Preparation and epitope characterization of monoclonal antibodies suitable for detection of Tomato y

免疫组化方法的具体步骤英文回答：Immunohistochemistry (IHC) is a widely used technique in biomedical research and clinical diagnostics to detect and visualize specific proteins or antigens in tissue samples. The technique involves several steps to achieve accurate and reliable results.1. Tissue preparation: The first step in IHC is to prepare the tissue sample. This involves fixing the tissue in a suitable fixative, such as formalin, to preserve its structure and prevent degradation of proteins. The fixed tissue is then embedded in paraffin or frozen for sectioning.2. Sectioning: The tissue sample is cut into thin sections using a microtome. Paraffin-embedded samples are sliced into thin sections (usually around 4-6 micrometers thick), while frozen samples can be sectioned even thinner(around 10-20 micrometers thick). These sections are mounted onto glass slides for further processing.3. Deparaffinization and rehydration: If the tissue sample was embedded in paraffin, the sections need to be deparaffinized to remove the wax. This is typically done by soaking the slides in xylene or a xylene substitute, followed by a series of alcohol washes to rehydrate the tissue.4. Antigen retrieval: In some cases, the antigens of interest may become masked or cross-linked during the fixation process, making them inaccessible to antibodies. Antigen retrieval methods, such as heat-induced epitope retrieval or enzymatic digestion, are used to unmask the antigens and improve their detection. This step is particularly important for formalin-fixed paraffin-embedded (FFPE) samples.5. Blocking: To prevent non-specific binding of antibodies, the tissue sections are incubated with blocking solutions. These solutions usually contain proteins, suchas bovine serum albumin (BSA) or milk, which occupy any available binding sites on the tissue and reduce background staining.6. Primary antibody incubation: The primary antibody, specific to the target antigen, is applied to the tissue sections and allowed to bind to the antigen of interest. The primary antibody can be monoclonal or polyclonal, depending on the experimental requirements. The incubation time and temperature may vary depending on the antibody and tissue type.7. Washing: After the primary antibody incubation, the tissue sections are washed to remove any unbound antibodies or other reagents. This step helps to reduce background staining and improve the specificity of the staining.8. Secondary antibody incubation: A secondary antibody is applied to the tissue sections, which recognizes and binds to the primary antibody. The secondary antibody is conjugated to an enzyme, fluorophore, or other detection molecule, allowing for visualization of the antigen-antibody complex.9. Washing: Similar to the previous washing step, the tissue sections are washed to remove any unbound secondary antibodies.10. Visualization: Depending on the detection molecule used, different visualization methods can be employed. For enzyme-conjugated secondary antibodies, a chromogenic substrate is added, resulting in the formation of a colored precipitate at the site of antigen-antibody binding. Fluorescently-labeled secondary antibodies can bevisualized using fluorescence microscopy.11. Counterstaining and mounting: To enhance the visibility of the stained tissue, counterstaining with dyes like hematoxylin or eosin can be performed. After counterstaining, the tissue sections are dehydrated and mounted with a coverslip using a mounting medium.中文回答：免疫组化（Immunohistochemistry，简称IHC）是一种在生物医学研究和临床诊断中广泛使用的技术，用于检测和可视化组织样本中的特定蛋白质或抗原。

A single- generation study 单项包括两代(生殖毒性)的研究 Acentric fragment 无着丝点片段Acridine orange 吖啶橙 Active metabolite 活性代谢产物 Additional test 附加试验 Adduct 加合物 ADME 吸引、分布、代谢、排泄 Administration period 给药期 Advers effect 不良反应 Against humanized proteins serum antibodies 抗人源蛋白血清抗体 Aginal smear 阴道涂片 Air righting reflex 空中翻正反射 Alkylating electrophilic cernter 浣化亲电子中心Allele 基因突变产生的遗传因子 Allergic reactions 过敏性反应(变应性反应) Altenative validated test 有效替代试验 Altered growth 生长改变 Ammoniun sulphide staining of the uterus 子宫硫化胺染色 Analogue 类似物(同系物) Analogue series of substance 同系物Analytical method 分析方法 Anaphase 分裂后期 Aneuploidy 非整倍体 Aneuploidy inducer 非整倍体诱导剂 Antigenic specificity 抗原特异性Art and ethical standards 技术和伦理标准Assessment of genotoxicity 遗传毒性评价 AUC 曲线下面积Auditory startle reflex 惊愕反射(听觉惊跳反射) Autoimmune 自身免疫 Autoradiographic assessment 放射自显影评价Autoradiography 放射自显影 Bacterial mutagenicity test 细菌致突变试验 Bacterial reverse mutation test 细菌回复突变试验 Bacterial strains 菌株 Bacterial test organisms 微生物试验菌 Base pairs 碱基对Base set of strains 基本菌株 Base substitution 碱基置换 Bioanalytical method 生物学分析方法 Bioavailability 生物利用度 Biological method 生物学意义Biotechnological products 生物技术产品 Biotechnoloty-derived pharmaceuticals 生物技术药物 Body burden 机体负担 Bone marrow cell 骨髓细胞 Bouin's fixation 包氏液固定Breakage of chromatid 染色单体断裂 Brealage of chromosome 染色体断裂 Bridging character 桥梁作用 C(time) 一定剂量、某一时间的浓度 Carcinogen 致癌物质Carcinogenesis 致癌性 Carcinogenic hazard 致癌性危害 Carcinogenicity bioassay 致癌性生物检测 Carcinogenicity potential of chemical 化合物的潜在致癌性 Carcinoginicity (oncogenicity) 致癌(致瘤) Cardiovascular 心血管 Case-by-case 个例 Cell proliferation 细胞增殖 Cell cultures 细胞培养 Cell line 细胞系 Cell membrane lipid 细胞膜脂质层 Cell replication system 细胞复制系统 Cell suspension 细胞悬液 Cell-mediated immunity 细胞介导的免疫 Cellular therapy 细胞治疗 Central nervous systems 中枢神经系统 Cerebral spinal fluid 脑脊液 Chemical nature 化学性质 Chinese hamster V79 cell 中国仓鼠V79细胞Chromatide 染色单体 Chromosomal aberration 染色体畸变 Chromosomal damage 染色体损伤 Chromosomal integrity 染色体完整性 Chronic toxicity testing 慢性毒性试验 Classfical biotransformation studies 经典的生物转化试验 Clastogen 染色体断裂剂 Clastogenic 致染色体断裂的 Clinical indication 临床适应证 Cloning efficiency 克隆形成率Closure of the hard palate 硬腭闭合 Cmax 峰浓度 Colony sizing 集落大小 Comparative trial 对比试验Complement binding 补体结合 Completely novel compound 全新化合物 Compound bearing structural alerts 结构可疑化合物 Concentration threshold 阈浓度 Concomitant toxicokinetics 相伴毒代动力学 Continuous treatment 连续接触 Corpora lutea 黄体 Corpora lutea count 黄体数 Cross-linking agent 交联剂 Culture condition 培养条件 Culture confluency 培养克隆率 Culture confluenty 培养融合 Culture medium 培养基 Cytogenetic change 细胞遗传学改变 Cytogenetic evaluation 细胞遗传学评价 Cytokines 细胞因子 Cytotoxicity 细胞毒Degradation 降解 Deletion 缺失 Descriptive statistics 描述性统计 Detection of bacterial mutagen 细菌诱变剂检测 Detection of clastogen 染色体断裂剂检测 Determination of metabolites 测定代谢产物 Developmental toxicity 发育毒性Direct genetic damage 直接遗传损伤 Distribution 分布DNA adduct DNA加合物DNA damage DNA损伤DNA repair DNA 修复DNA strand breaks DNA链断裂 Dose escalation 剂量递增 Dose dependence 剂量依赖关系 Dose level 剂量水平 Dose-limiting toxicity 剂量限制性毒性 Dose-raging studies 剂量范围研究 Dose-relatived mutagenicity 剂量相关性诱变性 Dose-related 剂量相关Dose-relatived cytotoxicity 剂量相关性细胞毒性 Dose-relatived genotoxic activity 剂量相关性遗传毒性 Dose-response curve 剂量-反应曲线 Dosing route 给药途径Embryo-fetal toxicity 胚胎-胎仔毒性 Endogenous components 内源性物质 Endogenous gene 内源性基因Endonuclease 核酸内切酶 Emdpmiclease release from lysosomes 溶酶体释放核酸内切酶End-point 终点 Epitope 抗原决定部位 Error prone repair 易错性修复 Escalation 递增Escherichia coli strain 大肠杆菌菌株 Escherichia coli 大肠杆菌Evaluation of test result 试验结果评价 Exaggerated pharmacological response 超常增强的药理作用 Exposure assessment 接触剂量评价 Exposure period 接解期 External metabolizing system 体外代谢系统F1-animals 子一代动物 False positive result 假阳性结果 Fecundity 多产 Fertility studies 生育力研究 Fetal abnormalities 胎仔异常 Fetal and neonatal parameters 胎仔和仔鼠的生长发育参数 Fetal development and growth 肿仔发育和生长 Fetal period 胎仔期 Fetotoxicity 胎仔毒性First pass testing 一期试验Fluorescence in situ hybridization(FISH) 原位荧光分子杂交 Foetuses 胎仔 Formulation 制剂 Frameshift mutation 移码突变 Frameshite point mutation 移码点突变 Free-standing 独立Fresh dissection technique 新鲜切片技术 Funtional deficits 切能缺陷 Functional test 功能试验 Functional indices 功能性指标 Fusion proteins融合蛋白 Gametes 配子 Gender of animals 动物性别 Gender-specific drug 性别专一性药物Gene knockout 基因剔除 Gene therapy 基因治疗 Gene mutation 基因突变 Genetic 遗传Genetic change 遗传学改变 Genetic damage 遗传学损伤 Genetic endpoint 遗传终点Genetic toxicity 遗传毒性 Genotoxic activity 遗传毒性作用 Genotoxic carcinogen 遗传毒性致癌剂 Genotoxic effect 遗传毒性效应 Genotoxic hazard 遗传毒性危害 Genotoxic potential 潜在遗传毒性 Genotoxic rodent carcinogen 啮齿类动物遗传毒性致癌剂 Genotoxicity 遗传毒性 Genotoxicity test 遗传毒性试验 Genotoxicity test battery 毒性试验组合 Genotoxycity evaluation 遗传毒性评价 Germ cell mutagen 生殖细胞诱变剂 Germ line mutation 生殖系统突变 GLP 临床前研究质量管理规范 Gross chromosomal damage 染色体大损伤 Gross evaluation of placenta 胎盘的大体评价 Growth factors 生长因子 Haemotoxylin staining 苏木素染色 Half-life 半衰期 Hematopoietic cells 造血细胞 Heptachlor 七氯化合物 Heritable 遗传 Heritable defect 遗传缺陷 Heritable disease 遗传性疾病 Heritable effect 遗传效应High concentration 高浓度Histologic appearance of reproductive organ 生殖器官的组织学表现 Histopathological chang 组织病理学改变 Homologous proteins 同系蛋白 Homologous series 同系 Host cell 宿主细胞 Human subjects 人体 Human carcinogen 人类致癌剂Human lymphoblastoid TH6cell 人成淋巴TK6细胞 Human mutagen 人类致突变剂 Humoral immunity 体液免疫 Immature erythrocyte 未成熟红细胞Immediate and latent effect 速发和迟发效应 Immunogenicity 免疫原性 Immunopathological effects 免疫病理反应immunotoxicity 免疫毒性 Implantation 着床 Implantation sites 着床部位 In vitro 体外 In vitro test 体外试验 In vivo 体内 In vivo test 体风试验Incidence of polyploidy cell 多倍体细胞发生率 Incisor eruption 门齿萌发 Independent test 独立试验 Individual fetal body weight 单个胎仔体重 Induced and spontaneous models of disease 诱发或自发的疾病模型Inducer of micronuclei 微核诱导剂 Inhalation 吸入 Inhibitor of DNA metabolism DNA代谢抑制剂 Intact animals 完整动物(整体动物) Internal control 内对照 Interphase nuclei 分裂间期细胞核 Intra-and inter-individual 个体与个体间 Isolated organs 离休器官Juvenile animal studies 未成年动物研究 Kinetic profile 动力学特点 Kinetics 动力学 Lactation 授乳、哺乳Large deletion event 大缺失事件 Late embryo loss 后期胚胎丢失 Level of safety 安全水平Libido 性欲 Life threatering 危及生命 Lipophilic compound 亲脂性化合物 Litter size 每窝胎仔数目 Live and deal conceptuese 活胎和死胎 Live offspring at birth 出生时存活的子代Local tolerance studies 局部耐受性研究 Local toxicity 局部毒性 Locu 位点 Long-termcarcinogenicity study 长期致癌性研究Loss of the tk gene tk基因缺失Major organ formation 主要器官形成 Male fertility 雄性生育力 Male fertility assessment 雄性生育力评价Mammalian sells 哺乳动物细胞 Mammalian species 哺乳类动物 Mammalian sell mutation test 哺乳动物细胞致突变试验 Marketing approval 上市许可 Maternal animal 亲代动物Mating behavior 交配行为 Mating period 交配期 Mating ratio 交配比例 Matrices 基质Maximum tolerated dose(MTD) 最大耐受剂量 Mechanism of genotoxicity 遗传毒性机制Mechanistic investigation 机制研究 Metabolic activation 代谢活化 Metabolic activation pathway 代谢活化途径 Metabolic activation system 代谢活化系统 Metabolism 代谢Metabolites profile 代谢物的概况 Metaphase 中期 Metaphase analysis 分裂中期相分析Metaphase cell 分裂中期相细胞 Micronucleus 微核 Micronucleus formation 微核形成Microtitre 微滴定 Mictotitre method 微滴定法 mimicking 模拟 Mitotic index 有丝分裂指数Molecular characterization 分子特性 Molecular technique 分子技术 Monitor 监测Monoclonal antibodies 单克隆抗体 Non-toxic compound 无毒化合物 Mouse lymphoma L5178Y cell 小鼠淋巴瘤L5178Y细胞 Mouse lymphoma tk assay 小鼠淋巴溜tk检测Mutagen 诱变原 Mutagenic carcinogen 诱变性致癌剂 Mutagenic potential of chemical 化合物的潜在致突变性 Mutant colony 突变体集落 Mutation 突变 Mutation induction in transgenes 转基因诱导突变Necropsy(macroscopic examination) 解剖(大体检查) Negative control 阴性对照 Negative result 阴性结果 Newcleated 有核 Non rodent 非啮齿类Non-clinical 非临床 Non-genotoxic carcinogen 非遗传毒性致癌剂 Non-genotoxic mechanism 非遗传毒性机制 Non-human primate 非人灵长类 Non-linear 非线性 No-toxic-effect dose level 无毒性反应剂量水平 Nucleated bone marrow cell 有核骨髓细胞 Nucleoside analogue 核苷酸同系物 Number of live and dead implantation 宫内活胎和死胎数 Numerical chromosomal aberration 染色体数目畸变 Numerical chromosome changes 染色体数目改变Oligonucleotide grugs 寡核苷酸药物 One ,twe,three generation studies 一、二、三子代研究Paraffine embedding 石蜡包埋 Parameter 参数 Parent compound 母体化合物 Parenteral 非肠道 Particulate material 颗粒物 Peripheral blood erythrocyte 外周血红细胞Pharmacodynamic effects 药效作用 Pharmacodynamics 药效学(药效动力学) Pharmacokinetic 药代动力学 Phenylene diamine 苯二胺 Physical development 身体发育 Physiological stress 生理应激 Pilot studies 前期研究 Pinna unfolding 耳廓张开 Plasmid 质粒 Plasminogen activators 纤维蛋白溶解酶原激活因子 Ploidy 整倍体 Point mutation 点突变 Polychromaticerythrocyte 嗜多染色红细胞 Polycyclic hydrocarbon 多环芳烃 Polymer 聚合物 Polyploidy cell 多倍体细胞 Polyploidy 多倍体 Polyploidy induction 多倍体诱导 Poorly soluble compound 难溶化合物 Positive control 阳性对照 Positive result 阳性结果 Post meiotic stages 减数分裂后期 Post-approval 批准后 Postcoital time frame 交配后日期Postimplantation deaths 着床后死亡 Postnatal deaths 出生后死亡 Postweaning development and growth 断奶后发育和生长 Potential 潜在性 Potential immunogenecity 潜在免疫原性Potential target organs for toxicity 潜在毒性靶器官Pre-and post-natal development study 围产期的发育研究 Pre-and postweaning survival and growth 断奶前后的存少和生长 Precipitate 沉淀期 Precision 精密度 Preclinical safety evaluation 临床前安全性评价 Predetermined criteria 预定标准 Prediction of carcinogenicity 致癌性预测Pregnant and lactation animals 怀孕与哺乳期动物 Preimplantation stages of the embryo 胚胎着床前期 Preliminary studies 预试验 Pre-screening 预筛选 Prevalence of abnormalities 异常情况的普遍程度 Primary active entity 主要活性实体 Priority selection 优先选择 Pro-drug 前体药物 Protocol modification 试验方案修改 Quantification of mutant 突变体定量 Racemate 消旋体 Radiolabeled proteins 放射性同位素标记蛋白 Radiolabelled compounds 放性性同位素标记化合物 Range-finding test 范围确定试验 Rate of preimplantation deaths 着床关死亡率 Rational study design 合理的试验设计 Receptor properties 受体性质 Recombinant DNA proteins DNA重组蛋白Recombinant DNA technology DNA重组技术 Recombination 重组 Recombinant plasma factors 重组血浆因子 Reduction in the number of revertants 回复突变数的减少 Relative plating efficiency 相对接种效率 Relative suspension growth 相对悬浮生长率 Relative total growth 相对总生长率 Relevant animal species 相关动物种属 Relevant dose 相关剂量Relevant factor 相关因素 Repeated-dose toxicity studies 重复剂量毒性研究 Reproductive toxicity 生殖毒性 Reproductive/developmental toxicity 生殖/发育毒性 Reverse mutation 回复突变 Reversibility 可恢复性(可逆性) Risk assessment 危险度评价 Rodent hematopoietic cell 啮齿类动物造血细胞 Route of administration 给药途径 Routine testing 常规试验S9-mix constituent S9混合液成分 Safeguards 安全监测 Safety pharmacology 安全药理学Safety margin 安全范围 Salmonella typhimurium 鼠伤寒沙门菌 Sampling time 采样时间Satellite groups 卫星组 Saturation of absorption 吸收饱和 Sensory functions and reflexes 感觉功能和反射Short term toxicity 短期毒性Short or medium-term carcinogenicity study 短或中期致癌性研究 Short treatment 短期处理 Sighting studies 预试验 Singledose(acute)toxicity 单剂量(急性)毒性 Single study design 单一研究设计 Site-specific targeted delivery 定位靶向释放 Small colony 小集落 Small colony mutant 小集落突变体Soft agar method 软琼脂法 Soluble genotoxic impurity 可溶性遗传毒性杂质 Solvent control 溶剂对照 Somatic cell 体细胞 Somatic cell test 体细胞试验 Species 种属 Specificity 特异性 Species specificity 种属特异性 Spindle apparatus 纺缍体 Stages of reproduction 生殖阶段Standard battery of test 标准试验组合Standard 3-test battery 标准三项试验组合 Standard battery 标准组合 Standard battery system 标准组合系统 Standard procedure 标准规程Standard protocol 标准试验方案Standard set of strains 标准菌株组Standard set of tests 标准试验组 Standard test battery 标准试验组合 Statistical evaluation 统计学评价 Steady-state levels 稳态浓度 Step-by-step 逐步 Stepwise process 阶梯式程序 Strain 品系 Structural changes 结构改变 Structural chromosomal aberration 染色体结构畸变 Subgroups 亚组Supravital staining 体外活动染色 Surface righting reflex 平面翻正反射 Survival 存活率suspension 悬浮物 Systemic exposure 全面接触 Target organs 靶器官 Target cell 靶细胞Target histidine genes 组氨酸目的基因 Target tissue 靶组织Target tissue exposure 靶组织接触 Teratogenic response 致畸胎反应 Terminal sacrifice 终末期处死 Test of carcinogenicity 致癌试验 Test approach 试验方法Test battery approach 试验组合方法 Test compound 受试物 Test model 试验模型 Test strategy 试验策略 Test systems 试验系统 Tester strain 试验菌株 Therapeutic 治疗 Therapeutic confirmatory 疗效确定 Therapeutic exploratory 疗效探索Therapeutic indication 治疗适应证 Time course 时程 Timing conventions 分段计时方法Tissue cross-reactivity 组织交叉反应 Tissue distribution 组织分布 Tissue exposure 组织接触Tissue uptake 组织吸收 Tk locus tk位点 Top concentration 最高浓度 Topical 局部的Topoisomerase inhibitor 拓朴异构酶抑制剂 Total erythrocyte 总红细胞Total litter loss 整窝丢失 Toxicity to reproduction 生殖毒性 Toxicokinetics 毒代动力学(毒物代谢动力学) Transgene 转基因 Transgenic animals 转基因动物 Transgenic plants 转基因植物Translocation 移位 Treatment regimen 实施方案 Tubal transport 输卵管运输 Tumor induction 肿瘤诱导 Tumor response 肿瘤反应 Tumor-related gene 肿瘤相关基因 Two or three phase approach 分段(二段或三段)研究 Two study design 分段(两段)研究设计Ovulation rate 排卵率 Unbound concentration 未结合浓度 Unexpected finding 非预期结果Unscheduled DNA synthesis(UDS) 程序外DNA合成 Unstable epoxide 不稳定过氧代物Whole blood 全血。

抗原表位鉴定方法的研究进展孙娟;王愉涵;刘艳;郭进露;武丽涛;李冬民【摘要】抗原决定簇又称为抗原表位,是抗原分子中决定抗原特异性的化学基团,也是蛋白质抗原性的基础.抗原抗体的特异性结合反应是针对特定的抗原表位而并非是完整的抗原,因此明确蛋白质的抗原表位对多肽和新型疫苗分子的设计及诊断试剂的发展具有重要意义.随着科技的发展,出现了一些新的鉴定单克隆抗体抗原表位的方法.本文在传统表位鉴定方法基础上简要综述了多技术结合鉴定抗原表位新方法.【期刊名称】《国外医学（医学地理分册）》【年(卷),期】2017(038)003【总页数】5页(P291-295)【关键词】抗原表位;多技术;鉴定方法【作者】孙娟;王愉涵;刘艳;郭进露;武丽涛;李冬民【作者单位】西安交通大学医学部基础医学院生物化学与分子生物学系,陕西西安710061;西安交通大学环境与疾病相关基因教育部重点实验室,陕西西安 710061;西安交通大学2013级临床医学五年制,陕西西安 710061;西安交通大学医学部基础医学院生物化学与分子生物学系,陕西西安 710061;西安交通大学环境与疾病相关基因教育部重点实验室,陕西西安 710061;西安交通大学2013级临床医学五年制,陕西西安 710061;西安交通大学医学部基础医学院生物化学与分子生物学系,陕西西安 710061;西安交通大学环境与疾病相关基因教育部重点实验室,陕西西安710061;西安交通大学2013级临床医学五年制,陕西西安 710061;西安交通大学医学部基础医学院生物化学与分子生物学系,陕西西安 710061;西安交通大学环境与疾病相关基因教育部重点实验室,陕西西安 710061;西安交通大学2013级临床医学五年制,陕西西安 710061;西安交通大学医学部基础医学院生物化学与分子生物学系,陕西西安 710061;西安交通大学环境与疾病相关基因教育部重点实验室,陕西西安 710061;西安交通大学医学部基础医学院生物化学与分子生物学系,陕西西安710061;西安交通大学环境与疾病相关基因教育部重点实验室,陕西西安 710061【正文语种】中文【中图分类】R751抗体一般仅与大分子抗原的某一部位反应，这一特异的部位称为抗原决定簇，也称为抗原表位，它是抗原分子中决定抗原特异性的化学基团。

Int J Clin Exp Med 2016;9(2):2211-2219 /ISSN:1940-5901/IJCEM0018414Original ArticleAlterations of expression profile ofcancer-related genes in co-cultured humanbone marrow-derived mesenchymal stromal cellswith brain glioma cells in vitro: propose concerns about oncological risk before clinical applicationRusen Zhu1, Chengjie Xu1, Xinggui Chen2, Rongyi Chen3, Liubo Lan4, Jinghong Li1, Yanqing Yin1, Yuansheng Liang11Department of Neurosurgery, Affiliated Hospital of Guangdong Medical University, Guangdong Medical University, Zhanjiang, Guangdong Province, China; 2Cancer Center, Affiliated Hospital of Guangdong Medical University, Guangdong Medical University, Zhanjiang, Guangdong Province, China; 3Department of Dermatology, Affiliated Hospital of Guangdong Medical University, Guangdong Medical University, Zhanjiang, Guangdong Province, China; 4Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang, Guangdong Province, ChinaReceived October 24, 2015; Accepted January 21, 2016; Epub February 15, 2016; Published February 29, 2016 Abstract: Background: Human bone marrow-derived mesenchymal stromal cells (hMSCs) may serve as ideal deliv-ery vehicles for gene therapy for human gliomas. The stability of hMSCs in culture had been concerned and studied, while the stability of these cells in glioma environment had been overlooked and remains unclear. In this study, we investigate the alterations of expression profile of cancer-related genes in hMSCs under the influence of glioma cells in vitro. Methods: HMSCs were obtained from normal adult persons and identified by analysis of distinct surface markers by flow cytometry and tests of their stemness, and hMSCs were co-cultured with U251 glioma cells and the expression profile of cancer-related genes were investigated by microarray assay, and the results of microarray were verified by Real-time quantitative RT-PCR. Results: The obtained hMSCs express distinct surface markers of mesenchymal stromal cells and could be induced to differentiate into neural lineage cells in vitro. Of the 440 can-cer-related genes covered by Oligo GEArray Human Cancer Microarray OHS-802, 16 were found to be significantly up-regulated (>3-fold) but none down-regulated in hMSCs co-cultured with U251 glioma cells. The up-regulations of some of those genes were confirmed by Real-time quantitative RT-PCR. The up-regulated genes include some important oncogenes. Conclusions: The present study is the first to show that the co-culture of hMSCs with human glioma cells lead to up-regulated expressions of some important oncogenes in hMSCs, the over-expression of which has been demonstrated to contribute to tumorigenesis. We propose concerns about oncological risk of hMSCs under the influence of glioma cells, and highlight the need for further studies before their clinical application as therapeutic vectors to treat human gliomas.Keywords: Mesenchymal stromal cells, glioma, gene therapy, tumorigenicity, cancer-related geneIntroductionMesenchymal stromal cells (MSCs) were dem-onstrated to be effective for delivering trans-genes to treat experimental gliomas based on their unique migratory properties within the central nervous system (CNS) and their good tropism to glioma cells [1]. The potential of MSCs for targeted gene delivery in the context of cancer is an exciting area of research that has gained considerable momentum in recent years, with studies reporting engineered MSCs specifically targeting multiple tumor types. However, concerns about the biosafety of MSCs must be addressed fully to allow MSC-mediated therapy for cancer to realize its full potential. The transformation potential of MSCs in culture had been concerned. Expansion of MSCs in vitro will be required for therapeutic applicationand so their stability in culture is paramount. The majority of studies have shown that human MSCs are stable, while murine MSCs are more prone to genetic transformation during in vitro culture, and may be capable of forming sarco-mas in vivo [2, 3]. Although transformation of human MSCs appears unlikely, and very rare, these studies certainly emphasize the impor-tance of stringent monitoring of MSCs before application in the clinical setting. However, there is still another important aspect of this problem, that is the transformation risk of human MSCs (hMSCs) in tumor environment, had not attracted much attentions. When applied to treat tumors hMSCs would leave the original living place and enter into the targeted tumor environment and the milieu of growth factors and inflammatory cytokines present in the tumor environment is very different from that in the original living place of hMSCs. It was reported that co-culture of human adipose tis-sue derived stem cells with human squamous cell carcinoma cells could cause significant changes of gene expressions and protein syn-thesis in human adipose tissue derived stem cells, which arouse worries about oncological risk [4]. hMSCs could serve as ideal genes vec-tor to treat human gliomas. For that application hMSCs would leave the bone marrow then reach and stay in the glioma environment, and interact with glioma cells. Whether malignant transformation of hMSCs would be induced in glioma environment is what we concern about here. To make a primary probe into this ques-tion we design this experiment. Since tumori-genesis could be attributed to genetic charac-teristics, we co-culture hMSCs with U251 glio-ma cells and investigated the alterations of expression profile of cancer-related genes in hMSCs, in order to find out whether there were any characteristics that could help in the evalu-ation of the risk of malignant transformation of hMSCs in human glioma environment. Materials and methodsIsolation of hMSCsTo isolate hMSCs, bone marrow aspirates were taken from the iliac crest of normal adult donors after informed consent and under a protocol approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical University. Nucleated cells were isolated by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient and resuspended in DMEM-F12 (Gibco BRL, Gaithersburg, MD, USA) plus 10% FBS, and incubated at 37°C with 5% CO2. After 24 hours, nonadherent cells were discarded, and adherent cells were thor-oughly washed twice with PBS and continued to incubate with the new medium. After that medi-um was changed every 3 days and cells were passaged once a week. Human glioma U251 cells were obtained from China Center for Type Culture Collection (Shanghai, People’s Republic of China) and maintained in DMEM-F12 plus 10% FBS at 37°C with 5% CO2.Epitope analysis of hMSCsObtained hMSCs were analyzed by flow cy-tometry. Detached cells were washed and re-suspended in PBS. Approximately 105 cells were incubated on ice for 30 min with conju-gated, monoclonal antibodies against CD34, and CD45 (Becton Dickinson, San Jose, CA, USA), CD73 (PharMingen, Uppsala, Sweden), and CD 166 (Ansell, Bayport, MN, USA). Nonspecific fluorescence was determined, using equal aliquots of cell preparation incu-bated with an-ti-mouse monoclonal antibodies (Becton Dickinson). Finally, the cells were assayed in a flow cytometer (FACSort, Beckton Dickinson), and the data analyzed with Cell- quest software (Becton Dickinson). Determination of hMSCs’ stemmnessAfter passages 2-5, the conversion of hMSCs into neurosphere-like structures was initiated. Specifically, cells were dissociated with 0.25% trypsin/0.01% EDTA and plated on uncoated flasks at a concentration of 1×105 cells/cm2 in serum-free DMEM/F12 medium containing B27/N2 (all from Gibco BRL, Gaithersburg, MD) plus 20 ng/mL of both epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), both from Sigma (St. Louis, MO) and incubated at 37°C with 5% CO2. The medium was changed once a week and growth factors were added twice a week. After 10-15 days, neurosphere-like structures could be observed. To induce differentiation in vitro, cell aggregates were plated on fibronectin-coated plates in B27 media containing 10% FBS. ImmunocytochemistryCells were fixed in 4% paraformaldehyde in PBS. Immunocytochemistry was carried out using standard protocols. Cell nuclei werecounterstained with 4,6-diamidino-2-phenylin-dole (DAPI; Roche Molecular Biochemicals, Mannheim, Germany). The antibodies used and their dilutions were: nestin, 1:500 (Chemicon International, Temecula, CA); GFAP, 1:100 (Chemicon International); NG2, 1:100 (Sigma); MAP2ab, 1:100 (Chemicon International); anti-rabbit IgG Cy3 conjugate, 1:100 (Chemicon International); and antimouse IgG FITC conju-gate, 1:100 (Sigma).Co-culture of hMSCs and U251 glioma cellsCo-culture of hMSCs and U251 glioma cells was performed in a transwell system. For that 2×104 hMSCs were seeded onto a polyester membrane transwell-clear insert (Corning, pore size 0.4 μm) while 2×104 U251 cells were seed-ed onto the bottom of a six-well cell culture plate. Cells were cultured up to five days in 4 ml DMEM-F12 plus 10% FBS per well and the medium was changed every 48 h. 2×104 hMSCs cultured alone in transwell inserts served as the control and were treated the same as the co-culture.Preparation of RNA samplesAfter co-cultured with U251 glioma cells or cul-tured alone (as control) for five days as described above, isolation of RNA samples in hMSCs were initiated. Total RNAs were isolated by using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA).Microarray expression analysisIn order to investigate the influence of glioma cells on the expression of cancer-related genes in hMSCs, we co-cultured hMSCs with U251 cells for five days, and the expression profile of cancer-related genes were investigated by microarray test, and the hMSCs cultured alone were treated as the control group. Using the TrueLabeling-AMP Linear RNA amplification kit (SuperArray Bioscience, Frederick, MD, USA), the mRNA was reversely transcribed to obtain cDNA and converted into biotin-labeled cRNA using biotin-16-UTP (Roche, Mannheim, Ger-many) by in vitro transcription. Prior to hybrid-ization, the cRNA probes were purified with the ArrayGrade cRNA cleanup kit (SuperArray Bioscience). The purified cRNA probes were then hybridized to the pretreated Oligo GEArray Human Cancer Microarrays OHS-802 (Super- Array Bioscience), which cover 440 cancer-related genes. Following several washing steps, array spots binding cRNA were detected using alkaline phosphatase-conjugated streptavidin and CDP-Star as chemiluminescent substrate. Chemiluminescence was detected by exposing the Array membranes to x-ray film. The image data were transformed into numerical data using the software GEArray Expression Analysis Suite (SuperArray Bioscience). The numerical data were then further evaluated with Microsoft Excel 2003. Data evaluation included back-ground correction (subtraction of minimum value) and median normalization. Data filtering criteria were as follows: at least one of the spot intensities to be compared had to be more than twice the background intensity, and the spot intensity ratios had to be higher than 3.0 (for overexpressed) or lower than -3.0 (for down- expressed).Real-time quantitative RT-PCRTo confirm the up-regulation of some of those oncogenes indicated by microarray, Real-time quantitative RT-PCR was performed. Primers were designed using the primer software (ver-sion 5.0, Whitehead Institute for Biomedical Research, Cambridge, MA, USA) under consid-eration of the special design criteria for real-time PCR primers. Primer sequences (forward, reverse) and lengths of the amplified products were as follows: β-actin (5’-CCTGTACGC-CAACACAGTGC-3’, 5’-ATACTCCTGCTTGCTGATC- C-3’, 211 bp); KIT (5’-TTGTCATCAGCCAC- CATCCT-3’, 5’-CCAGTCCATACCTCCCTCTCTT-3’, 121 bp); CAPNS1 (5’-GATAGCGACACCACAGG- CAAG-3’, 5’-GAGATTTGAAGGCACGGAACAT-3’, 283 bp); TK1 (5’-GTGATTCTCGGGCCGATGT- T-3’, 5’-GCGAGTGTCTTTGGCATACTTGAT-3’, 114 bp); MMP-1 (5’-CGCACAAATCCCTTCTACC-3’, 5’-CTCTGTCGGCAAATTCGTAAG-3’, 103 bp); CC- ND1 (5’-TGGCGTTTCCCAGAGTCATC-3’, 5’-CC- ACCTCCCTTCAACACTTCC-3’, 195 bp); CDC20 (5’-TCCCTGGCAGTCCAATGTCC-3’, 5’-GGAGACC- AGAGGATGGAGCAC-3’, 135 bp); RELA (5’-CG- CTGCATCCACAGTTTCCA-3’, 5’-GCACAGCATTCA- GGTCGTAGTCC-3’, 151 bp); STC1 (5’-GAAG- CCTGCTGGAATGTGAT-3’, 5’-GGGATGTGCGTTT- GATGTG-3’, 237 bp). The RNA samples were reversely transcribed using the MMLV-reverse transcriptase (Promega). An oligo-dT primer was used for priming the reverse transcription. Beta-actin was chosen as the reference gene for normalization of the results. The quantita-tive real-time PCR was performed using aRotor-Gene 3000 thermal cycling system (Corbett Research, Sidney, Australia) with Syber green (Molecular Probes, Eugene, OR, USA) as the detection system. The results were ana-lyzed with the Rotor-Gene 6.0 software (Corbett Research) and Microsoft Excel 2003.ResultsIdentification of hMSCsApplied hMSCs were identified by analysis of distinct surface markers in flow cytometry and tests of their stemness. Flow cytometric analy-sis of hMSCs with monoclonal antibodies against CD34, CD45, CD73, and CD166. hMSCs were negative for CD34 and CD45 and positive for CD73 and CD166 (Figure 1).Applied hMSCs could be converted into cells with characteristics of NSCs in vitro, which formed cell aggregates that were morphologi-cally similar to NSC-derived neurospheres (Figure 2A ) and highly expressed the neural stem cell marker nestin (Figure 2B). And theFigure 1. Epitope analysis of human bone marrow-derived mesenchymal stromal cells (hMSCs). Flow cytometric analysis of cultured hMSCs with monoclonal antibodies against CD34, CD45, CD73, and CD166. hMSCs were nega -tive for CD34 and CD45 and positive for CD73 and CD166.cells forming the neurospherelike structures could be induced to differentiate into neural lin-eage cells in vitro (Figure 3).Microarray expression analysishMSCs were co-cultured with U251 glioma cells for five days, and the expression profile of can -cer-related genes were investigated by microar-ray assay. Of the 440 cancer-related genes cov-ered by Oligo GEArray Human Cancer Microarray OHS-802, 16 were found to be significantly up-regulated (>3 fold) but none down-regulated inhMSCs co-cultured with U251 glioma cells compared to that in hMSCs cultured alone (Figure 4; Table 1). The up-regulated genes in hMSCs co-cultured with U251 cells as indicat -ed by microarray assay include some important oncogenes, the over-expressions of which have been demonstrated to have a positive role in tumorigenicity.Real-time quantitative RT-PCRTo confirm the results of microarray assay, wechose 8 important oncogenes among those up-Figure 2. Characteristics of human bone marrow-derived mesenchymal stromal cells (hMSCs) -derived neural stem-like cells. A. Representative sequence of phase-contrast photomicrographs of neural stemlike cells 1, 5, 10, and 15 days after conversion from hMSCs. B. hMSCs-derived neural stemlike cells form neurosphere-like structures, andshow high nestin expression. The nuclei were counterstained with DAPI (blue).Figure 3. In vitro differentiation of human bone marrow-derived mesenchymal stromal cells (hMSCs)-derived neural stemlike cells into astroglial, oligodendroglial, and neuronal cell types. After 7-10 days in differentiation conditions, the cells were stained for markers for astrocytes (GFAP), oligodendrocytes (NG2), or neurons (MAP2ab). Nuclei were counterstained with DAPI (blue).regulated genes revealed by microarray assay to be further tested by Real-time quantitative RT-PCR. Real-time quantitative RT-PCR re- vealed that expression of KIT, CAPNS1, TK1, MMP1, CCND1, CDC20, RELA, and STC1 mRNA were all up-regulated in hMSCs co-cultured with U251 glioma cells compared to that in hMSCs cultured alone, and the magnitude of up-regulation were: KIT, 4.84; CAPNS1, 4.21; TK1, 2.69; MMP1, 8.34; CCND1, 8.94; CDC20, 4.88; RELA, 5.17; STC1, 1.82. DiscussionHuaman MSCs (hMSCs) represent an optimal cellular vector for gene therapy to treat human gliomas. Nevertheless, research about the influence of human glioma cells on hMSCs is still not in the focus. When server as delivery vector to treat glioma hMSCs would leave the original living place and enter into the targeted tumor environment. What kind of changes would happen to hMSCs under the influence of glioma cells is hardly know. What we concern One of the most important characteristics of malignant cells is uncontrolled cell growth. The KIT, CAPNS1, and TK1 genes were found to be up-regulated in hMSCs co-cultured with U251 cells. The up-regulation of all these genes was demonstrated to be a positive factor in cell pro-liferation and to contribute to carcinogenesis. KIT functions as the receptor for stem cell fac-tor (SCF) and this interaction is essential for regulation of proliferation and survival. Up- regulation of KIT signaling has been associated with oncogenic transformation in cells express-ing the molecule [5]. Capns1 is a regulatory subunit of Calpain, functions in anti apoptosis and its depletion is coupled to increased sensi-tivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells. Calpain System is elevated during transforma-tion; it is required for autophagy and survival of cancer cells and plays a key role in metastatic cell migration and angiogenesis [6]. Thymidine kinase 1 (TK1) is a key enzyme involved in thesynthesis of DNA precursors and therefore cellFigure 4. Biotin-labeled cRNA probes were synthesized from total RNA of hMSCs cultured alone or total RNA of hMSCs co-cultured with U251 cells and hybridized to the membrane of Oligo GEArray Human Cancer Microar-ray OHS-802 spotted with 440 gene-specific oligonucleotide fragments. After hybridization, the membrane was incubated with phosphatase-conjugated streptavidin, and the signal was visualized with CDP-Star and exposed to x-ray film.about here is the bio-safety of hMSCs in aspect of tumori-genic risk in glioma environ-ment. The present study is the first to show that the co-culture of hMSCs with human glioma cells lead to a signifi -cant change in the cancer-related gene expression pro-file of hMSCs.The microarray analysis re- vealed that 16 cancer-rela- ted genes were found to be significantly up-regulated in hMSCs co-cultured with U251 cells compared to that in hMSCs cultured alone. In particular, the up-regulated genes include 8 important oncogenes: KIT, CAPNS1, TK1, MMP1, CCND1, CDC20, RELA, and STC1, which are involved in cell grow/prolifera-tion, cell invasion/metastasis, cell cycle, and signal trans-duction pathways. The up-reg-ulation of all these 8 onco-genes mRNA was confirmed by Real-time quantitative RT- PCR.proliferation-dependent Thymidine kinase, a cell cycle-dependent marker, can be detected in the serum of patients diagnosed with many different types of cancer. Serum levels of thymi-dine kinase have also been shown to reflect the progression of cancer as well as an indication of the efficacy of chemotherapeutic interven-tion [7].Invasive and metastatic behavior is another important characteristic feature of malignant cells. During tracing glioma cells, hMSCs pres-ent good migration ability in the CNS. This prop-erty, however, would contribute to the malig-nancy of this cell population once they obtained uncontrolled cell growth ability, which should be concerned about. Matrix metalloproteinas-es (MMPs) have long been associated with cell invasion and metastasis, MMPs are up-regulat-ed in almost every type of human cancer, and their expression is often associated with poor survival [8]. MMP1 was found to be up-regulat-ed in hMSCs co-cultured with U251 cells, which may imply enhanced invasion and metastasis abilities in these cells under the influence of glioma cells.The connection between the cell cycle and can-cer is obvious: cell cycle machinery controls cell proliferation, and cancer is a disease of inap-propriate cell proliferation. The genes CCND1 and CDC20, which are closely involved in cell cycle process, were found to be up-regulated in hMSCs co-cultured with U251 cells. CCND1 is an important regulator of cell cycle progression and can function as a transcriptional co-regula-tor; the overexpression of CCND1 has been linked to the development and progression of cancer [9]. CDC20 is an essential cell-cycle regulator required for the completion of mitosis and serves as an integrator of multiple intracel-lular signaling cascades that regulate progres-sion through mitosis [10]. Overexpression of CDC20 has been observed in cancers, which is associated with impairment of the spindle assembly checkpoint, resulting in chromosom-al instability in cancer cells [11, 12].NF-kappaB signaling controls a number of genes involved in immuno-inflammatory re-sponses, cell cycle progression, inhibition of apoptosis and cell adhesion, thus promoting carcinogenesis and cancer progression [13]. RelA, the p65 subunit of NF-kappaB, was found to be up-regulated in hMSCs co-cultured with U251 cells. RelA has diverse functions but it is often associated with conferring resistance to programmed cell death [14]. Aberrant activa-tion of RELA is associated with many human cancers and the anti apoptotic function of RELA has been found to reduce the efficacy of many common cancer therapies [15].The genes STC1 involved in cell surface recep-tor linked signal transduction was found to be up-regulated in hMSCs co-cultured with U251 cells. There is growing evidence that altered STC1 expression patterns may have a role in human cancer and enhanced STC1 gene expression has been found in a number of can-cers [16].Taken together, we found up-regulation of a number of cancer-related genes in hMSCs co-cultured with U251 cells and the up-regulated genes include some important oncogenes, the over-expression of which had been demonstrat-ed to contribute to tumorigenesis. Additional studies will be needed to understand the pos-sible biological significance of these alterations of gene expression. Though no final conclusion could be made yet that tumorigenic potential in hMSCs would be induced under the influence of glioma cells, our findings still propose con-cerns about that, and highlight the need for fur-ther studies on this problem before their clini-cal application as therapeutic vectors to treat human gliomas.AcknowledgementsThis study was supported in part by the grants from: Natural Science Foundation of Guangdong Province, China (2015A030313525); Medical Scientific Research Foundation of Guangdong Province, China (No. B2009197); Starting Research Fund for Scholars with Doctoral Degree from Guangdong Medical University; Research Fund for Scholars with Doctoral Degree from Affiliated Hospital of Guangdong Medical University; Science and Technology Planning Project of Zhanjiang City, Guangdong Province, China.Disclosure of conflict of interestNone.AbbreviationshMSCs, Human bone marrow-derived mesen-chymal stromal cells; FBS, Fetal bovine serum;DMEM, Dulbecco’s modified Eagle’s medium; PBS, Phosphate buffer saline.Address correspondence to: Dr. Rusen Zhu, Depart- ment of Neurosurgery, Affiliated Hospital of Guang-dong Medical University, Guangdong Medical Uni-versity, 57 Renmin Avenue, Zhanjiang, Guangdong Province, China. 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The Final Examination of Immunology (A)Ⅰ. Concepts ( 2.5 marks / concept, totally 20 marks )1. Epitope2. Superantigen3. Positive selection4. Idiotype5. APC6. Hypersensitivity7.Fab8. McAbⅡ. Fill each of the blank spaces with suitable words. (0.5 mark/blank space, totally10 marks)1.Macrophage-derived cytokines include , , ,and .2.Cells with the capacity to induce cytotoxicity of the target cell are, and .plement can be activated directly by microbes throughpathway and pathway at the early stage of infection. MASP1 andMASP2 are involved in the pathway. D factor is involved inthe pathway.4.FcγR is usually found on the surface of , , andcells5．Cytokines like and induce Th1 and inhibit Th2 responses, while and induce Th2 and inhibit Th1 responses.Ⅲ. For each of the following questions, choose the ONE BEST answeror completion. (0.5 mark / question, totally 20 marks)1. PhagocytosisA. Is carried by cells of the adaptive immune systemB. Is restricted to macrophagesC. Is important in bacterial infectionsD. Is a process that dose not involve energyE. Results in division of the cell2. Molecules directly involved in NK cell mediated killing includeA. Muramyl dipeptideB. GranzymeC. ComplementD. IFN-gammaE. Superoxide3. Dendritic cells are characterized byA. The presence of TCRB. Expression of CD3C. Expression of IgM moleculesD. Their ability to release histamineE. Their interface between the innate and adaptive immune systems4. C3bA. Is chemotacticB. Is an anaphylatoxinC. Opsonizes bacteriaD. Directly injures bacteriaE. Is the inactive form of C35. B cell are distinguished from T cells by the presence ofA. CD4B. Surface IgC. CD8D. CD3E. Class I MHC antigen6. Specific antibodies are readily detectable in serum following primarycontact with antigen after:A. 10 minB. 1 hC. 5–7 daysD. 3–5 weeksE. Only following a second contact with antigen7. Protection against microorganisms inside cells is provided by:A. T-cellsB. AntibodyC. C3bD. C1qE. The membrane attack complex8. Activation of the alternative pathway involvesA.C1qB. C1sC. C2D. C3E. C49. The infection in the uterus can be diagnosed based on the elevated levelof antigen specific ___ .A. IgGB. IgMC. IgAD. IgDE. IgE10. Viral replication within cells is inhibited directly byA.IFN-αB.TNF-αC. IL-1D. IL-4E. IL -1311. Which of the following gene clusters do not contribute to antigenbindingA. VLB. CLC. VHD. DE. J12. T cell antigen receptorA. Recognizes conformational epitopes on the native antigenB. Has Ig light chainsC. Is made up of a heavy chain and beta-2 microglobulinD. Are associated with Igαand Igβto form a complexE. Recognize epitopes on linear peptides associated with MHCmolecules13. The class I MHC processing pathway primarilyA. Processes antigens that are present in the cytosol.B. Processes antigen from the extracellular environmentC. Generates peptides, complexes them with class I MHC molecules forpresentation to helper T cellsD. Generates peptides, complexes them with class I MHC molecules forpresentation to NK cellsE. Is involved in the process of the antibody response14. Cytokines responsible for immunosuppression includeA. IL-1B. IL-2C. IFN-γD. TGF-βE. TNF-α15. ___ express CD3 and CD4 moleculesA. Ts and Tc cellsB. Th1 and Th2 cellsC. NK and B cellsD. TH1 and Tc cellsE. All T cells16. Cell with specific killing effects isKB. NKC. NeutrophilsD. CTLE. Mφ17. CD8+ T cellsA. Can be classified into Th1 and Th2 subgroups based on biologicalfunctionsB. Do not produce IFNγC. Can recognize and kill virus infected cellsD. Can bind free virusE. Do not require direct cell to cell contact with their targets for killing18. B cell don’t expressA. CD2B. MHC-ⅠmoleculeC. MHC-ⅡmoleculeD. CR2E. LFA-319. Factors may influence the induction of an immune response includeA. The nature of an antigenB. The route of administrationC. The dose of antigenD. Maturity of the immune systemE. All of the above20. A given Ig isotype isA. A heavy chain variant encoded by allelic genes.B. A light chain constant region encoded by allelic genes.C. Present in all normal individuals.D. A collection of hypervariable region epitopes recognized by ananti-idiotype.E. Monoclonal.21. IgG:A. Appears early in the immune response.B. Kills bacteria directly.C. Is important in mucosal immunity.D. Usually exists as a pentamer.E .Opsonizes bacteria.22. IgA in seromucus secretions:A. Has no J-chain.B. Has no secretory piece.C. Is dimeric.D. Cannot bind to virus.E. Activates the classical complement pathway.23. Which CD molecule can bind with HIV specifically?A.CD2B.CD4C.CD8D.CD21E.CD2824. The secondary, but not the primary, immune response is based onA. MemoryB. The bonus effect of multivalencyC. Complement activationD. Mast cell degranulationE. Clonal selection25. Which of the following is true?A. CD4 — MHC-ⅠB. CD8 — MHC-ⅡC. CTLA-4 —B7D. CD40 — CD58E. CD2 — ICAM-126. The cytokine which is most involved in the class switch to IgEproduction isA. IL-1B. IL-2C. IL-3D. IL-4E. IL-527. The C3 convertase in classical pathway of complement activation isA.C3bBbB.C4b2bC.C4a2bD.C4b2b3bE.C3bnBb28. The molecules mediating signal transduction following antigenbinding to cell surface immunoglobulin on a B-cell are called:A. Ig FcB. Ig-alpha and Ig-betaC. MHCD. CD4E. CD329. A severe depression of CD4+T cell subset in man is likely to result inA.Severe depression of humoral immune responseck of development of skin delayed hypersensitivity reactionsC.Both A and BD.Neither A nor Bck of complement30. Cytokines always actA. By binding to specific receptors.B. In an autocrine fashion.C. At long range.D. Antagonistically with other cytokines.E. Synergistically with other cytokines.31. T cell-derived cytokinesA. Are antigen-specific products of T cell activationB. Are stored in the resting T cell and released on activationC. Are MHC-restricted in their effectsD. Influence the class of antibodies produced by B cellE. All of the above32. In the complement systemA. Alternative pathway relys on antibodyB. Classical pathway is best activated by bacterial endotoxinC. C1 is the first enzyme complex in the classical pathwayD. Both the alternative and classical pathway converge at C2E. The membrane attack complex is made up of C3a and C5a33. Ig with binding site for C1q areA. IgE and IgDB. IgG and IgMC. IgM and IgAD. IgA and Ig GE. IgM and IgE34. Prior to class switching, B-cells expressA. IgA aloneB. IgA and IgGC. IgM and IgDD. IgD aloneE. No surface Ig35. Interactions between _____ are not restricted by MHC moleculesA. Th cell & dendritic cellB. NK cell & target cellC. Tc cell & target cellD. Macrophage & Th cellE. Th cell & B cell36. A major factor regulating the adaptive immune response isA. The granulocyteB. Complement membrane attack complexC. C-reactive proteinD. Antigen concentrationE. Platelet37. Penicillin can causeA. TypeⅠhypersensitivityB. TypeⅠ& ⅡhypersensitivityC. TypeⅠ, Ⅱ&ⅢhypersensitivityD. TypeⅠ, Ⅱ, Ⅲ & Ⅳ hypersensitivityE. TypeⅡ & Ⅲhypersensitivity38. Which of the following is produced by CD4+ Th1 Cells?A. IL-2 receptorB. IL-8C. C3D. IL-4E. IL-1239. Rhesus hemolytic disease of the newborn involvesA. IgE.B. Antibody to cell surfaces.C. Soluble immune complexes.D. Cytokine release from T-cells.E. Stimulatory antibodies.40. InterferonsA. Are found only in mammalian speciesB. Are divided into 5 main familiesC. Inhibit virus replication in the target cellD. Only affect infected cellsE. Are specific for individual virusesⅣ. Answer questions (30 marks)1.Please simply describe the biological functions of antibodies. (3 marks)2.Please simply describe the functions of the important surface moleculesof macrophages. (5 marks)3.Please simply describe the general process of CD4+ T cell mediateimmune response. (6 marks)4.Please describe the structure, distribution and functions of HLA-Ⅱmolecules. (6 marks)5.Please compare the components and mechanisms of type Ⅱand Ⅲhypersensitivity. (10 marks)The Answers for the Final Examination of Immunology(A)Ⅰ. Concepts (2.5 marks / concept, totally 20 marks)1. Epitope: the smallest unit of an antigen to which an antibody or cell can bind.2. Superantigen: molecules that polyclonally activate a large fraction of the T cells by binding to MHC class II molecules and T-cell receptors.3. Positive selection:thymocytes in the cortex of thymus with TCRs with a moderate affinity for self-MHC molecules on epithelial cells are allowed to develop further. Others undergo apoptosis and die. MHC – restriction.4. Idiotype: are unique antigenic determinants associated with antigen binding sites of antibodies and are the results of the different amino acid sequences which determine their specificities.5. APC: is a cell that displays peptide fragment of protein antigens, in association with MHC molecules, on its surface and activatesantigen-specific T cells.6. Hypersensitivity: refer to the tissue damage or functional disturbance that occurs as an overreaction by the immune system to a variety of antigens encountered on a second occasion.7. Fab: fragment antigen binding. Complete L chain paired with the VH and CH1 domains of the H chain8. McAb:monoclonal antibodies which are produced by a single clone of B cell hybridoma and are homogenous in structure and specificity.Ⅱ. Fill each of the blank spaces with suitable words. (0.5 mark/blank space, totally 10 marks)1. TNFα, IL-1, IL-6, IL-8, IL-12 (位置可互换)2. Tc, NK, Macrophage, neurophile，eosinophile，(其中任选三个,位置可互换)3. Alternative, MBL, MBL, Alternative4. NK, Macrophage, neurophile，B cell, eosinophile，pletellete (其中任选四个, 位置可互换)5. IFNγ，IL-12, IL-4, IL-10Ⅲ. For each of the following questions, choose the ONE BEST answer or completion. (0.5 mark / question, totally 20 marks)1. C2. B3. E4. C5. B6. C7. A8. D 9 .B 10. A11. B 12. E 13. A 14. D 15. B16. D 17. C 18. A 19. E 20. C21. E 22. C 23. B 24. A 25.C26. D 27. B 28. B 29. C 30. A31. D 32. C 33. B 34. C 35. B36. D 37. D 38. A 39. B 40. CⅣ. Answer questions (30 marks)1.Please simply describe the biological functions of antibodies. (3 marks) Biological functions of Ab1)○.5Bind specific with antigens.2)○.5complement activation3)○.5Opsonization4)○.5ADCC5)○.5Mediating type І hypersensitivities:6)○.5Placental transfer and mucosal transfer2.Please simply describe the functions of the important surface moleculesof macrophages. (5 marks)(1).○1Molecules involved in phagocytosis: ○1Scavenger receptors, mannose receptors, LPS receptor (CD14), complement receptors, Fc receptors (FcγRⅢ / CD16, et al) （列举三个以上给1分）(2). ○1Molecules involved in signaling of activaton: ○.5TLR-4 (toll-like eceptor-4), TLR-2(3). ○1Molecules involved in antigen presentation: ○.5MHC class Ⅱmolecules and B7 (after activation), MHC class Ⅰmolecules3.Please simply describe the general process of CD4+ T cell mediateimmune response. (6 marks)Antigen recognition of T cell: Class Ⅱ molecules are load with exogenous peptides in an endosomal compartment and are recognized by CD4 helper Tcell○2The activation of T cell: signal 1: the engagement of the TCR; signal 2: B7 on APC bind to CD28 on the T cell○2Effects: Act on Mφand cause their activation; Mediate type Ⅳhypersensitivity; Help B cell growth and Ab production.(答出两种效应即给分)○24. Please discribe the structure, distribution and functions of HLA-Ⅱmolecules. (6 marks)Structure: ○.5heterodimers of α and βglycoprotein chains, ○.5α1 and β1 form the binding groove.Distribution: ○1expressed on APC Function: 1. ○1Antigen processing and presenting (exogeneous pathway): 2. ○1Involve in transplant rejection 3. ○1Regulation of immune response 4. ○1Involve in T cell development in the thymus5. Please compare the components and mechanisms of type Ⅱand Ⅲhypersensitivity. (10 marks)Type Ⅱhypersensitivity Type Ⅲhypersensitivity Ag ○1cellular antigen ○1mostly are soluble Ag Ab ○1IgG , IgM ○1IgG , IgM Mechanism 1. ○1 Cell lysis by complement activation via classical pathway 2. ○1ADCC by NK cell, M φand neutrophiles via Fc γR on these cells 3. ○1Opsonization via CR and Fc γR on phagocytes 1. ○1Complement activation leads to production of C3a 、C5a→activation of basophiles →Tissue edema 2．○1Neutrophile infiltration →tissue damage3．○1Platelet activation →Microthrombi formation。