LAIR-1/LAIR-2(CD305/CD306)与配体结合亲和力的比较

目的:LAIR-1/LAIR-2(CD305/306)与配体结合亲和力的比较。方法:应用不同浓度混合的LAIR-1和LAIR-2融合蛋白,同时或先后与配体表达细胞孵育后,用特异性LAIR-1或LAIR-2单克隆抗体(mAb)进行免疫荧光染色和流式细胞术(FCM)分析,比较LAIR-1和LAIR-2融合蛋白与细胞结合的百分率的变化及相互竞争情况。结果:LAIR-1和LAIR-2膜型配体的分布较广泛,人和小鼠LAIR受体与配体的结合具有相互交叉的特点。LAIR-2能够明显阻断LAIR-1与其膜型配体的结合,而LAIR-1对LAIR-2与其配体的结合无影响。结论:LAIR-1和LAIR-2可能结合相同的膜型配体,但LAIR-2结合配体的亲和力明显高于LAIR-1结合的亲和力。此结果为深入探讨LAIR家族免疫调节的分子机制提供重要的实验依据。     

人CD112(Nectin2/PRR2)单克隆抗体的制备和鉴定

目的：表达和纯化CD112 胞膜外区重组蛋白, 制备针对CD112胞膜外区的单克隆抗体（mAb）, 并且研究CD112的分布.方法：采用基因重组技术在真核系统表达CD112-Fc融合蛋白, 亲和层析法进行蛋白纯化;纯化的CD112-Fc融合蛋白免疫BALB/c小鼠, 杂交瘤技术建立分泌CD112 mAb的杂交瘤细胞株, 间接ELISA、 Western blot及FCM鉴定mAb 的特异性.用流式细胞术（FCM）检测 CD112 的细胞系分布.结果：构建了高效真核表达载体pIg3C-CD112, 表达并纯化了CD112-Fc融合蛋白, 其纯度大于 90%.以纯化重组蛋白为免疫原共制备9株分泌抗CD112 mAb的杂交瘤细胞株（命名为FMU-CD112.1 ～FMU-CD112.9）, 制备腹水并纯化 mAb.FMU-CD112.1、 3、 6和8能用于Western blot, FMU-CD112.1、 4、 6和8可用于 FCM.CD112细胞分布检测显示该分子主要分布在正常上皮及内皮细胞系、巨核细胞谱系和部分T细胞系, 并高表达于上皮及内皮来源的肿瘤、胶质瘤等细胞系.结论：成功地表达纯化了CD112-Fc融合蛋白并建立了稳定分泌CD112 mAb的杂交瘤细胞株, 为CD112的功能研究打下坚实的基础.     

抑制性受体LAIR-1对巨核谱系分化的调节作用

 目的 研究抑制性受体LAIR-1在巨核谱系细胞的表达及其对巨核细胞分化的调节作用。方法 采用流式细胞术和激光共聚焦显微镜检测LAIR-1在巨核谱系细胞的表达；免疫磁珠法纯化脐血CD34+细胞，体外无血清培养系统诱导CD34+细胞向巨核细胞分化，观察LAIR-1被抗体或配体交联激活后对巨核细胞分化的影响。结果 LAIR-1表达于人骨髓CD34+CD41a+和CD41a+CD42b+细胞以及脐血CD34+CD41a-和CD34+CD41a+细胞；脐血CD34+细胞向巨核细胞分化过程中，LAIR-1表达在倍性为2N和4N的不成熟CD41a+细胞上；交联激活LAIR-1分子能够抑制脐血CD34+细胞向巨核细胞的分化。结论 LAIR-1可能是参与巨核细胞早期分化发育的一种新的负调控分子。     

人CD226分子胞膜外区D1和D2真核表达载体的构建、表达和鉴定

目的 构建含有人CD226分子胞膜外区结构域1(D1)和结构域2(D2)的真核表达载体,表达并纯化重组蛋白.方法用特异性引物分别扩增CD226 D1和D2的基因,定向插入真核表达载体pSecTag2B-Fc,进行酶切和DNA测序鉴定,重组质粒瞬时转染293T细胞,收集上清,纯化蛋白及SDS-PAGE和Western blot鉴定表达产物.结果利用PCR方法克隆出CD226胞膜外区D1和D2的基因,并正确插入pSecTag2B-Fc载体中,真核表达载体瞬时转染293T细胞,Western blot结果证实转染293T细胞的培养上清液中含有hCD226D1-Fc和hCD226D2-Fc融合蛋白,培养上清经亲和层析柱纯化,可获得较高纯度的重组蛋白.结论 成功的构建了分泌型融合蛋白hCD226D1-Fc和hCD226D2-Fc真核表达载体,获得纯度较高的融合蛋白,为进一步探讨其配受体的相互作用机制奠定了基础.     

人LAIR-1/CD305基因启动子的生物信息学分析

目的:研究人LAIR-1/CD305启动子及其5′上游调控序列。方法:通过检索NCBI中的人类基因组数据库,获得LAIR-1的转录本序列及翻译起始位点上游2500bp的序列。利用Promoter2.0等软件预测LAIR-1的启动子序列,然后利用MatInspector等软件对启动子序列的转录因子结合位点和启动子功能模块进行预测。结果:LAIR-1基因的核心启动子区位于翻译起始密码子上游的600～200bp区域内。在转录调控区发现了一些重要的转录因子结合位点,并预测到13种启动子功能模块。结论:LAIR-1基因的表达可能受到多种转录因子和5′端上游调控序列的调控。     

沉默LAIR-1表达对卵巢癌细胞增殖功能的影响

沉默白细胞相关免疫球蛋白样受体1(LAIR-1)在卵巢癌H08910细胞的表达,探讨其对卵巢癌细胞增殖功能的影响。构建LAIR-1的shRNA慢病毒表达载体,转染人浆液性卵巢癌细胞系H08910。RT-PCR和Western blot检测H08910细胞中LAIR-1分子的沉默效率;采用PI染色法和MTT法检测沉默LAIR-1表达对HO8910细胞周期及增殖功能的影响。成功建立沉默LAIR-1分子表达的稳定HO8910细胞株。与阴性对照组比较,转染LAIR-1-shRNA慢病毒载体的HO8910细胞中LAIR-1的mRNA及蛋白水平表达均明显降低(P<0.05)。LAIR-1-shRNA组G1期细胞数目明显低于CON组和NCshRNA组(P<0.05);沉默LAIR-1分子表达后,LAIR-1-shRNA HO8910细胞的增殖率明显高于NC-shRNA HO8910组和空白对照组(P<0.05)。沉默卵巢癌细胞HO8910LAIR-1分子表达可明显增强细胞增殖的能力,提示卵巢癌细胞表达的LAIR-1可能对癌细胞的生物学功能发挥抑制作用。     

含3C蛋白酶切位点的人CD226胞膜外区Ig融合蛋白的真核表达及应用

目的：构建含3C蛋白酶酶切位点的人CD226(PTA1)分子胞膜外区Ig融合蛋白编码基因的真核表达载体，并进行表达和初步鉴定。方法：将人CD226分子的胞膜外区基因，克隆入含3C蛋白酶靶序列的Ig真核表达载体p-3c—Ig中。测序证实后，转染COS7细胞并进行瞬时表达。表达产物经亲和层析件纯化，并通过免疫荧光染色及3C蛋白酶酶切反应进行鉴定结果：经表达和纯化获得CD226胞膜外区的Ig融合蛋白。该融合蛋白可与表达于ECV304细胞表面的CD226配体有效结合。同时，融合蛋白的Fc段亦经3C蛋白酶切除，从而获得CD226胞膜外区的真核表达分子。结论：成功地获得了含有3C蛋白酶酶切位点的人CD226分子胞膜外区Ig真核表达产物，为进一步对CD226分子进行结构和功能研究，以及X线结晶衍射提供了重要条件。     

四环素调控的小鼠LAIR-1/CD305转基因小鼠的初步建立

目的：建立四环素调控的小鼠LAIR-1/CD305转基因小鼠,为进一步研究mLAIR-1分子的体内功能奠定基础.方法：构建pBI-5-mLAIR-1载体,显微注入B6D1F1受精卵,PCR检测新生小鼠基因组DNA中LAIR-1与荧光素酶（Luciferase）基因.将mLAIR-1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含盐酸强力霉素（Dox）的培养基进行培养,检测细胞裂解液中荧光素酶活性.将荧光素酶表达依赖Dox小鼠与C57BL/6交配,采用PCR对子代鼠进行检测.结果：共获得9只首建鼠,其目的基因表达高度依赖Dox,并得到其中5只首建鼠的F1代小鼠.结论：获得了四环素调控的小鼠LAIR-1转基因小鼠,可用于该分子体内功能的研究.     

The regulation of inhibitory receptor LAIR-1 on the differentiation of megakaryocytic lineage

目的 研究抑制性受体LAIR-1在巨核谱系细胞的表达及其对巨核细胞分化的调节作用.方法 采用流式细胞术和激光共聚焦显微镜检测LAIR-1在巨核谱系细胞的表达;免疫磁珠法纯化脐血CD34+细胞,体外无血清培养系统诱导CD34+细胞向巨核细胞分化,观察LAIR-1被抗体或配体交联激活后对巨核细胞分化的影响.结果 LAIR-1表达于人骨髓CD34+ CD41a+和CD41a+ CD42b+细胞以及脐血CD34+ CD41a -和CD34+ CD41a+细胞;脐血CD34+细胞向巨核细胞分化过程中,LAIR-1表达在倍性为2N和4N的不成熟CD41a+细胞上;交联激活LAIR-1分子能够抑制脐血CD34+细胞向巨核细胞的分化.结论 LAIR-1可能是参与巨核细胞早期分化发育的一种新的负调控分子.     

人工高表达LAIR-1对人肝癌细胞系SMMC-7721细胞增殖、凋亡和侵袭的影响

研究人白细胞相关免疫球蛋白样受体1(leukocyte-associated immunoglobulin-like receptor 1,LAIR-1)对人肝癌细胞系SMMC-7721细胞增殖、凋亡和侵袭的影响,探讨LAIR-1的表达在肝癌中的作用。以LAIR-1慢病毒表达载体(LV-LAIR-1)转染SMMC-7721细胞,建立稳定高表达LAIR-1的细胞株LAIR-1+SMMC-7721。采用流式细胞术(flow cytometry,FCM)、Western blotting、RT-PCR和激光扫描共聚焦显微技术(laser scanning confocal microscopy,LSCM)等方法鉴定LAIR-1分子在SMMC-7721细胞的表达水平;采用CCK-8试剂盒、FITC Annexin V凋亡试剂盒和Transwell侵袭实验分别检测LAIR-1对SMMC-7721细胞增殖、凋亡和侵袭的影响。检测结果显示,经LV-LAIR-1转染的SMMC-7721细胞高水平表达LAIR-1分子;功能实验结果显示,与实验组细胞相比,LAIR-1的表达对细胞增殖无明显影响(P>0.05),但是能够明显促进细胞凋亡(P<0.05),并能够显著抑制细胞的侵袭能力(P<0.05)。上述研究结果提示,LAIR-1分子的表达可能是影响肝癌细胞生物学特性的一个重要因素。     

LAIR-2单克隆抗体的制备、鉴定和夹心ELISA方法的建立

为了制备LAIR 2单克隆抗体并建立夹心ELISA方法。应用淋巴细胞杂交瘤技术制备抗LAIR 2单克隆抗体 (mAb )。采用间接免疫荧光染色和流式细胞术鉴定LAIR 2的细胞分布。辣根过氧化物酶 (HRP )标记LAIR 2mAb ,并建立检测LAIR 2的夹心ELISA方法。用重导向杀伤实验 (RCA )鉴定LAIR分子参与调节杀伤功能的关系。结果成功地制备了 3株特异识别LAIR 2的mAb ,1株mAb (3G4 )能够识别LAIR 1和LAIR 2的共同表位 ,但不能在重导向杀伤实验中抑制CD16诱导的杀伤功能。夹心ELISA方法检测LAIR 2的敏感性为 5ng/ml,为LAIR 2的分布及定量检测提供了方法     

The inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1) is expressed at high levels by human naive T cells and inhibits TCR mediated activation

Human leukocyte-associated Ig-like receptor-1 (LAIR-1) is a transmembrane glycoprotein with a single extracellular Ig-like domain and a cytoplasmic tail containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs). It is constitutively expressed on the majority of human mononuclear leukocytes and functions as an inhibitory receptor. In this study, we show that freshly isolated peripheral blood T cells are heterogeneous in their expression levels of LAIR-1. We have found that naive T cells express the highest levels of LAIR-1, even more than memory cells. The cross-linking of LAIR-1 inhibits T cell receptor (TCR) mediated signals in freshly isolated human naive T cells and whole populations of CD4⁺ or CD8⁺ T cells. TCR cross-linking increased cell surface expression of LAIR-1 in a process that requires p38 MAP kinase and ERK signaling. Altogether, these results indicate that LAIR-1 is capable of negatively regulating T cell functions, and its high level of expression by naive T cells suggests that it may function at an early stage in the development of an immune response.     

Tumor-expressed collagens can modulate immune cell function through the inhibitory collagen receptor LAIR-1

Many tumor types over-express collagens, what correlates with enhanced metastatic capacity and unfavorable clinical outcome. This is generally explained by the importance of collagens in creating a microenvironment that supports tumor cell survival and enhances cell migration. Importantly, collagens act as ligands for the inhibitory receptor LAIR-1, which inhibits the function of multiple types of immune cells. Here we propose a new role for tumor expressed collagens and show that these structural proteins can be exploited by tumor cells to inhibit immune responses through an interaction with LAIR-1. We show that both LAIR-1-Fc fusion proteins and LAIR-1 expressing cells bind to transmembrane collagens expressed by tumor cells. Interference with collagen expression by specific knock-down of prolyl 4-hydroxylase diminishes LAIR-1 binding to tumor cells, demonstrating the specificity of the interaction. Consistently, both transmembrane collagens and extracellular collagens produced by multiple tumor cell types can activate LAIR-1. Furthermore, overexpression of collagen XVII on target cells results in diminished NK cell cytotoxic activity. Thus tumor-expressed collagens can bind and trigger immune inhibitory signaling via LAIR-1, suggesting that collagens indeed may affect tumor immune evasion.     

Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization

Ligation of very late antigen (VLA)-4 (α₄β₁ integrin) with a cross-linked anti-α₄ subunit monoclonal antibody (mAb) triggered a biphasic Ca²⁺ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca²⁺ response. Tumor necrosis factor-α-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca²⁺ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca²⁺ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca²⁺ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (～ 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca²⁺ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca²⁺ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca²⁺.     

人CD28-Fc融合蛋白在真核细胞中的表达与纯化

目的建立稳定表达CD28-Ig融合蛋白系统,提供用于研究的CD28-Ig融合蛋白。方法构建高表达的真核细胞表达载体,利用FAD批准的、可用于生产人用重组蛋白药物的CHO细胞进行表达,MTX加压筛选出稳定表达的细胞株。蛋白A纯化获得电泳纯融合蛋白。结果经RT-PCR,SDS-PAGE及Western印迹鉴定,CHO细胞上清中纯化的蛋白是CD28与Ⅰg的融合蛋白,并获得纯度较高的融合蛋白。结论成功建立CD28-Ig融合蛋白表达体系,并获得稳定表达融合蛋白的细胞株,为T细胞活化第二信号系统的基础研究奠定了基础。     

抑制ECV304细胞内EOLA1基因表达后效应观察

目的观察抑制人脐静脉内皮细胞系ECV304细胞内内皮高表达脂多糖相关因子1（endothe-lial-overexpressed lipopolysaccharide-associated factor 1,EOLA1）基因表达后细胞生长的变化。方法构建EGFP-EOLA1融合蛋白表达载体pEGFP-N2/EOLA1,转染ECV304细胞,G418压力筛选获得稳定表达株;设计靶点特异性的寡核苷酸,连接到经BamHⅠ和HindⅢ酶切线性化的pSlincer3.1/H1质粒上。转染重组质粒到稳定表达EGFP-EOLA1融合蛋白的ECV304细胞,检测靶基因的抑制情况,观察EOLA1表达被抑制后细胞生长的改变。结果抑制EOLA1表达后ECV304细胞生长明显减慢。结论EOLA1基因在细胞内参与了细胞生长的调控。     

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAb) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAb against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogeneic, as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence on B7-1 expression.