蓖麻毒素的毒性作用机制及其在肿瘤治疗中的应用

蓖麻毒素对哺乳动物细胞具有很强的毒性,属于Ⅱ型核糖体失活蛋白。其A链是活性链,可使核糖体失活,从而使蛋白质合成受阻,细胞死亡。基于蓖麻毒素的细胞毒性,人们将蓖麻毒素及其A链应用于肿瘤治疗领域,目的在于特异性杀伤肿瘤细胞,并减小对正常细胞的毒性。本文就蓖麻毒素的分子结构、毒性机制以及近年来在肿瘤治疗领域的研究进展作一综述。     

免疫毒素的临床应用研究

免疫毒素（Immunotoxins，简称ITs）是一种新型的靶向药物，由抗肿瘤相关抗原的抗体和毒素通过化学键偶联而成。组成免疫毒素的毒素大都为糖蛋白，基本上分为两类，一类是单链毒素，如：白树素等，由之构建的免疫毒素无非特异性毒性；另一类为双链免疫毒素，如白喉毒素、蓖麻毒素，由A链和B链通过二硫键连接而成，A链是“弹头”，有细胞毒作用；B链为糖蛋白，其上有半乳糖结合位点等结构，能与细胞膜L半乳糖残基、内皮生长因子样受体、巨球蛋白受体样分子等发生特异性结合，引起非特异性毒性，并能促使A链内化，加强、加快A链的细胞毒作…     

人胃低分化腺癌细胞系的建立及其单克隆抗体的研制

本文报道自胃癌患者转移的淋巴结获得了体外培养细胞系为人胃低分化腺癌细胞,已传110代,冻存的细胞易复苏,异种移植成功率高,细胞恶性程度高。该细胞免疫的BALB/C小鼠的B淋巴细胞与BALB/C小鼠骨髓瘤细胞(SP_2/O)进行融合,共获得2株能分泌抗人胃癌单克隆抗体杂交瘤细胞株,分别命名为GC—3A5和GC—1A5,融合率为70.83％,阳性率为2.35％,经免疫荧光及双P免疫酶标组化法试验对胃癌细胞均为阳性,腹水效价1:400,GC—3A5McAb为IgM抗体,GC—1A5为IgG_1抗体。     

不同免疫球蛋白启动子控制的白喉毒素A链基因对淋巴瘤细胞的抑制作用

目的 比较免疫球蛋白κ轻链启动子/增强子和免疫球蛋白重链启动子/增强子控制的白喉毒素A链基因对淋巴瘤细胞生长抑制.方法 将白喉毒素A链基因或细菌β半乳糖苷酶基因与免疫球蛋白启动子/增强子相连构建成白喉毒素A链真核表达载体pcDNA3IgκDTA、pcDNA3IgHDTA或细菌β半乳糖苷酶真核表达载体pcDNA3IgκLacZ、pcDNA3IgHLacZ.由脂质体介导将质粒转染至产或不产免疫球蛋白的真核细胞中,检测β半乳糖苷酶基因在不同细胞中的表达水平,并进一步检测白喉毒素A链基因的表达对转染细胞的生长抑制作用.结果 由免疫球蛋白κ轻链启动子/增强子或免疫球蛋白重链启动子/增强子控制的β半乳糖苷酶基因只能在产免疫球蛋白(κ轻链型)细胞株CA46中表达.当β半乳糖苷酶基因与质粒pcDNA3IgκDTA或pcDNA3IgHDTA共转染时,β半乳糖苷酶基因的表达只在CA46细胞中被抑制.质粒pcDNA3IgκDTA、pcDNA3IgHDTA的表达均能明显抑制CA46细胞的生长,而对照质粒pcDNA3IgκLacZ或pcDNA3IgHLacZ对CA46细胞则无明显作用,并且pcDNA3IgκDTA的抑制作用较pcDNA3IgHDTA更强.结论 由免疫球蛋白κ轻链启动子/增强子或免疫球蛋白重链启动子/增强子控制的白喉毒素A链基因的表达能特异性地杀伤产免疫球蛋白(κ轻链)的肿瘤细胞.并且免疫球蛋白κ轻链启动子/增强子较免疫球蛋白重链启动子/增强子有更强的启动转录活性.     

治疗肿瘤的新途径——免疫毒素

免疫毒素是指与植物毒素或细菌毒素共价结合的细胞结合性抗体或抗原。所用的毒素可以是整个分子或具有毒性的多肽组分。常用的是蓖麻蛋白,它有一条毒性多肽链(A链),与一条细胞结合性多肽链(B链)相接。B链是一种凝集素,可与细胞表面含半乳糖的糖蛋白或糖脂结合。A链可通过某种尚未充分了解的机理接近细胞浆。不少结合性物     

基于HPV16的新型伪病毒对DNA特异性B细胞的杀伤研究

目的 构建基于人乳头状病毒(human papilloma virus,HPV)16型的新型伪病毒,并检测其对DNA特异性B细胞(R4A)的杀伤效应.方法 利用昆虫杆状病毒表达系统表达病毒蛋白,在体外变性与复性过程中将病毒蛋白包装B细胞特异性启动子IgK控制下的白喉毒素(Diphtheriatoxin,DT)A链(DT-A)基因表达型质粒DNA,形成伪病毒.转染R4A细胞,荧光显微镜和流式细胞仪检测其杀伤效应.酶联免疫法检测R4A细胞分泌抗ds-DNA抗体的滴度.结果 转染R4A细胞48 h后,荧光显微镜和流式细胞仪成功检测到新型伪病毒对R4A细胞的杀伤效应,R4A细胞分泌抗ds-DNA抗体的滴度明显降低.结论 基于HPV16的新型伪病毒能有效特异杀伤R4A细胞,并降低抗ds-DNA抗体的滴度,为系统性红斑狼疮的治疗提供了理想的策略.     

CIK细胞联合顺铂对胃癌SGC-7901/DDP细胞的体外杀伤作用

目的 探讨细胞因子诱导的杀伤(CIK)细胞联合顺铂(DDP)对胃癌耐顺铂细胞株SGC7901/DDP及亲代敏感细胞株SGC-7901的体外杀伤作用.方法 体外培养SGC-7901/DDP及SGC-7901细胞,分为对照(A)组、DDP作用(B)组、CIK细胞作用(C)组和CIK细胞联合DDP(D)组.MTT法、RT-PCR法和Western blot法分别检测肿瘤细胞增殖、mdr-1基因mRNA表达和P-糖蛋白(P-gp)蛋白表达.结果 与SGC-7901细胞相比,DDP对SGC-7901/DDP细胞的杀伤率明显降低(P＜0.05),耐药指数为1.73.在SGC-7901/DDP细胞中,C、D组杀伤作用明显高于A组,且与SGC-7901细胞比较差异有统计学意义(P＜0.05).与B、C组相比,D组对SGC-7901/DDP细胞的体外杀伤活性明显升高,逆转倍数为1.41.C、D组SGC-7901/DDP细胞中mdr-1基因mRNA、P-gp蛋白表达均明显低于A组(P＜0.05).结论 CIK细胞与DDP的联合应用使SGC-7901/DDP细胞的生长受到明显抑制,其可能与CIK细胞下调mdr-1基因mRNA、P-gp蛋白表达及增加SGC-7901/DDP细胞对DDP的敏感性有关.     

B7-1基因修饰的胃癌细胞诱导抗胃癌主动免疫研究

目的　探讨B7 1基因转染胃癌细胞是否具有抗肿瘤主动免疫增强作用。方法　采用重组腺病毒载体将B7 1基因导入人胃癌细胞SGC 790 1,G4 18阳性克隆筛选 ,流式细胞分析显示B7 1的表达 ;将携带B7 1基因的胃癌细胞 (命名为SGC 790 1/B7 1)接种于C5 7BL/ 6小鼠背部皮下 ,观测其致瘤性能 ;SGC 790 1/B7 1致敏的小鼠对野生型瘤细胞是否具有免疫保护作用 ;用SGC 790 1和SGC 790 1/B7 1细胞分别经腹腔免疫小鼠 ,得到腹腔浸润淋巴细胞及致敏脾细胞 ,MTT法检测其体外杀伤实验。结果　B7 1基因在胃癌细胞中获得高表达 ;SGC 790 1/B7 1诱导的CTL(cytotoxicTlymphocyte)对SGC 790 1的杀伤活性显著高于野生型SGC 790 1诱导的CTL对相同靶细胞的杀伤活性 (P <0 0 5 ) ;SGC 790 1/B7 1诱导的CTL对SGC 790 1/B7 1的杀伤率显著高于对野生型SGC 790 1的杀伤率 (P <0 0 5 )。结论　B7 1促进抗胃癌CTL的增殖、活化 ,能诱导抗胃癌主动免疫功能。     

Gelonin的免疫抑制作用

由单克隆抗体与细胞毒物质组成的免疫毒素已引起极大关注。毒素中以蓖麻毒素为最常用,它由二条肽链组成,起细胞毒作用的是A 链。从植物Geloniummultiflorum 种子分离得分子量为30道尔顿的天然植物蛋白质gelonin 也具有抑制蛋白质合成的作用,其结构及生物学性质与A 链相似,但体内毒性比蓖麻毒A链明显为低,可与抗体组成免疫毒素。体外实验表明,在培养初期加入gelonin 可抑制小鼠脾细胞对PHA、ConA 和LPS 的反应,抑制程度取决于gelonin 浓度以及所加进的促有丝分裂剂。当gelonin 为2μg/ml 时,     

丝裂霉素C的免疫结合物对胃癌的导向治疗作用

用葡聚糖Ｔ－７０作中介，制成鼠抗人胃癌单克隆抗体ＭＧｂ２－丝裂霉素Ｃ结合物。该结物的体外试验显示对靶细胞具选择杀伤作用，体内实验治疗结果表明，该结合物组的肿瘤抑制剂明显地高於丝裂霉素Ｃ组，当α－重组人干扰素与免疫结合物合并治疗时，α－重组人干扰素对免疫结合物具有增效作用。     

Fate of an antibody-ricin A chain conjugate administered to normal rats

The pharmacokinetics and catabolism of ricin A chain, a mouse monoclonal antibody (LICR-LOND-Fib 75) and a disulphide linked conjugate of the two have been studied following their intravenous administration to normal rats. Results indicate that the conjugate was removed from the circulation much more rapidly than the antibody but less quickly than the free ricin A chain. Disappearance of the conjugate from the circulation appeared to be biphasic with an early rapid initial phase followed by a much more rapidly than the antibody but less quickly than the free ricin A chain. Disappearance of the conjugate from the circulation appeared to be biphasic with an early rapid initial phase followed by a much slower phase. The fate of a conjugate with a 125I iodide label in the antibody component was compared with that of a conjugate similarly labelled but in the ricin A chain component. The results indicate that breakdown of the conjugate involves both cleavage of the disulphide linkage and complete catabolism of the whole conjugate molecule with the release of 125I iodide. Rapid cleavage of the disulphide bond in the vasculature does not appear to be responsible for the initial rapid disappearance of the conjugate from the circulation.     

Selective cytotoxicity against human tumor cells by an anti-gastric cancer monoclonal antibody-mitomycin C conjugate

An anti-gastric cancer monoclonal antibody, MGb2, was chosen to prepare MGb2-mitomycin C (MMC) conjugate. Four to five molecules of MMC were introduced into each molecule of antibody with the antibody activity well retained. The conjugate showed a highly selective cytotoxicity upon human gastric cancer cells KATO-III. In the 48-h exposure test, the cytotoxic effect of MGb2-MMC upon target cells was similar to that of free MMC, but much greater than that of normal mouse immunoglobulin-MMC conjugate. Instead, the MGb2-MMC showed a statistically less cytotoxic effect upon non-target cells. Imaging and biodistribution studies indicated that the MGb2 was still well localized in tumor tissue after its conjugation with MMC.     

IgE-immunotoxins. I. IgE-intact ricin

An IgE immunotoxin consisting of rat IgE myeloma protein, IR 162, conjugated via the heterobifunctional linking agent N-succinimidyl-3-(2-pyridyldithio)propionate to intact ricin was synthesized and evaluated. The capacity of this IgE-immunotoxin to bind to rat basophilic leukemia cells (RBL cells) and to inhibit RBL cell incorporation of [3H]leucine was assessed. The IgE-intact ricin conjugate sensitized RBL cells for histamine release after treatment with anti-IgE with a time-course of sensitization and dose-response equivalent to native IgE. Intact ricin and IgE-intact ricin were both cytotoxic to RBL cells as assessed by [3H]leucine incorporation. Lactose (50 mM) competed with intact ricin binding and toxicity such that more than 100 ng/ml ricin (8 times its IC50 in the absence of lactose) was required for ricin to kill RBL cells in the presence of lactose. Lactose (50 mM) was not able to fully inhibit 1-100 ng/ml IgE-ricin immunotoxin killing of RBL cells. Saturation of RBL cell IgE receptors by preincubation with IgE totally inhibited IgE-intact ricin-induced toxicity, in the presence of lactose, indicating that toxicity required IgE Fc receptor binding.     

Target ricin by coupling to an anti-macrophage monoclonal antibody

By altering the receptor binding specificity of the highly potent natural toxin ricin, a macrophage specific immunotoxin was developed. Ricin ordinarily does not demonstrate cell type specificity and is capable of binding and entering cells through galactose containing receptors resulting in rapid cell death. A murine anti-rat peritoneal macrophage IgGl monoclonal antibody, B-6, was developed to serve as a target specific carrier for ricin. By covalently binding monoclonal antibody B-6 and reversibly binding lactose to ricin, a new biologically active hybrid toxin possessing macrophage specificity was developed. When P3X63-Ag8.653 myeloma cells, which served as an nonspecific target cell type, and macrophages were treated with the ricin conjugate over a broad range of concentrations and various time periods, the conjugate demonstrated substantially greater toxicity toward macrophages than myeloma cells even though both cell types responded similarly to treatments with unconjugated ricin. It was also observed that ricin was considerably more toxic to macrophages when conjugated to monoclonal antibody B-6 than unconjugated ricin. Through ricin-antibody conjugation a high degree of specificity and toxicity can be attained potentially suitable for anti-tumor reagents and immuno-modulators.     

Generation of ammodytoxin-anti-cathepsin B immuno-conjugate as a model for delivery of secretory phospholipase A2 into cancer cells.

Ammodytoxin C (AtxC) is a toxic secreted phospholipase A2 (sPLA2) from the venom of Vipera ammodytes snake. To evaluate its potential to kill cancer cells, the toxin was cross-linked to the monoclonal antibody against cathepsin B which endocytoses upon binding to cathepsin B, an antigen overexpressed on the plasma membrane of cancer cells. A photo-reactive derivative of AtxC, possessing the same biological activity as the native toxin, was reacted with antibodies to form a covalent immuno-conjugate. In conditions of the cytosolic redox potential, AtxC was gradually released from the conjugate due to reduction of the disulfide bond in the spacer arm of the cross-linker. The phospholipase activity of the preparation reached maximum in 10min and then decreased gradually. As demonstrated by fluorescence microscopy, the immuno-conjugate targeted Caco-2 colon adenocarcinoma cells but was very slowly internalized, the likely reason of only slight cytotoxicity being observed. Despite the lack of a clear cytotoxic effect of AtxC-antibody conjugate on Caco-2 cells, we demonstrated in this work a new methodology for the targeted delivery of (toxic) sPLA2 into cells, promising in research or therapy.     

Colloidal gold-based immunochromatographic assay for detection of ricin.

A rapid immunochromatographic assay was developed to detect ricin. The assay was based on the sandwich format using monoclonal antibodies (Mabs) of two distinct specificities. One anti-ricin B chain Mab (1G7) was immobilized to a defined detection zone on a porous nitrocellulose membrane, while the other anti-ricin A chain Mab (5E11) was conjugated to colloidal gold particles which served as a detection reagent. The ricin-containing sample was added to the membrane and allowed to react with Mab (5E11)-coated particles. The mixture was then passed along the porous membrane by capillary action past the Mab (1G7) in the detection zone, which will bind the particles that had ricin bound to their surface, giving a red color within this detection zone with an intensity proportional to ricin concentration. In the absence of ricin, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of ricin was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 100 pg/ml.     

阿霉素与胃癌单克隆抗体交联物的体内外抗肿瘤作用

以氧化葡聚糖法制备了阿霉素(ADM)与胃癌单克隆抗体(MoAb)3H11的交联物3H11-DEX-ADM,ADM与3H11克分子比为73:1。经ELISA法测定,交联物的抗体活性保留86%。体外细胞毒试验显示,3H11-DEX-ADM对胃癌细胞BGC823的杀伤作用比游离ADM明显增强,其IC₅₀分别为1.0μg/ml及3.75μg/ml。在荷瘤裸鼠治疗实验中3H11-DEX-ADM能显著抑制肿瘤生长,抑制率为51.5%(P<0.05),明显高于ADM组及其非特异性抗体交联物(NI gG-DEX-ADM)组,后者的抑制率分别为21%及24%。表明3H11-DEX-ADM对肿瘤具有选择杀伤作用。将3H11-DEX-ADM与丝裂霉素C(MMC)-3H11交联物(3H11-HSA-MMC)联合应用,细胞素实验未能显示协同或相加作用;游离ADM与MMC联合亦呈相似结果。     

Silica coating magnetic nanoparticle-based silver enhancement immunoassay for rapid electrical detection of ricin toxin

We developed a novel silica coating magnetic nanoparticle-based silver enhancement immunoassay (SEIA) for ricin toxin (RT) rapid electrical detection using interdigitated array microelectrodes (IDAMs) as electrodes. This novel system was developed by taking advantage of the separation and enrichment properties of magnetic nanoparticles (MNPs) and the catalytic properties of gold nanoparticles (GNPs). In this system, MNPs labeled with anti-ricin A chain antibody 6A6 were used to capture ricin and GNPs labeled with anti-ricin B chain antibody 7G7 were used as detectors. To enhance the electrical signal, the catalytic properties of GNPs were used to promote silver reduction. In the presence of ricin, a sandwich structure was formed which could be separated by a magnetic field. The sandwich complex was then transferred to IDAMs. The silver particles bridged the IDAM gaps and gave rise to an enhancing electrical signal that was detected by conductivity measurements. The results showed that the sensitivity of the SEIA for ricin electrical detection was five times greater than that of conventional colorimetric sandwich ELISA. Once the antibody used for detection was coated on the plates or MNPs, our system was three times more rapid than colorimetric sandwich ELISA. This rapid and sensitive detection system provides promising new potential for ricin detection.     

Translocation of ricin across polarized human bronchial epithelial cells

Due to widespread availability, toxicity, and potential for use as a bioterrorism agent, ricin is classified as a category B select agent. While ricin can be internalized by a number of routes, inhalation is particularly problematic. The resulting damage leads to irreversible pulmonary edema and death. Our study describes a model system developed to investigate the effects of ricin on respiratory epithelium. Human bronchial epithelial (HBE) cells were cultured on collagen IV-coated inserts until polarized epithelial cell monolayers developed. Ricin was added to the apical or basal medium and damage to the cell monolayer was then assessed. Within a few hours after exposure, the cell monolayer was permeable to paracellular passage of the toxin. A mouse anti-ricin antibody neutralized ricin and prevented cellular damage as long as the antibody was present before the addition of toxin. These studies suggested that effective therapeutic agents or antibodies neutralizing ricin biological activity must be present at the apical surface of epithelial cells. The in vitro system developed here provides a method by which to screen potential therapeutics for protecting lung epithelial cells against ricin intoxication.     

Ricin A immunotoxins of IgG and Fab of anti-CALLA monoclonal antibody: Effect of water soluble long-chain SPDP on conjugate yield,immunoselectivity and cytotoxicity

The water soluble long-chain crosslinker, sulfo-succinimidyl-6-[3'-(2-pyridyldithio)-propionamido]hexanoate (S-LC-SPDP) was used to prepare ricin A chain (RTA) immunotoxins constructed with whole IgG and Fab fragments of the anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody. In this study, a) S-LC-SPDP modification efficiencies of whole IgG and Fab, b) conjugation yields of the immunotoxins prepared and c) in vitro immunoreactivity and cytotoxicity of immunotoxins constructed were examined. IgG-RTA and Fab-RTA immunotoxins were prepared with 67.3% and 57.0% conjugation yields, respectively. These long spacer intermolecular linked immunotoxins were selectively immunoreactive and cytotoxic against to immunogenic Daudi cells but little or no-binding and cytotoxic against to antigen K562 cells. Both IgG-RTA and Fab-RTA immunotoxins were 210- and 45-fold more active than intact RTA in vitro, respectively.