免疫学概论及分子生物学在免疫学中的应用

1、Fang Li, Guizhi Cheng, Hong Qu, Bing Wang, Jie Ma, Shiying wang Dept. of Immunology College of basic medical sciences Dalian Medical University Tel: 86110294 ,I. Introduction To Immunology II. Application of Molecular Biology in Immunology,CHAPTER I,Introduction To Immunology,Immunity,Immune system,

2、Theory,Application,Immunology,Definition : Immunology is the study of immunity in this broader sense and of the cellular and molecular events that occur after an organism encounters microbes and other foreign macromolecules,The Immune System is the name of a collection of Molecules, Cells and Organs

3、 whose complex interactions form an efficient system that is usually able to protect an individual from both outside invaders and its own altered internal cells.,中枢免疫器官 （Central lymphoid organ）,淋巴细胞发育成熟的场所 骨髓（bone marrow）、 胸腺（thymus）,免疫器官 immune organs,T细胞免疫缺陷,先天性胸腺发育不全 （DiGeorge 综合症）,This is a meta

4、phase（中期） cell that has been hybridized with the probe for DiGeorge which is caused by a microdeletion on chromosome 22. The probe is a dual-color mixture of two seperate probes for chromosome 22. The green signal is an internal control and is located at 22q13. It allows for quick identification of

5、both #22 chromosomes. The red signal is located at the DiGeorge region at 22q11.2. Since both 22s have the red signal in this cell there is not a microdeletion within the DiGeorge region and this patient would not have DiGeorge syndrome.,无胸腺的裸鼠是T细胞选择性缺陷的天然动物模型,Athymic, or nude, mouse (nu/nu: on chro

6、mosome 11 ),This defect leads to absence of the thymus or a vestigial thymus and cell-mediated immunodificiency. Can not easily survive, mortality : 100% within 25 weeks; 50% within 2 weeks after birth.,Skeletal preparations from wild type and Tbx1 mutant embryos. The mutant embryo on the right has

7、abnormalities in the pharyngeal(咽的) arch-derived skeletal structures in the head. This mutant provides a mouse model of the human DiGeorge syndrome,Athymic nude mice are kept behind a barrier with HEPA filtered air; autoclaved fee, bedding, water and cages. A research assistant uses aseptic procedur

8、es to protect these mice from organisms in the environment,Tumors formed by Rat1 cells,肿瘤组织的原位移植（SOI） 取皮下种植生长的前列腺癌肿瘤 1mm1mm的组织碎块 麻醉小鼠 显微外科手术 裸鼠下腹部做一1cm的纵切口， 暴露前列腺 将1块PC-3-GFP组织碎块植入裸鼠前列腺上,20天 25天 30天,CONTROL-3,EXPERIMENT-3,final,p.s：桔黄色空心箭头所指为转移的腰淋巴结； 桔黄色实心箭头所指为原发肿瘤； 白色空心箭头所指为肠系膜淋巴结转移； 白色实心心箭头所指为胰腺转移

9、,CONTROL-3,EXPERIMENT-3,final,外周免疫器官 （Peripheral lymphoid organ）,淋巴细胞定居和发挥功能的场所 脾（spleen）、淋巴结（lymphoid nodes） 黏膜相关淋巴组织（MALT）,单核巨噬细胞,（monocytes and macrophages）,免疫细胞 immune cells,抗体 （antibody）,主要组织相容性复合体 （MHC）,T 细胞受体TCR,免疫分子 immune molecules,1,2,3,4,5,免疫记忆 疫苗与抗感染免疫,免疫记忆,（Immunological memory）,经历了某种微生

10、物感染之后短时间内一般不会再次被同一种微生物感染。这种现象称为免疫记忆。,记忆细胞,Immunological memory makes vaccination against infectious diseases possible!,疫苗（VACCINE）,Mycobacterium tuberculosis.,脊髓灰质炎 Polio,狂犬病Rabies,天花Smallpox virus,白喉 diphtheria,Hepatitis A,Hepatitis B,接种疫苗后21天内的反映情况,免疫记忆 Immunologic memory,A B 白喉vaccine D E,Anti-白喉

11、antibody against白喉,免疫缺陷 肿瘤,变态反应(超敏反应) 自身免疫性病 移植免疫,临床免疫及免疫病理 ,Hypersensitivity 超敏反应,Immunodeficiency 免疫缺陷,Autoimmune diseases 自身免疫病,Cancer of the immune system 免疫系统肿瘤,Tumor Immunology 肿瘤免疫,I Hypersensitivity 超敏反应,Eczema 湿疹,An atopic eczema reaction on the back of a knee of a child allergic to rice an

12、d eggs,Asthma,DC, Th2, B, Mast cell Epithelium, Eos IL-4, IgE,Allergen,Serum sickness,Patients with hypersensitivity vasculitis with typical skin rash.,T、B 细胞联合免疫缺陷 Severe Combined Immunodeficiency，SCID,II Immunodeficiency 免疫缺陷,HIV 感染淋巴细胞,Immunodeficiency 免疫缺陷,HIV budding from human lymph tissue (TE

13、M x133,335),Immunodeficiency 免疫缺陷,Lesions on the stomach of a patient with Kaposis sarcoma,Figure - Kaposis Sarcoma,Kaposis sarcoma (skin).,Skin showing AIDS-associated Kaposis sarcoma,Immunodeficiency 免疫缺陷,Hairy leukoplakia （黏膜白斑） of tongue in AIDS,Oral thrush鹅口疮.,Candida and herpes simplex in AIDS

14、,Severe angular cheilitis in AIDS 口角炎,Immunodeficiency 免疫缺陷,RA 患者手指关节实质性损伤,III Autoimmune diseases 自身免疫病,SLE 患 者 脸 部 的 蝴 蝶 斑,Autoimmune diseases 自身免疫病,Systemic lupus erythematosis 系统性红斑狼疮,(Ach: acetylcholine) 乙酰胆碱,ankylosing spondylitis (B27),强直性脊柱炎,Destruction of pancreatic islet -cells by infiltra

15、ting T-cells in the Nonobese diabetic (NOD) mouse,Application of Molecular Biology in Immunology,MOLECULAR BIOLOY,Seek to explain the relationships between the structure and function of biological molecules and how these relationships contribute to the operation and control of biochemical processes.

16、,Practical applications in the development of new and safe products such as therapeutics, vaccines and foodstuffs, and in the diagnosis of genetic disease and in gene therapy,Modern molecular biology:,一、新细胞因子的cDNA克隆技术,Protein FMVL.,传统的蛋白质到核酸途径,差异显示技术克隆细胞因子,利用同一细胞在不同条件下表达免疫分子的差异克隆新细胞因子，例如T细胞活化前低表达细胞因

17、子，活化后则高表达细胞因子，利用DD-PCR、RDA、SSH等技术可克隆T细胞活化后高表达的基因，其中可能含有新细胞因子cDNA.,现代的核酸到蛋白质途径,Interleukin-10. Chain: l. ( 150 residues ) - 9 helices Cytokine synthesis inhibitory factor. Interleukin-10 receptor alpha chain. Chain: r Fragment. ( 205 residues ) - 2 helices, 12 strands : extracellular domain,(-),IL-10

18、,(+),PHA,Expressing,mRNA,cDNA,Revere transcript,回收片段，克隆化,PHA刺激的U937细胞 IL-10抑制的U937细胞 PHA刺激的U937细胞 Tester cDNA with adaptor-1 Driver cDNA (超量) Tester cDNA with adaptor-2,Suppression Subtractive Hybridization,SSH 示意图,GTTCCCAATCTGAAGTGAAGCCGAGCTGGGCGAGAAGTAGGGGAGGGCGGTGCTCCGCCG 60 CGGTGGCGGTTGCTATCGCTT

19、CGCAGAACCTACTCAGGCAGCCAGCTGAGAAGAGTTGAG 120 GGAAAGTGCTGCTGCTGGGTCTGCAGACGCGATGGATAACGTGCAGCCGAAAATAAAACA 180 M D N V Q P K I K H TCGCCCCTTCTGCTTCAGTGTGAAAGGCCACGTGAAGATGCTGCGGCTGGATATTATCAA 240 R P F C F S V K G H V K M L R L D I I N CTCACTGGTAACAACAGTATTCATGCTCATCGTATCTGTGTTGGCACTGATACCAGAAAC 300 S

20、 L V T T V F M L I V S V L A L I P E T CACAACATTGACAGTTGGTGGAGGGGTGTTTGCACTTGTGACAGCAGTATGCTGTCTTGC 360 T T L T V G G G V F A L V T A V C C L A CGACGGGGCCCTTATTTACCGGAAGCTTCTGTTCAATCCCAGCGGTCCTTACCAGAAAAA 420 D G A L I Y R K L L F N P S G P Y Q K K GCCTGTGCATGAAAAAAAAGAAGTTTTGTAATTTTATATTACTTTTTAGTT

21、TGATACTAA 480 P V H E K K E V L - GTATTAAACATATTTCTGTATTCTTCCAAAAAAAAAAAAAAAAAAAAAAAAAAA 534 CKLF1的完整cDNA序列和编码的蛋白质序列 (GenBank AF096895),CKLF1独特的功能 广谱的体内、外趋化活性 对造血祖/干细胞的促增殖作用 对骨骼肌细胞的促增殖分化作用,新细胞因子CKLF1（Chemokine-like factor 1),( Biochem J 2001, 357, 127135 ),A,B,小鼠骨骼肌基因注射10天后注射部位组织学图像（HE 132） A pcDI组；

22、B pcDI-CKLF1组,A,D,C,B,pCDI-hCKLF1 裸 基 因 注 射 小 鼠 肺 部 病 变,A: 对照；B: 2周； C：3周；D：4周,CKLF1的病理作用,CKLF1特殊的组织分布,成人组织低表达或不表达 胚胎组织和肿瘤组织高表达，变异体形式多 CKLF1 在肿瘤组织中的特征性分布预示着该基因在肿瘤发病中可能起重要作用,二、基因突变 MUTAGENESIS OF CLONED GENES,Delection mutagenesis Site-directed mutagenesis PCR mutagenesis,利用点突变技术（ Site-directed mutag

23、enesis ）定向改变某（个）些氨基酸，以产生具有不同生物学活性的新的突变体。,Site-directed mutagenesis,Changing one or a few nucleotides at a particular site Annealing a mutagenic primer to a template followed by complementary strand synthesis by a DNA polymerase. (PCR),IL-8 为重要的炎症介质 (急性炎症反应,类风湿性关节炎),趋化因子介导的炎症是机体对外来刺激的一种病理性免疫反应，与若干重大临

24、床病患的产生和发展密切相关(炎症、自身免疫性疾病等)。,The role of IL-8 signaling in the tumor microenvironment. Tumor-derived IL-8 has the capacity to exert profound effects on the tumor microenvironment. For example, secretion of IL-8 from cancer cells can enhance the proliferation and survival of cancer cells through autoc

25、rine signaling pathways. In addition, tumor-derived IL-8 will activate endothelial cells in the tumor vasculature to promote angiogenesis and induce a hemotactic infiltration of neutrophils into the tumor site. Although IL-8 can promote cell invasion and migration, the capacity of IL-8 to induce tum

26、or-associated macrophages to secrete additional growth factors will further increase the rate of cell proliferation and cancer cell invasion at the tumor site.,IL-8-CXC家族趋化因子, 最重要的中性粒细胞趋化因子，以高亲和力结合受体CXCR1/CXCR2，介导中性粒细胞引起的炎症反应。,endogenic/ectogenicsstimulation,Mast cell,neutrophil,Acute inflammation,m

27、onocyte,IL-8 R,IL-8,Chain: A ( 71 residues ) - K&S: 1 helix, 3 strands Chain: B ( 71 residues ) - K&S: 1 helix, 3 strands Chain: C ( 19 residues ),C,SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS,IL-8,1 2 3 4 5 6 7 8 9 10111213141516171819 3132,GP,(3132),目的: 筛选出高受体亲和力而无生物活性

28、的突变体,利用点突变技术（ Site-directed mutagenesis ）定向改变某（个）些氨基酸，以产生具有不同生物学活性的新的突变体。,作为CXCR1/CXCR2 受体拮抗剂,Site-directed mutagenesis,Changing one or a few nucleotides at a particular site Annealing a mutagenic primer to a template followed by complementary strand synthesis by a DNA polymerase. (PCR),Genetic code

29、,Second position,First position (5 end),Third position (3 end),U C A G,U C A G,U C A G U C A G U C A G U C A G,Phe UUU Ser UCU Tyr UAU Cys UGU Phe UUC Ser UCC Tyr UAC Cys UGC Leu UUA Ser UCA Stop UAA Stop UGA Leu UUG Ser UCG Stop UAG Trp UGG Leu CUU Pro CCU His CAU Arg CGU Leu CUC Pro CCC His CAU Ar

30、g CGC Leu CUA Pro CCA Gln CAA Arg CGA Leu CUG Pro CCG Gln CAG Arg CGG IIe AUA Thr ACU Asn AAU Ser AGU IIe AUC Thr ACC Asn AAC Ser AGC IIe AUG Thr ACA Lys AAA Arg AGA Met AUG Thr ACG Lys AAA Arg AGG Val GUU Ala GCU Asp GAU Gly GGU Val GUC Ala GCC Asp GAC Gly GGC Val GUA Ala GCA Glu GAA Gly GGA Val GU

31、G Ala GCG Glu GAG Gly GGG,SAKELRCQCIKTYSKPFHPKFIKELRVIESGP,1 2 3 4 5 6 7 8 9 10111213 14 15 16 171819 3132,agt gct aaa gaa ctt aga tgt cag tgc ata aag aca tac tcc aaa cct ttc cac ccc aaa ttt atc aaa gaa ctg aga gtg att gag agt gga cca,11K,11R (精氨酸),AAG,CGT CGC CGA CGG AGA AGG,赖氨酸 苏氨酸 酪氨酸,甘氨酸 脯氨酸,5-c

32、tt aga tgt cag tgc ata AAG aca tac tcc aaa cct ttc cac-3,5-ctt aga tgt cag tgc ata AGG aca tac tcc aaa cct ttc cac-3,cDNA,About 40 bp G+C% (Tm) mutant site in the middle of the primer,DNA polymerase,dNTP,95C,62C,72C,95C,62C,72C,30 cycles,DpnI,DpnI 5-GmA TC-3 3-C T mAG-5,Methylase甲基化酶: adds a methyl

33、group to a C (dcm) or A (dam) base within the same recognition sequences in the cellular DNA. dam add a methyl group to As N6 in “GATC” sequence.,Nick,A-T-T-G-A-A-T-G-T-C-A-T-C-C-A-T-G,5,3,5,3,T-A-C-C-G-G-T-A-T-A-C-C-G-G-A-A-T,5,3,Agarose gel electrophoresis,23.1 9.4,6.6,4.4,2.3 2.0,0.56,M,M,1 2,M:

34、marker 1.parent DNA 2.mutant DNA,Transformation,Plate,Isolation of DNA,Sequencing,Expression of mutant protein,Assaying for function,SAKELRCQCIKTYSKPFHPKFIKELRVIESGP,1 2 3 4 5 6 7 8 9 10111213 14 15 16 171819 3132,agt gct aaa gaa ctt aga tgt cag tgc ata aag aca tac tcc aaa cct ttc cac ccc aaa ttt at

35、c aaa gaa ctg aga gtg att gag agt gga cca,11K,11R (精氨酸),AAG,CGT CGC CGA CGG AGA AGG,赖氨酸 苏氨酸 酪氨酸,甘氨酸 脯氨酸,5-ctt aga tgt cag tgc ata AAG aca tac tcc aaa cct ttc cac-3,5-ctt aga tgt cag tgc ata AGG aca tac tcc aaa cct ttc cac-3,111213 3132,IL-8-K11R （赖氨酸/精氨酸）,IL-8-T12S （苏氨酸/丝氨酸）,IL-8-Y13F （酪氨酸/苯丙氨酸）,IL-

36、811R12S13F,IL-8-K11R / G31P （甘氨酸/脯氨酸）,IL-8-K11R / P32G,IL-8-K11R / 31P32G,IL-8-K11R / G31P: Highest binding lowest activity As a receptor antagonist,Mutants-,三、基因免疫 (gene immunization),DNA Vaccines,The Third Vaccine Revolution,Plasmid vaccine,DNA疫苗是有目的的将DNA质粒（plasmid）导入人体（vaccinee ）。 质粒携有编码蛋白的基因，体内低

37、效率感染细胞，表达抗原，引起免疫反应。 *DNA-mediated or DNA-based immunization ，因为目的不是产生对DNA分子本身的抗体，而是得到由受者细胞表达的蛋白。,Plasmid vaccine,Gene for antigenic protein,antigenic protein,antigenic peptides,Antigen binding causes cytotoxic T cells to multiply and attack inoculated cells,Memory T cell,Activated cytotoxic T cell,Antigen binding triggers release of antibodies,Memory B cell,Activate