肌萎缩蛋白参与淋巴细胞的活化及免疫突触的形成

目的：探讨在神经肌接头中发挥重要作用的膜表面糖化蛋白-肌萎缩蛋白是否参与免疫突触的形成，进而影响淋巴细胞的活化。方法：首先通过RT-PCR和FACS分别从mRNA水平和蛋白水平观察肌萎缩蛋白在各种免疫细胞中的表达，并经过共聚焦显微镜观察其是否参与免疫突触的形成，应用构建的反义质粒，研究其是否影响淋巴细胞的抗原特异性和非特异性活化。结果：肌萎缩蛋白广泛表达于T细胞、B细胞、不成熟DC、成熟DC及巨噬细胞中，且参与了免疫突触的形成。当淋巴细胞活化后其表达含量无明显上升，但其表达被下调后，不管是对特异抗原引起的淋巴细胞活化还是对非特异抗原引起的淋巴细胞活化都具有显著的抑制作用。结论：组成性表达于各种免疫细胞中的肌萎缩蛋白参与了免疫突触的形成，并影响了抗原特异性和抗原非特异性的淋巴细胞的活化。     

集聚蛋白在免疫细胞中的表达及其意义

为研究在神经突触中发挥重要作用的突触调节蛋白集聚蛋白是否在免疫细胞中表达并影响淋巴细胞的活化。首先通过RT-PCR、荧光免疫组织化学和FACS分别从mRNA水平和蛋白水平观察集聚蛋白在各种免疫器官、细胞中的表达;通过实时定量PCR检测分析集聚蛋白的表达与淋巴细胞活化的关系;并构建针对集聚蛋白的反义质粒,从而进一步研究下调集聚蛋白的表达对淋巴细胞活化的影响。结果发现集聚蛋白广泛表达于脾脏、胸腺、淋巴结等多种免疫器官中并在T细胞、B细胞、imDC、mDC及巨噬细胞中均有表达,其表达含量与淋巴细胞的活化状态密切相关,且其表达被下调后淋巴细胞的活化显著被抑制。因而组成性表达于各种免疫细胞中的集聚蛋白可被诱导性上调,并参与了淋巴细胞的活化。     

细胞表面β-1,4-半乳糖基转移酶-Ⅰ在免疫突触形成中的作用

目的:研究细胞表面β-1,4-半乳糖基转移酶-I(β-1,4-galactosyltransferase I,β-1,4-GalT-I)在免疫突触形成中的作用。方法:以抗CD3、CD28单克隆抗体活化Jurkat细胞,应用实时荧光定量PCR及流式细胞术检测分析β-1,4-GalT-I的表达变化,激光共聚焦显微镜观察β-1,4-GalT-I在Jurkat细胞活化前后的表达与定位;将Jurkat细胞与SEB负载的Raji细胞共培养以构建Jurkat-Raji细胞免疫突触,观察β-1,4-GalT-I在免疫突触中的定位。结果:β-1,4-GalT-I mRNA表达水平随着Jurkat细胞的活化逐渐增高,在细胞活化后36小时达到高峰;活化后细胞表面β-1,4-GalT-I分子的表达较静止状态高;共聚焦显微镜显示在活化的Jurkat细胞表面及Jurkat-Raji细胞免疫突触部位β-1,4-GalT-I表达信号增强且其分布发生重排和簇集,且与CD3分子共定位,与CD28分子部分共定位。结论:Jurkat细胞表面的β-1,4-GalT-I分子参与了免疫突触的形成。     

γδT细胞杀肿瘤细胞过程中溶酶体运动及肌动蛋白纤维、T细胞受体重排特征研究

目的观察γδT细胞溶解肿瘤靶细胞过程中溶酶体运动特征，了解T细胞受体、肌动蛋白纤维在效应，靶细胞接触区域的分布特征，探讨TST细胞抗肿瘤生物学活性的可能机理。方法Lysotracker Red-DND99标记的TST细胞与人恶性黑色素瘤MeWo细胞共同培养，激光共聚焦显微镜实时观察御细胞杀伤肿瘤细胞过程中溶酶体运动过程及其特点。TST细胞与MeWo细胞共同培养后甲醛固定，以rhodsmine phalloidin标记肌动蛋白纤维或anti-TCRδ1-FTTC抗体标记T细胞受体，激光共聚焦显微镜观察TST细胞及肿瘤细胞之间免疫突触形成特征。结果TST细胞可快速识别并集聚于肿瘤细胞周围。在与肿瘤细胞直接接触的10min内，将胞内溶酶体成分“倾吐”于肿瘤细胞后随之离开。肿瘤细胞受大量TST细胞攻击后约60min，出现细胞膜皱缩、变圆，约80min死亡。在上述效应，靶细胞接触作用过程中，肌动蛋白纤维及T细胞受体均匀分布于TST表面，不出现在效应，靶细胞接触区域富集现象。结论TST细胞通过与肿瘤细胞直接接触，释放其细胞内溶酶体内含物直接杀伤肿瘤细胞。与细胞毒性T淋巴细胞不同，TST细胞与肿瘤细胞之间不形成典型的免疫突触。     

激活的人外周T淋巴细胞表达水通道蛋白AQP3

目的:探讨水通道蛋白(Aquaporin,AQP)在人外周T淋巴细胞的表达和生理学意义.方法:利用逆转录PCR和免疫印迹技术检测AQP0～9 mRNA和蛋白在静止的和经活化的T淋巴细胞的表达.结果:静止的淋巴细胞无AQP0-9表达,而活化的T淋巴细胞表达AQP3 mRNA和蛋白.结论:T淋巴细胞激活过程可能需要AQP3转运水和甘油的功能.     

新的人突触相关蛋白(FRG4)抗原表位分析及抗体制备

目的：分析一个新的人突触相关蛋白(FRCA)抗原表位并制备其多克隆抗体。方法：从人胎肝文库PCR扩增获得FRCA基因全长cDNA序列；通过生物信息学分析，预测FRG4编码氨基酸序列的二级结构、抗原决定簇、功能结构域，并进行了多序列比对；采用固相多肽合成法合成了FRCA抗原多肽，并免疫家兔；用免疫组化检测蛋白在人肝癌细胞HepG2细胞中的表达。结果：通过生物信息学分析选取抗原13肽PKLVKEEVFWRNY，制备兔抗人FRCA多克隆抗体。高效液相色谱检测显示抗体纯度达82．79％，抗体滴度为1：16000，Western blot证实该抗体具有较好的反应性和特异性，免疫组化证实其主要在HepG2细胞胞浆中表达。结论：成功制备了新的人突触相关蛋白(FRCA)多克隆抗体。     

免疫突触与T细胞活化

淋巴细胞活化是适应性免疫应答的关键因素,免疫突触的形成是T细胞识别抗原、增殖和活化的关键步骤,是机体细胞免疫应答和体液免疫应答的重要组成部分.干预免疫突触形成,可望用于预防治疗与免疫相关的疾病.     

免疫突触与T细胞活化关系的研究

免疫突触是免疫识别中T细胞与APC间形成的超分子活化簇的结构,其形成过程涉及多种接头蛋白、共刺激分子的相互作用,与细胞骨架的活化以及黏附分子的作用有关。突触形成将提高TCR与MHC-肽的相互作用、促进T细胞信号转导相关分子的相互作用、有利于T细胞效应功能的发挥。目前关于免疫突触与T细胞活化关系的研究已成为免疫学研究的焦点问题之一,这问题的解决将进一步丰富免疫学理论,为肿瘤及自身免疫性疾病的免疫治疗提供治疗靶点。     

人CD4~+T淋巴细胞活化相关miRNA的筛选及其靶分子鉴定

目的:筛选与CD4~+ T淋巴细胞活化相关的miRNA,确定其靶分子.方法:用抗CD3的单免隆抗体(mAb)和抗CD28的mAb诱导Jurkat细胞活化,利用miRNA芯片检测并筛选出活化前后表达丰度有显著变化的microRNA,用定量RT-PCR证实筛选出的miRNA在Jurkat细胞活化的差异表达.设计引物构建miRNA的表达载体.利用牛物信息学软件预测miRNA的靶分子,将靶分子mRNA的3'UTR克隆入pGL3Luciferase报告基因载体中.观察转染的miRNA对报告基因表达的影响.结果:流式细胞术(FCM)证实了抗CD3的mAb和抗CD28的mAb可以诱导Jurkat细胞的活化.经miRNA芯片检测表明,Jurkat细胞活化前后有5种miRNA表达水平明显下降,定量RT-PCR证实了miRNA芯片的结果.成功构建了部分预测靶分子的荧光素酶报告基因载体.双荧光素酶报告系统检测了miR-181c对靶分子的干扰作用.结论:miR181c与人CD4~+ T淋巴细胞的活化有着密切的关系,CD69可能足miR-181c的一个靶分子.     

Caveolae与淋巴细胞信号传导

淋巴细胞表面ＧＰＩ－锚固蛋白是一类重要的信号分子，功能配体或ＭｃＡｂ交联ＧＰＩ－蛋白，淋巴细胞表面多种ＧＰＩ－蛋白聚集，随之膜内陷形成囊泡化ｃａｖｅｏｌａｅ即非典型ｃａｖｅｏｌａｅ传导细胞活化信号，ｃａｖｅｏｌａｅ是细胞ＧＰＩ－蛋白信号传导的结构功能单位。     

Surface Antigen Changes Accompanying Lymphocyte Activation

It is here shown that some noncongenic anti-H-2 antisera produced by using normal spleen cells at the immunogen can contain cytotoxic antibody activity against activated, but not resting, lymphocytes of unrelated H-2 haplotype strains. Reactivity was noted against lymphocytes activated in vitro with T- or B-cell mitogens, or in vivo by antigenic stimulation. Analysis of the results indicated that the anti-activated lymphocyte activity was most likely related to production of anti-Ala-1 ('activated lymphocyte antigen') alloantibodies. This appears to occur as a result of donor lymphocyte activation during production of the anti-H-2 antisera, using Ala-1-incompatible donor and recipient combinations, where the donor cells are competent to recognize and react against host H-2 incompatibilities. These results confirm the finding that lymphocyte activation can be accompanied by the expression of an antigen that is either absent or weakly expressed on resting lymphocytes.     

Contrasting cytoskeletal regulation of MHC class II peptide presentation by human B cells or dendritic cells

MHC class II-mediated antigen presentation by B lymphocytes or dendritic cells (DC) initiates CD4+ T lymphocyte activation. In B lymphocytes, MHC class II peptide presentation has been characterised by recruitment of MHC class II, F-actin and lipid rafts to the B cell-T cell immunological synapse. We now show that MHC class II engagement in B lymphocytes induced lipid raft-independent Rho and Rac activation and that inhibition of either Rho-GTPase activation or actin polymerisation in the B cell abrogated T cell activation without altering B cell-T cell conjugate formation. Short-hairpin RNA studies excluded a role for the Cdc42 effector Wiskott-Aldrich syndrome protein. In contrast, antigen presentation by DC was Rho-GTPase-independent although actin was recruited to the DC-T cell interaction site. Moreover, actin depolymerisation in the DC significantly increased T cell activation without altering the number of DC-T cell conjugates. Finally we show that stable recruitment of HLA-DR to the site of the immunological synapse is not a uniform observation in DC and demonstrate reduced HLA-DR expression at the site of microtubule organising centre polarization. Therefore although actin accumulates in DC and B lymphocytes at the immunological synapse with antigen-specific T lymphocytes, this does not reflect comparable functional roles of their actin cytoskeletons in antigen presentation.     

Activity dependent removal of agrin from synaptic basal lamina by matrix metalloproteinase 3

Agrin is a heparan sulfate proteoglycan, which plays an essential role in the development and maintenance of the neuromuscular junction. Agrin is a stable component of the synaptic basal lamina and strong evidence supports the hypothesis that agrin directs the formation of the postsynaptic apparatus, including aggregates of AChRs, and junctional folds. Changes in the distribution of agrin during synaptic remodeling, denervation and reinnervation reveal that agrin can be quickly and efficiently removed from the synaptic basal lamina in a regulated manner. In order to fully understand this mechanism we sought to identify those molecules that were responsible for the removal of agrin. Matrix Metalloproteinases (MMPs) were the most likely molecules since MMPs are involved in the regulation of the pericellular space, including the cleavage of matrix proteins. In particular, MMP3 has been shown to be effective in cleaving heparan sulfate proteoglycans. Antibodies to MMP3 recognize molecules concentrated in the extracellular matrix of perisynaptic Schwann cells. MMP3 specific phylogenic compounds reveal that active MMP3 is localized to the neuromuscular junction. Purified recombinant MMP3 can directly cleave agrin, and it can also remove agrin from synaptic basal lamina. MMP3 activity is itself regulated as activation of MMP3 is lost in denervated muscles. MMP3 null mutant mice have altered neuromuscular junction structure and function, with increased AChRs, junctional folds and agrin immunoreactivity. Altogether these results support the hypothesis that synaptic activity induces the activation of MMP3, and the activated MMP3 removes agrin from the synaptic basal lamina.     

蛋白聚糖在发育期大鼠大脑皮质的表达

目的 观察蛋白聚糖(agrin)在生后大脑皮质不同发育阶段的表达，探讨其与大脑皮质发育的关系。方法 免疫组织化学方法。结果 Agrin在生后不同发育阶段的表达变化明显。生后第1d，agi4n的表达很低，第2d开始迅速上升，6d时达到高峰，以后逐渐下降，第4周时降至成年鼠水平。并且，agi4n的表达与大脑皮质各层神经元发育顺序不尽一致。结论 Agrin是一种与大脑皮质发育有关的蛋白聚糖，它对神经元的发育成熟可能有重要作用。     

Regulation of acetylcholine receptor clustering by the tumor suppressor APC

At the developing neuromuscular junction, motor neuron-derived agrin triggers the differentiation of postsynaptic membrane into a highly specialized structure, where the nicotinic acetylcholine receptors (AChRs) are aggregated into high-density clusters. Agrin acts by activating the muscle-specific kinase MuSK and inducing coaggregation of the 43-kDa protein rapsyn with AChRs on muscle cell membrane. The signaling mechanism downstream of MuSK is poorly defined. We report here that the mouse tumor suppressor protein adenomatous polyposis coli (APC) has a role in AChR clustering and that the Wnt/beta-catenin pathway may crosstalk with agrin signaling cascade during synapse formation.     

人B细胞活化相关基因BC-1514全长cDNA的克隆与原核表达

目的 克隆与B细胞活化相关的新基因及其原核表达。方法 采用差异显示反转录PCR（DDRT-PCR）技术对人扁桃体活化和静止B细胞mRNA的差异表达进行分析。差异显示的片段经过Northern杂交验证后,作为探针进行入活化B细胞cDNA文库的筛选,将所获得的阳性克隆的编码区经PCR扩增后克隆到原核表达载体pGEX-5X-1中,重组质粒经酶切,测序鉴定后转化大肠杆菌BL-21,以IPTG诱导表达融合蛋白。结果 以在活化B细胞高表达的EST32为探针,经3轮筛选人活化B细胞文库获得一个新的全长为1514bp的cDNA克隆（命名为BC-1514）,重组的BC-1514蛋白可在E.coli中以融合蛋白的形式有效表达,其表达量约占细菌总蛋白量的14.3%左右,BC-1514cDNA的GenBank的登录号为AF304442。结论 获得了1条新的与B细胞活化相关的cDNA克隆并在E.coliBL-21中得到了有效表达。     

Activation of lymphocytes by BAT and anti CTLA-4: comparison of binding to T and B cells.

In this study we compare the binding and immune stimulatory properties of BAT and anti CTLA-4 monoclonal antibodies (mAbs). Both antibodies were previously shown to manifest effective immune responses against tumor cells. We have described that BAT antibody, produced against Daudi, a B lymphoblastoid cell line, binds and activates T cells. In this paper we demonstrate that anti CTLA-4, produced against the T-cell activation determinant CTLA-4, binds also to B lymphoblastoid cell lines like Daudi and Raji. Both antibodies do not bind resting B cells. BAT binds resting T lymphocytes as well as activated T lymphocytes, whereas anti CTLA-4 binds only activated T cells. Competitive binding experiments indicate that the binding sites of BAT and anti CTLA-4 on activated T cells are distinct. We have studied the in vitro stimulatory effect of BAT and anti CTLA-4 on lymphocytes cultured with or without tumor cells. In contrast to BAT that increased the proliferation of lymphocytes that have been cultured with tumor cells, anti CTLA-4 did not synergize with tumor cells to enhance lymphocyte proliferation.     

Equine T lymphocytes express MHC class II antigens

Six anti-HLA class II mouse monoclonal antibodies (mAbs) were used in conjunction with a rat monoclonal antibody raised against horse lymphocytes to define class II major histocompatibility complex (MHC) molecules in the horse. By utilizing an ELISA assay and complement dependent lymphocytotoxicity assay, five out of the six anti-HLA class II antibodies and the rat anti-horse monoclonal antibody were found to react with a high percentage of peripheral blood lymphocytes. Flow cytometry demonstrated a variable antigen density on peripheral blood lymphocytes and clear evidence for expression by lymphocytes that carried no detectable surface immunoglobulin. None of the antibodies reacted with equine platelets. The mAbs immunoprecipitated an antigenic complex of Mr 29,000-33,000 from horse lymphocytes. It appears that the distribution of MHC class II antigens in the horse is different from that in man but is similar to that in the dog, since MHC class II antigens are expressed on resting peripheral blood lymphocytes which lack membrane-bound immunoglobulins. Correlations between the distribution of MHC class II antigens on lymphocyte subpopulations and their role in immunological phenomena may contribute to our understanding of the functional properties of these molecules.