抗胶质瘤细胞单克隆抗体SZ-38及其识别抗原的生化特征

本文以人脑胶质瘤细胞系SHG-44为抗原,利用杂交瘤技术建立了一株恒定地分泌抗胶质瘤细胞单克隆抗体(McAb)的杂交瘤细胞株SZ-38。McAb SZ-38与9/10胶质瘤细胞系发生强结合反应。而与淋巴细胞、ABO型红细胞等所有正常血液细胞及绝大多数被检测肿瘤细胞系无反应。经免疫转移电泳及免疫沉淀法鉴定,该McAb识别抗原为胶质瘤细胞膜上Mw47,000糖蛋白。应用SPA-Sepharose 4B提纯MeAb SZ-38,再把纯化抗体交联于Sepharose 4B,以McAb亲和层析法提取SZ-38抗原,经SDS-PAGE证实其有较高的纯度。     

抗人肺癌细胞单克隆抗体的制备及其特性的研究

用人肺鳞癌细胞LTEP-78细胞系免疫Blab/c小鼠获得3株抗人肺癌细胞的单克隆抗体杂交瘤系。其中BLTI-01株经六次克隆化培养,体外传代8个月以上。BLTI-01与白细胞抗原及血型抗原基本上无交叉反应;与骨髓细胞无交叉反应;与癌胚抗原和胎甲球蛋白不相关;与肺鳞癌、肺腺癌细胞系及部分其它肿瘤细胞呈阳性反应。     

抗HSV-1淋巴细胞杂交瘤的建立及应用单克隆抗体对HSV分型

应用HSV-1 SM44株感染的BHK-21细胞及提取的感染细胞膜蛋白抗原免疫Balb/c小鼠。以免疫小鼠脾细胞与小鼠骨髓瘤细胞系Sp_2/0融合,经ELISA筛选仅与HSV-1反应的阳性克隆及克隆化,建立了一株产生抗HSV-1型特异性McAb的杂交瘤细胞系(Mad-2)。经ELISA,IF及抗原吸收试验证明,该细胞系的培养上清及制备的小鼠腹水均与HSV-1呈特异性反应,但不与HSV-2反应。ELISA测定McAb Mad-2的腹水效价为10~(-7)Ig亚类鉴定为IgG-1。应用Mad-2和抗HSV型共同性McAb 1A12及抗HSV-2型特异性McAb CH-A9,对43株临床HSV分离株做了ELISA及IF分型。结果表明,28株口唇、眼部感染分离株和1株宫颈炎分离株均与1A12和Mad-2反应,不与CH-A9反应,为HSV-1。余14株富颈炎分离株均与1A12和CH-A9反应,而不与Mad-2反应,为HSV-2。ELISA和IF分型的结果完全一致。本研究提出作者应用的一套抗HSVMcAb有可能作为HSV型别鉴定的标准试剂。     

BALB/c小鼠作为单纯疱疹病毒1型感染模型的免疫学分析

在单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)小鼠感染及其相关研究中,临床病理和免疫学指标对其分析具有重要技术意义。本研究观察了HSV-1在不同条件下感染BALB/c小鼠后的多个免疫学指标,包括外周血单核细胞(peripheral blood mononuclear cell,PBMC)群体中树突细胞比例及功能、血清中和抗体水平、PBMC中HSV-1抗原特异性T细胞水平,以及潜伏感染期小鼠神经组织中CD8^＋ T细胞浸润情况。结果显示,HSV-1毒株Mckrae、17＋以角膜及滴鼻途径感染3周龄及6周龄BALB/c小鼠后,小鼠PBMC中树突细胞数量增加,并显示出刺激病毒抗原特异性T细胞增殖的能力。病毒感染后35 d,小鼠PBMC中未检测到白细胞介素4(interleukin 4,IL-4)^＋抗原特异性T细胞,但能检测到低水平的γ干扰素(interferon γ,IFN-γ)^＋抗原特异性T细胞;小鼠血清中未检测到或仅能检测到低水平的中和抗体。HSV-1以皮下及足垫注射途径感染BALB/c小鼠90 d后,足垫感染途径较皮下感染诱导出更高水平的血清中和抗体,PBMC中可检测到IL-4^＋及IFN-γ^＋抗原特异性T细胞,但不同毒株及小鼠周龄之间出现T细胞反应程度差异。组织病理学结果表明,各组小鼠三叉神经组织中均有CD8^＋ T细胞浸润。这些结果提示,不同HSV-1毒株以不同途径感染不同周龄BALB/c小鼠后,均可刺激树突细胞成熟及呈递病毒抗原,但血清中和抗体及PBMC中病毒抗原特异性T细胞水平在不同毒株、感染途径及小鼠周龄之间有差异。     

B型肉毒毒素重链抗原表位的预测及表位合成肽抗原性的检测

目的:预测B型肉毒毒素蛋白抗原表位,研究表位合成肽的生物学活性。方法:利用生物信息学,结合Anthwine分析软件及网络平台,预测B型肉毒毒素蛋白重链的B细胞抗原表位。人工合成4条(P1,P2,P3,P4)表位多肽,与抗B型肉毒毒素类毒素的马血清反应;采用腹腔注射的方式用合成肽免疫BALB/c小鼠,检测免疫血清与合成肽的结合;对免疫小鼠腹腔注射B型肉毒毒素。结果:合成的多肽能与抗B型肉毒毒素的类毒素的马血清结合;多肽免疫小鼠后能产生针对多肽的抗体,其中P1、P3、P4产生的抗体对受毒素攻击的小鼠具有保护作用。结论:成功预测了抗原表位,合成的表位多肽具有较好的抗原性。     

小鼠胎肝基质细胞体外扩增骨髓来源的LTC-IC

基质细胞是胎肝造血微环境的主要成分,参与造血干/祖细胞的自我更新、增殖分化的调控。为了研究小鼠胎肝基质细胞在造血微环境中的功能,采用转染SV40大T抗原基因的方法建立了小鼠胚胎期第12.5天（Embryonic-day 12.5, E12.5d）胎肝基质细胞系A4、B3,并进一步鉴定基质细胞系的一般细胞生物学特性和造血支持功能。结果：A4、B3为细胞形态、生长行为以及表面分子表达不同细胞系,二者均可维持骨髓源长期培养启动细胞（Longterm cultureinitiating cell,LTC-IC）至少4周并且有不同程度的扩增LTC-IC能力,其中B3扩增LTC-IC的能力是A4的83倍。外源性细胞因子组合SCF+IL-3+IL6+Epo在本实验体系中不影响LTC-IC数量的维持和扩增。暗示E12.5d胎肝造血微环境中基质细胞的功能是不同的,其机制有待进一步研究。     

嵌合NY-ESO-1 DNA疫苗诱导黑色素瘤小鼠模型抗肿瘤免疫的研究

NY-ESO-1作为一种肿瘤抗原,具有较强的免疫抗原性,并且已成为肿瘤候选疫苗之一。但由于目前大多应用NY-ESO-1多肽以及蛋白质疫苗,其临床试验效果欠佳,亟需更为有效的NY-ESO-1抗原设计的肿瘤免疫治疗出现。该研究的目的是探索将NY-ESO-1与具有增强免疫效应的五种因子分别重组成嵌合蛋白抗原,使其更有效地被加工、转运和呈递,以期找到最佳的基因佐剂,增强NY-ESO-1作为肿瘤治疗性DNA疫苗的免疫效果。采用电脉冲体内细胞高效转入的方法对C57BL/6小鼠进行DNA免疫,发现用编码NY-ESO-1或连接有HSP70的嵌合体质粒免疫可诱导强烈的NY-ESO-1特异性Ig G1反应。NY-ESO-1连接泛素的质粒免疫小鼠主要诱导NY-ESO-1特异性Ig G2a反应,表明此基因佐剂诱导强的Th1免疫反应。与其他嵌合NY-ESO-1质粒免疫相比,NYESO-1连接泛素的质粒免疫小鼠,有效地保护小鼠对有NY-ESO-1表达的B16F10黑色素瘤细胞系的挑战作用,证明强的Th1免疫反应对预防和治疗肿瘤具有重要作用。去除调节性T细胞(regulatory T cells,Treg)可进一步增强泛素-NY-ESO-1嵌合DNA疫苗治疗黑色素瘤的作用。此外,Ub-NY-ESO-1质粒结合编码异源黑素瘤抗原GP100和TRP-2的质粒免疫可诱导对抗含有NY-ESO-1表达的B16F10黑色素瘤的协同抗肿瘤免疫疗效。该研究结果表明,编码泛素-NY-ESO-1嵌合抗原的质粒DNA疫苗或与编码其他相关黑色素瘤抗原的质粒的联合可能是有效的治疗黑色素瘤的疫苗。     

转化相关蛋白p86的鉴定

以转化细胞Rat3-3免疫大鼠,获得单克隆抗体E5(MeAb E5)。其对应抗原耐热,是分子量为86kD的蛋白质(P86)。它在c-Ha-ras癌基因转化的细胞上均有较高的阳性表达,而在其相应的非转化细胞上含量甚微。以人工合成的反义c-Ha-ras寡聚核苷酸As-Ⅱ处理Rat3-3细胞,该细胞生长受抑(44.2％),,此对p86的表达亦降低(39.1％)。P86在五种人胃癌细胞系中均有较高表达,在原发胃癌及其转移灶中阳性率达82.6％。p86不仅是一种与C-Ha-ras转化相关的蛋白,而且为胃癌相关抗原。     

两株不含c-di-GMP代谢相关基因的重组卡介苗菌株引起的免疫应答对不同毒力的结核菌株的抵抗作用

菌膜是一种生物膜,作为一种类似于结核分枝杆菌感染过程中出现的某些特征的结构,如抗生素耐药性和感染的长期性,以及作为感染豚鼠的体液反应抗原的来源,在结核病领域重新引起了人们的兴趣。有充分的证据表明,在其他细菌中,第二信使c-di-GMP调节从浮游细胞到生物膜形成的转变。在这项工作中,作者使用牛分枝杆菌活疫苗BCG来确定与c-di-GMP代谢相关的基因缺失是否会影响与巨噬细胞的相互作用,在小鼠细胞系和小鼠中诱导免疫应答的能力,以及在表面膜的生长过程中如何改变蛋白谱。     

细胞工程

<正> 852628 限定伴随人类T细胞表面糖蛋白的T细胞白血病单克隆抗体SN2[英]/Seon,B.K.…//J.Immunol.-1984,132(4)-2039～2095[译自DBA,1984,3(13),84-06314]从一个白血性T细胞系,MOLT-4的膜分离得糖蛋白,经纯化,在弗氏完全佐剂中制备成白血病抗原给小鼠腹腔注射。用聚乙二醇使免疫小鼠的脾细胞与小鼠骨髓瘤细     

CAGE, a novel cancer/testis antigen gene, promotes cell motility by activation ERK and p38 MAPK and downregulating ROS

We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels.     

Perturbation of gastric mucosa in mice expressing the temperature-sensitive mutant of SV40 large T antigen. Potential for establishment of an immortalised parietal cell line

Gastric parietal cells have a unique secretory membrane system that undergoes a profound transformation when the parietal cell is stimulated to secrete acid. Understanding this process has been hindered by the lack of an immortalised parietal cell line. Here we have explored a strategy for the development of a parietal cell line by the generation of transgenic mice bearing the temperature-sensitive mutant of the SV40 large T antigen (SV40 tsA58) under the control of the regulatory sequences of the gastric H+/K+ ATPase beta-subunit (H/Kbeta-tsA58). Three H/ Kbeta-tsA58 transgenic mouse lines were established, namely 218, 224 and 228, all of which expressed the tsA58 T antigen in the gastric mucosa. Unexpectedly, the gastric mucosae of all lines were hypertrophic indicating that the temperature-sensitive large T antigen was partially active at 37 degrees C. Immunofluorescence together with light and electron microscopic studies revealed that mature parietal and zymogenic cells were absent in H/Kbeta-tsA58 transgenic lines 218 and 224, and small undifferentiated cells were the dominant cell type in the gastric units. On the other hand, a few mature parietal cells were detected in line 228 together with an increased proportion of undifferentiated cells and, normally rare, pre-parietal cells. As line 228 represented a rich source of pre-parietal cells, gastric cells from line 228 were isolated and cultured at 33 degrees C, the permissive temperature for tsA58. Gastric epithelial cells, expressing the T antigen, were maintained in culture for over 6 weeks. Upon a temperature shift to 39 C the cultured gastric cells developed characteristics of differentiated parietal cells, including the presence of a nascent canaliculus and dramatically increased production of the gastric H+/K+ ATPase beta-subunit. Therefore, this system shows the potential to generate an immature parietal cell line that can be induced to differentiate in vitro.     

Natural antibody to a human bladder carcinoma cell line

Summary Sera from 144 healthy blood donors were screened for the presence of antibody against the bladder carcinoma cell line RT4 by means of an immune adherence assay. Only one serum (9241) demonstrated high activity with a titer of 1/124. Qualitative and quantitative absorption analyses were carried out with a wide variety of cells to characterize the specificity of the reaction. Cells used for absorption were both cultured and non-cultured and normal and malignant. The reactivity was absorbed by the bladder cancer cell lines RT4 and 5637 and the lung cancer cell line SK-LC-LL. In addition, surgically obtained urothelium from a patient with transitional cell carcinoma abrogated reactivity. No other absorbing cells removed the reactivity of serum 9241 for RT4. The antigen defined by this serologic reaction is heat-stable and trypsin-resistant, as demonstrated by using heated or trypsinized RT4 cells for absorption.This study shows that naturally occurring antibody directed toward cell surface antigens of tumor cells is present in some normal individuals. This finding is important in the evaluation of a possible causal relationship between the presence of antibody specific for tumor cell antigens and the development of neoplasia.     

Novel tumor-associated antigen of hepatocellular carcinoma defined by monoclonal antibody E4-65

A monoclonal antibody, E4-65, produced by immunizing mice with SMMC-7721 cells, a human hepatocellular carcinoma (HCC) cell line, was used to identify and characterize an unreported HCC-associated antigen. Indirect immunofluorescence studies showed that E4-65 antibody reacted with five out of eight HCC cell lines, but not with 10 non-HCC tumor cell lines or a normal liver cell line. Using immunohistochemical examination, E4-65 antigen was detected on the cell membranes and in the cytoplasm of human liver tumor tissues, but was not found in most other tumors, or normal adult or fetal tissues, except for a weakly positive reaction in tissues of the digestive system. Western blot analysis showed that E4-65 antibody bound to a 45 kDa protein in the human HCC cell line and tissue lysates. Enzyme treatment and lectin blotting did not detect the carbohydrate chain in E4-65 antigen. This HCC-associated protein represents a potentially useful target for diagnoses and immunotherapy of human HCC.     

视黄酸对胃癌细胞周期的调控

Retinoic acid can induce growth inhibition and apoptosis, and regulate cell cycle in many types of cancer cell lines. In this study, we investigated the role of all-trans retinoic acid (ATRA) and its mechanism of action in human gastric cancer cell lines. Our results demonstrated that ATRA effectively inhibited growth in three of four gastric cancer cell lines by induction of G0/G1 arrest, and did not induce apoptosis in four gastric cancer cell lines. In RA-sensitive cell lines, ATRA-induced G0/G1 arrest is associated with down regulaton of c-myc and hyperphosphorylated Rb expression, and up regulation of p21WAF1/CIP1 and p53 expression. There were no significant changes in cyclin D1 or CDK4 expression induced by ATRA. Futhermore, expression of these genes were not regulated by ATRA in ATRA-resistant gastric cancer cell line. These results indicate that growth inhibition, rather than apoptosis, is correlated with G0/G1 arrest of these cell lines, more important molecules related cell cycle, including c-myc, p21WAF1/CIP1, p53 and Rb, are involveed in regulation of cell cycle in gastric cancer cells.     

视黄酸对胃癌细胞周期的调控

视黄酸(RA)能够抑制许多类型癌细胞生长、诱导细胞凋亡和调节细胞周期。本文研究了全反式视黄酸(ATRA)对人胃癌细胞的作用机理。结果表明,ATRA通过诱导细胞滞留在G_0/G_1期而显著抑制胃癌细胞生长,但ATRA不能诱导胃癌细胞凋亡;ATRA调控细胞周期与c-myc、磷酸化Rb水平的下调和p21~(WAF1/CIP1)、p53水平的上调有关,而cyclinD_1和CDK_4水平没有明显变化。在RA抗性细胞中,ATRA不能调节这些基因表达。结果证实,ATRA对胃癌细胞生长抑制与其诱导细胞滞留在G_0/G_1期有关,而与细胞凋亡的诱导无关,许多重要的、与周期相关的分子,包括cmyc、p21~(WAF1/CIP1、p53和Rb等参与细胞周期的调控。     

Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.     

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.