calreticulin expression on human peripheral blood cells. Although much is known about C1q receptors and binding molecules there are still many questions regarding their role in vivo.     

Platelet interactions with C1q in whole blood and in the presence of immune complexes or aggregated IgG.

Previous studies identified specific receptors for C1q on human blood platelets in purified systems using monomeric C1q. To assess the physiologic potential of platelet C1q receptors, C1q binding was evaluated in whole blood and in the presence of immune complexes or aggregated IgG. Blood was obtained from healthy volunteers and collected directly into EDTA (1 vol 100mM EDTA:9 vol whole blood) and purified, 125I-labeled C1q or 125I-C1q associated with albumin-anti-albumin immune complexes. Samples were incubated at 22 or 37 degrees C for 60 min, and total cell bound C1q and platelet associated C1q were quantified. Platelet-bound, monomeric C1q or immune complex-associated C1q represented 40-50% of total peripheral blood cell-associated C1q. C1q binding was unaffected by the incubation temperature, but the preincubation of 125I-C1q with immune complexes enhanced binding two- to threefold. This binding was partially inhibited by preincubating platelets with either the collagen-like amino-terminal fragments of C1q (c-C1q) or a monoclonal antibody Fab fragment recognizing platelet Fc receptors. A more complete inhibition was achieved if platelets were preincubated with both agents. Similar observations were made using washed platelets and 125I-C1q associated with aggregated IgG. The role of C1q and platelet C1q receptors in enhancing aggregated-IgG binding to platelets was further supported by experiments demonstrating increased 125I-aggregated IgG binding to platelets not only after preincubation of 125I-aggregated IgG with C1q but also following platelet preincubation with C1q. These data suggest that C1q receptors may participate in the localization and presentation of C1q-associated immune complexes on the platelet surface and demonstrate that platelets contribute significantly to the C1q binding activity of peripheral blood.     

Enhanced Binding and Degradation of the C1q Subcomponent of Complement by Thioglycollate-Stimulated Guinea Pig Peritoneal Macrophages

Expression of C1q receptors on the plasma membrane of thioglycollate-stimulated guinea pig peritoneal exudate macrophages increased 1.54 times as compared to unstimulated controls. A Scatchard plot of the binding of 125I-C1q to the cells revealed that the binding is a result of an increase in the number of receptors and not to an increased affinity of the receptors. Thioglycollate-activated macrophages were found to be 1.6 times more active than nonactivated macrophages in the binding of 125I-C1q at 4 degrees C. The enhanced binding of 125I-C1q by activated peritoneal macrophages was reflected in an increase in the amount of 125I-C1q degraded by these cells as compared to resident peritoneal macrophages. This suggests that stimulation of phagocytic cells leads to an increase in the expression of C1q receptors and to a concomitant increase in the uptake and degradation of C1q.     

A rosette assay for the determination of C 1 q receptor-bearing cells.

A rosette assay for the identification of cells with receptors for C 1 q is described. Glutaraldehyde-treated bovine erythrocytes bound C 1 q specifically, and the reagent thus prepared provided a valid indicator for rosette formation mediated by C 1 q receptors. The presence of these receptors on the membrane of a subset of human peripheral lymphocytes (mainly non-G cells) and on B-derived lymphoblastoid cells was confirmed. Rosette formation was dependent on the number of C 1 q molecules bound per indicator cell and was specifically inhibited by soluble native C 1 q and pepsin-resistant C 1 q fragments. These data, together with the reduced binding activity of C 1 r-C 1 s-associated C 1 q, indicated that the C 1 q binding sites for lymphoid membranes are expressed on the collagen-like moiety, C 1 q rosette formation provided a simple new procedure for fractionation of human lymphocyte populations and separation from phagocytes that do not express receptors for C 1 q.     

Binding of Complement Proteins C1q and C4bp to Serum Amyloid P Component (SAP) in Solid Contra Liquid Phase

Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy. Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')₂ anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate that firm binding of C1q and C4bp to SAP requires that SAP is presented on a solid phase, that C1q and C4bp react with sites distinct from the heparin binding site, and that C1q and collagen I share binding sites on SAP. Immobilized native SAP, aggregated SAP and SAP-heparan-sulphate complexes induced no detectable complement activation.     

C1q-binding proteins and C1q receptors

Aggregated or immobilized complement C1q induces cellular responses in many different cell types. C1q-induced cellular responses may be involved in host defense and in protection against autoimmunity because C1q-deficient humans have infectious complications and a very high incidence of autoimmune disease. The search for the C1q receptor(s), which has been ongoing for 25 years, has led recently to the recognition that proteins identified as binding to C1q may be divided into two groups: C1q-binding molecules that are normally intracellular; and cell surface C1q receptors.     

cC1q-R (calreticulin) and gC1q-R/p33: ubiquitously expressed multi-ligand binding cellular proteins involved in inflammation and infection

The first component of complement, C1, is a multi-molecular complex comprising of C1q and the Ca(2+)-dependent tetramer C1r(2)-C1s(2). The traditional role of C1q within the complex is that of recognition signal-a signal, which is instantly converted into a highly specific intramolecular proteolytic activation of the C1r(2)-C1s(2) tetramer thereby triggering activation of the classical pathway. Another important function of C1q is its ability to bind to a wide range of cell types resulting in the induction of cell-specific biological responses. These cells include polymorphonuclear leukocytes, monocytes, lymphocytes, dendritic cells, endothelial cells and platelets. Interaction of C1q with endothelial cells and platelets, for example, leads to cellular activation followed by release of biological mediators and/or expression of adhesion molecules, all of which contribute, directly or indirectly to the inflammatory process. These specific responses are mediated by the interaction of C1q with C1q binding proteins or receptors on the cell surface. To date, four types of putative C1q binding cell surface expressed proteins/receptors have been described. These include cC1q-R/CR, or calreticulin (CR), a 60 kDa protein, which is also known as collectin receptor; gC1q-R/p33, a 33 kDa homotrimeric protein; C1q-Rp (CD93), a 120 kDa, O-sialoglycoprotein; and CR1 (CD35), the receptor for C3b. Although the specific role of each of these molecules in a given C1q-mediated cellular response is yet to be worked out, all of them may, in one form or another, participate in the inflammatory processes associated with vascular or atherosclerotic lesions, autoimmune diseases, or infections. The main focus of our laboratory for the past 20 years has been to elucidate the structure and function of cC1q-R/CR and gC1q-R/p33, both of which have been isolated and characterized on the basis of their ability to bind C1q. The purpose of this article is therefore to provide an up to date overview of these two proteins with particular emphasis on their unique structural and functional features, their multi-faceted nature and most importantly their role in infection and inflammation.     

Binding of soluble immune complexes to Raji lymphocytes. Role of receptors for complement components, C1q and C3-C3b.

We have found that, although the binding of particulate antigen-antibody complement complexes such as EAC to lymphoblastoid Raji cells is mediated largely through receptors for C3b, the binding of complement-containing soluble complexes such as those prepared with aggregated human IgG (AHG) occurs also via receptors for C1q. Evidence supporting this conclusion included: (1) Binding of AHG to Raji cells takes place after incubation in EDTA serum; (2) Binding of AHG does not occur in C1q deficient EDTA serum but does take place after addition of C1q; (3) The extent of binding of AHG in EDTA serum is a function of the amount of C1q present; (4) Raji cells can bind up to 5-4 times 10(5) molecules of 125I C1q per cell which can be blocked by unlabelled C1q; (5) AHG pre-incubated with C can bind to a T-cell line MOLT, which lacks receptors for C3b but possesses receptors for C1q to the same extent as Raji cells; (6) Immunoassays for immune complexes in human sera yield similar results whether Raji cells, MOLT cells or C1q precipitation is used for assay; (7) EAC-Raji cell rosettes can be inhibited with inulin-treated, C1q deficient serum containing C3b or C3d whereas binding of AHG or immune complexes in patient samples to Raji or MOLT cells is not inhibited by this reagent. We conclude that receptors for C1q on certain B and T lymphocytes may play an important role in physiologic functions of lymphocytes depending on binding of soluble immune complexes to their surfaces.     

Complement components, regulators and receptors are produced by human monocyte-derived dendritic cells

Complement and dendritic cells (DCs) are essential components of innate immunity. Both participate in local inflammation and moreover have roles in the initiation of the acquired immunity response and in the maintenance of tolerance. Recent studies have demonstrated the ability of DCs to synthesize C1q, C3, Factor I, Factor B and complement receptors 3 and 4. In this study, we demonstrate that human DCs are a source of other soluble complement proteins including C1q, C4b binding protein (C4BP), C7 and C8. Complement receptors (CR)1 and the CD18 chain (common for CR3 and CR4) were also present on DCs while CR2 was not detected.