合成人IgE肽抗血清抑制I型变态反应的实验研究

作者设计合成了５个人ＩｇＥ多肽片段，分别交联载体蛋白后免疫小鼠，诱生的抗血清２个在ＥＬＩＳＡ中对人与鼠ＩｇＥ分子有交叉反应，３个无反应，５个抗血清均显示有中等程度的被动皮肤过敏反应（ＰＣＡ）抑制作用，被测试的２个抗血清在大鼠肥大细胞被协致敏试验中也显示了抑制作用，研究结果初步表明，采用人ＩｇＥ分子受体结合部位的适当残基序列合成肽疫苗，免疫动物所诱导的抗体可抑制１型变态反应。     

抗IgE抗体的研究进展

I型变态反应性疾病 ,包括哮喘、过敏性鼻炎、特应性皮炎及食物过敏等 ,在过去的 2 0年中发病率逐年上升 ,全世界约有 1.0～ 1.5亿人患有哮喘 ,每年可导致约 18万人死亡[1] 。估计全球为防治哮喘的经济代价已超过用于结核病和艾滋病的总和[2 ] 。目前 ,比较成熟的抗过敏疗法及脱     

IgE与其高亲和力受体FcεRⅠ的相互作用

IgE在I型变态反应中起着及其重要的作用,其与效应细胞上的IgE高亲和力受体(FcεRI)结合,从而触发细胞内一系列信号转导,使肥大细胞发生脱颗粒并释放大量炎性介质是哮喘等过敏性疾病的重要发病机制。本文主要针对IgE及其受体FcεRI的生物学特点以及它们相互结合这一重要步骤的结构研究进行综述。     

人郎罕细胞上的FcεRⅠ

人郎罕细胞能表达ＩｇＥ的高亲和性受体一ＦｃεＲＩ，这一新发现使人们对此受体的功能有了新的认识，同时也为进一步研究郎罕细胞和其他抗原提呈细胞在特应性疾病的发生中所起的病理生理作用提供了新的方向。     

Ⅰ型变态反应动物模型内源性抗IgE抗体的诱导及其对血清IgE水平的抑制作用

２５只白兔用卵清蛋白为抗原经全身和鼻粘膜局部免疫，４ｗ后其中１７只动物血清卵清蛋白特异性ＩｇＥ抗体上升到免疫前的５倍以上，并经抗原刺激鼻粘膜诱发出打喷嚏样反应、流清涕等变应性鼻炎样临床症状。这１７只动物用作Ⅰ型变态反应动物模型，其中９只进一步用制备的异种基因型ＩｇＥＦｃε片段为抗原免疫。２ｗ后在血清中检测到较高浓度的抗ＩｇＥ抗体，同时伴有血清总ＩｇＥ抗体浓度下降，上述变化持续观察１０ｗ无明显改变。提示异常增高的ＩｇＥ抗体水平可通过诱导机体产生内源性抗ＩｇＥ抗体的方式抑制。     

人FcεRIα亚基的细胞外区的克隆表达和抗血清的制备及功能

为获得有生物活性的FcεRIα亚基细胞外区及多克隆抗体 ,并探知其功能 ,构建FcεRIα亚基的细胞外区的原核表达载体PBAD/gIIIA/FcεRIα,经表达、纯化后用斑点杂交法检测其活性。纯化制品免疫兔子获得抗血清 ,并研究抗血清对FcεRI的作用。结果发现 ,经原核表达出的FcεRIα亚基的细胞外区能与IgE结合 ;获得具有特异性抗FcεRI的抗血清 ,可能抑制嗜碱粒细胞脱颗粒。实验提示原核表达系统能产生有生物学活性的FcεRIα亚基的细胞外区 ,用其制备的抗血清能够识别FcεRIα亚基的细胞外区 ,可能抑制嗜碱粒细胞脱颗粒 ,为研究FcεRIα表达调节、对其的封闭作用及研究过敏性疾病的发病机制奠定物质基础     

FcεRⅠ受体α亚基的重组表达、单克隆抗体的制备及特异性鉴定

目的通过基因重组的方法表达IgE高亲和力受体FcεRⅠα亚基，并用重组蛋白制备单克隆抗体。方法用RT-PCR的方法从过敏性疾病病人外周血嗜碱性粒细胞调取FcεRⅠα蛋白基因，经T-A克隆、亚克隆至pET28a（＋）原核表达载体，将重组质粒转化到大肠杆菌BL．21．STAR（DE3），IPTG诱导表达FeaRIOt亚基蛋白，通过Ni-NTA亲和层析纯化融合蛋白；并用其免疫BALB／c小鼠，运用杂交瘤技术制备抗FeaRIOt亚基单克隆抗体，用细胞免疫荧光法对单克隆抗体的特异性进行鉴定。结果成功调取了人IgE高亲和力受体FcεRⅠα亚基的基因，且测序正确；构建原核表达质粒FcεRⅠα-pET28a（＋）；成功建立4株稳定分泌抗FcεRⅠα亚基的单克隆抗体杂交瘤细胞株，分别命名为G101、C22、G113、G42。通过细胞免疫荧光实验证实，4株单克隆抗体均能特异性结合人嗜碱性粒细胞表面的FcεRⅠα亚基。结论成功利用基因重组技术制备了FcεRⅠα亚基蛋白，制备了4株效价高、特异性好的抗FcεRⅠα亚基单克隆抗体，为FcεRⅠα亚基及其抗体在过敏性疾病中的生物学功能研究奠定了基础。     

正常和过敏性哮喘儿童外周血FcεRⅡ阳性细胞与IgE关系的探讨

本文应用单克隆抗体间接免疫荧光染色和流式细胞仪分析技术，对１８名正常儿童和１３名过敏性哮喘儿童外周血淋巴细胞膜表面低亲和力ＩｇＥ受体（ＦｃεＲⅡ）进行测定。结果显示：ＦｃεＲⅡ主要分布于外周血Ｂ淋巴细胞表面。过敏性哮喘儿童外周血单个核细胞（ＰＢＭＣ）和Ｂ细胞中ＦｃεＲⅡ阳性细胞数高于正常对照，并与血清ＩｇＥ浓度呈显著相关。结果表明ＦｃεＲⅡ参与过敏性哮喘儿童体内ＩｇＥ合成的调节过程。     

人郎罕细胞上的FcεRⅠ

人郎罕细胞能表达ＩｇＥ的高亲和性受体－ＦｃεＲⅠ，这一新发现使人们对此受体的功能有了新的认识，同时也为进一步研究郎罕细胞和其他抗原提呈细胞在特应性疾病的发生中所起的病理生理作用提供了新的方向。     

新鲜羊膜致I型超敏反应的变应原性研究

研究新鲜人羊膜的变应原性及其致敏后发生I型超敏反应的可能性。建立豚鼠全身主动过敏实验模型。分新鲜羊膜组、新鲜蛋清组(阳性对照)和PBS液组(阴性对照),每组10只豚鼠。观察豚鼠在致敏期和激发后的反应,采用化学荧光法检测外周血组胺含量,血液流变分析系统检测4项血液流变学指标(全血高切变率黏度、全血低切变率黏度、血浆黏度、红细胞聚集指数)。致敏期间各组豚鼠的体重变化无明显差别(P>0.05);激发后羊膜组豚鼠与阴性组表现一致,无异常反应;羊膜组外周血组胺含量及4项血液流变学指标均与阴性对照无明显差别(P>0.05),与阳性对照有显著性差异(P<0.01)。经规范化无菌处理后的新鲜羊膜,一般不具有变应原性,不会引起I型超敏反应。     

saRNA调控NIS基因介导131I对肝癌HepG2细胞体外抑制作用的实验研究

目的 探索通过小激活RNA(saRNA)调控钠碘转运体(NIS)基因特异性表达介导肝癌核素显像和治疗的可行性,为RNA激活(RNAa)技术介导核素基因治疗肿瘤的应用提供实验依据.方法 saRNA的设计合成:通过基因BLAST比对,设计与合成三条针对NIS基因的saRNA序列;细胞转染:以LipofectaminTM 2000为saRNA载体,将saRNA转染到肝癌HepG2细胞后72h,采用Western blotting分析NIS蛋白的表达水平,筛选出RNAa效率最高的saRNA,并进一步分析saRNA转染后不同时相NIS的表达水平,以及saRNA浓度-NIS表达水平的浓度-效应关系;体外核素摄取实验:体外培养条件下,评价转染肝癌HepG2细胞对125I的摄取;细胞生长增殖抑制实验:体外培养条件下,评价131 I对转染肝癌细胞的生长抑制率.结果 saRNA可上调NIS基因表达水平,其中saRNA482序列上调NIS表达水平最高;saRNA上调NIS的峰值出现在转染后72h,NIS维持高表达水平在10d以上;且NIS上调水平随saRNA浓度增高而增高.体外培养条件下,转染细胞与未转染细胞的摄碘水平差别明显,其中最高者摄碘较对照细胞高出64倍.体外培养条件下,相对未转染组肝癌细胞,131I对转染组HepG2细胞的生长抑制率为60.7％.结论 本研究设计合成的靶向NIS基因的saRNA可上调肝癌细胞NIS的表达水平,增强肝癌细胞摄取放射性碘的能力,研究还证实,通过RNAa调控NIS基因表达可介导131I治疗肝癌的潜力.     

Harnessing engineered antibodies of the IgE class to combat malignancy: initial assessment of FcɛRI‐mediated basophil activation by a tumour‐specific IgE antibody to evaluate the risk of type I hypersensitivity

Background IgE antibodies, sequestered into tissues and retained locally by the high‐affinity IgE receptor, Fc?RI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor α (FRα), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FRα and cannot cross‐link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross‐link FRα‐MOv18‐IgE‐receptor‐Fc?RI complexes on basophils to cause type I hypersensitivity. Objective To assess the propensity for MOv18 used in a therapeutic setting to cause Fc?RI‐mediated type I hypersensitivity. Methods As validated readouts of the potential for MOv18 to cause Fc?RI‐mediated type I hypersensitivity we measured release of a granule‐stored mediator from a rat basophilic leukaemia cell line RBL SX‐38 stably transfected with human tetrameric (αβγ2) Fc?RI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. Results Serum FRα levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FRα, or of healthy volunteer (n=14) or ovarian carcinoma patient (n=32) sera, did not induce RBL SX‐38 cell degranulation. Exposure to FRα‐expressing ovarian tumour cells at target‐to‐effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n=6), or cancer patients, despite detectable levels of circulating FRα (n=5). Conclusion and Clinical Relevance These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, Fc?RI‐mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre‐clinical studies. Cite this as: S. M. Rudman, D. H. Josephs, H. Cambrook, P. Karagiannis, A. E. Gilbert, T. Dodev, J. Hunt, A. Koers, A. Montes, L. Taams, S. Canevari, M. Figini, P. J. Blower, A. J. Beavil, C. F. Nicodemus, C. Corrigan, S. B. Kaye, F. O. Nestle, H. J. Gould, J. F. Spicer and S. N. Karagiannis, Clinical & Experimental Allergy, 2011 (41) 1400–1413.     

雌二醇对人类成骨细胞HOS TE85增殖及Ⅰ型胶原产生的影响

雌激素替代疗法对更年期骨质疏松的预防及治疗效果显著,但作用机理不很明确,本研究通过体外培养的人类成骨细胞ＨＯＳＴＥ８５,观察雌二醇对成骨细胞增殖及Ⅰ型胶原产生的影响。方法：在体外培养的人类成骨细胞ＨＯＳＴＥ８５培养液中,加入雌二醇（０．０１－１０ｎｍｏｌ／Ｌ）,分别培养２４、４８、７２及９６ｈ,于结束培养前６ｈ,加入３Ｈ－胸腺脱氧核苷检测细胞增殖;或于细胞开始培养时,加入３Ｈ－脯氨酸检测Ｉ型胶原产生。结果：在２４ｈ时,雌二醇刺激细胞增殖,但与对照组无显著差异;在４８、７２及９６ｈ时,雌二醇抑制细胞增殖。但无明显量效关系。在４８、７２ｈ时,雌二醇明显刺激细胞Ｉ型胶原产生,量效关系显著,在９６ｈ时。也能刺激Ｉ型胜原产生。结论：雌二醇对人类成骨细胞ＨＯＳＴＥ８５增殖具有短期刺激,长期抑制的作用,对Ｉ型胶原产生具有促进作用,这可能是其临床治疗作用的主要机制。     

Hydrocortisone enhances total IgE levels--but not the synthesis of allergen-specific IgE--in a monocyte-dependent manner.

Recently, hydrocortisone (HC), when combined with human IL-4, has been reported to increase IgE levels in supernatants (SN) of in vitro cultured leucocytes. In this study we investigated the influence of HC on allergen-specific IgE synthesis. Moreover, we examined the relevance of different cell types in this respect. Peripheral blood mononuclear cells (PBMC), T-cell depleted PBMC, CD14-depleted PBMC and highly purified B cells from 10 allergic (birch pollen and/or grass pollen) patients and five non-allergic individuals were investigated. The cells were incubated with HC and/or recombinant human IL-4 (rIL-4) for 8 days. A considerable increase of total IgE was observed in HC/rIL-4-stimulated cultures compared with rIL-4 alone, HC alone or non-stimulated cultures. We demonstrate that this effect depends on the presence of monocytes in in vitro cultures. These results were seen in every experiment, irrespective of healthy or atopic state of the blood donor. The increase of IgE could not be attributed to a rise of birch pollen-and/or grass pollen-specific IgE in patients allergic to these allergens, as shown by IgE-immunoblot. Radio-allergosorbent test (RAST) investigations of HC/rIL-4-stimulated cells cultures from allergic and non-allergic patients confirmed that HC/rIL-4-induced elevated IgE production was also not due to increased production of IgE, specific for important aero-allergens (pollens, house dust mite or animal dander). Therefore we conclude that newly synthesized IgE is not specific for allergens, but that sequential isotype switching in human B cells leads to increased polyclonal IgE production.     

Inhibition of murine IgE and immediate cutaneous hypersensitivity responses to ovalbumin by the immunomodulatory agent leflunomide

Leflunomide has been identified as an immunoregulatory and anti-inflammatory compound. Allergic disease is characterized by elevated serum IgE levels, production of allergen-specific IgE and the release of inflammatory mediators from mast cells and granulocytes. Here we demonstrate, using an in vivo murine model, the ability of leflunomide to down-regulate levels of total and allergen-specific serum IgE production. Mice receiving leflunomide (45 mg/kg) orally at the time of primary immunization with ovalbumin adsorbed to aluminium hydroxide adjuvant, showed a reduction in total serum IgE levels of 95%, 41% and 32% following primary, secondary and tertiary immunizations, respectively (P < 0.05). When leflunomide was administered both at the time of primary and subsequent immunizations, reductions in total and specific serum IgE levels of > 80% and > 38%, respectively, were observed (P < 0.05). Administration of leflunomide to mice which had already developed an IgE response resulted in reductions in total and specific serum IgE levels of > 80% and > 45%, respectively (P < 0.05). Following leflunomide treatment, animals failed to develop immediate cutaneous hypersensitivity responses when challenged intradermally with allergen. Down-regulation of immunoglobulin production was not restricted to IgE, since levels of allergen-specific IgG1 and IgG2a in serum were also reduced. The finding of significant reductions in total and allergen-specific IgM suggests that the mechanism of action does not involve selective inhibition of immunoglobulin class switching. A loss in production of the T helper cell-derived B cell differentiation factor IL-5 may account for the reduction in immunoglobulin levels. In adoptive transfer experiments leflunomide did not induce tolerance in allergen-reactive Th2 populations, contrary to animal disease models of transplantation and autoimmunity, where leflunomide was shown to induce tolerance in the effector T cell population.     

Soluble form of the human high-affinity receptor for IgE inhibits recurrent allergic reaction in a novel mouse model of type I allergy

The recombinant soluble form of the human high-affinity receptor for IgE α subunit (sFc?RIα) is able to block the binding of IgE to the cell surface Fc?RI and prevent the activation of mast cells and basophils. To evaluate its anti-allergic effects in vivo, we established a novel model for type I allergy by transplanting antigen-specific IgE-secreting hybridomas into syngeneic mice. The hybridomas continuously produced anti-2,4,6-trinitrophenyl IgE in vivo, and ear swelling responses could be elicited upon applying picryl chloride to the ears of these mice, which reached their maximum 1–2 h after the antigen challenge. The second swelling response, in extent comparable to the first response, was induced by the second antigen challenge several days later. When the sFc?RIα was intravenously administered before the first challenge and periodically betweeen the first and the second challenges, the ear swelling response to the second challenge, but not to the first challenge, was suppressed almost completely. Immunohistochemical examination revealed that treatment with the sFc?RIα blocked the binding of IgE to the cell surface IgE receptors after the first challenge. Thus, our results indicate that the sFc?RIα suppresses recurrent allergic reactions by preventing IgE binding to the cell surface Fc?RI.     

模拟肽对LPS诱导CD14的表达

本文观察PMB及其模拟肽处理前后FITC-LPS与J774A1的结合能力及CD14表达,并检测培养上清中细胞因子TNF-α、IL-6的含量变化。结果发现①FITC-LPS分别与PMB、peptide 1孵育后,细胞膜平均通道荧光显著减少,LPS与J774A1的结合能力显著降低;②100 ng/ml LPS刺激J774A1 3 h后CD14阳性率明显增加;LPS分别与PMB和peptide 1预孵育后可显著降低LPS刺激J774A1细胞膜CD14表达;③LPS刺激J774A1分泌TNF-α和IL-6显著增加,LPS分别与PMB和petide 1预孵育后能显著减少细胞因子TNF-α和IL-6分泌。结果证实多粘菌素B及其模拟肽(peptide 1)可能通过下调J774A1 CD14表达,降低细胞因子水平来减少LPS诱导的炎症反应。     

2,4,6—三硝基氯苯诱发小鼠迟发型变态反应肝损伤模型的复制及某些中药的保护作用观察

昆明种小鼠经腹腔注射大剂量环磷酰胺处理后,用2,4,6—三硝基氯苯(PC)两次外涂腹部致敏,再用PC经皮肝穿刺攻击,可诱发迟发型变态反应(PC—DTH)性肝损伤,其血清ALT、AST显著升高,病理解剖见肝脏明显肿大,肝细胞变性、坏死,汇管区炎细胞浸润。具有免疫调节活性的中药复方肝得宁、乙肝宁对该模型有显著治疗作用。