免疫治疗PPT精品课件

1、免疫治疗ImmunotherapyGene therapy can be broadly defined as the transfer of genetic material into a cell to transiently (短暂) or permanently(永久) alter the cellular phenotype(表型). 第一节 基因治疗 (gene therapy)分子外科The idea is simple; the practice is not.As you might guess from the low success rates of vector tra

2、nsfection, this is a dicey procedure, and the techniques are still crude, at best. Germ Line Therapy生殖细胞治疗Somatic Gene Therapy体细胞治疗Introduction of a nucleic acid or target gene (transgene) directly into cells is referred to as transfection(转染). transduction (转导) refers to the introduction of a trans

3、gene into a cell through a viral vector system. *Gene transfer can be performed by transfection or transduction of target cells in vitro and then administration of the modified cells (修饰的细胞) to an animal or patient. *In vivo gene transfer is accomplished by direct transfection or transduction of tar

4、get cells in the patient. Strategies for Delivering Therapeutic Transgenes into Patients(a) target cells are removed from the patient, (b) target cells are selected for, (c) transduced with a recombinant retrovirus coding the gene of interest (d) expanded to obtain a therapeutically useful number of

5、 transduced number of cells, and (e) transplanted to the patient.Schematic of ex-vivo gene therapy protocolADA(腺苷脱氨酶）基因突变（常染色体隐性遗传）引起免疫缺陷AMPIMP肌苷腺苷ADA腺苷 脱氧腺苷dAMPdADPdATP抑制核苷酸还原酶，阻碍DNA合成T、B 细胞增殖障碍腺苷脱氨酶 (adenosine deaminase ) ADA 重度联合免疫缺陷症（Severe Combined ImmunoDeficiency ） The first case for gene the

6、rapy in the world is SCIDOKT3抗体,IL-2ADAADAADAmonocyteT cell从1990年转移ADA基因到现在的大部分基因治疗临床试验都是先从病人体内获得某种细胞（例如T淋巴细胞），进行培养，在体外完成基因转移后，筛选成功转移的细胞扩增培养，然后重新输入患者体内。这种效果较为可靠，称其为体外（ex vivo）基因治疗。 受治患儿为4岁女孩，于1990年9月14日开始接受白细胞透入，用梯度分离患儿离体血细胞得到单核细胞，培养这些细胞并刺激T淋巴细胞分化，与带正常ADA基因的载体共培养数日，然后将T细胞输回患儿体内。受治患儿在其后的10个半月中共接受7次自体

7、细胞输注，患儿免疫功能增强，临床症状改善。ADA基因校正的T细胞相当于正常人的20-25，其后六个半月未接受基因治疗，然后三至五个月接受一次基因治疗。1991年1月起第二例患儿（9岁女孩）接受同样基因治疗并取得类似疗效。两例患儿在接受基因治疗奏效后较少感染，从密闭环境转人正常人生活，并均已入学。 1994年美国科学家利用经过修饰的腺病毒为载体，成功地将治疗遗传性囊性纤维化病的正常基因cfdr 转入患者肺组织中。这种直接往人体组织细胞中转移基因的治病方法叫做体内（in vivo）基因治疗。 cfdrA new gene is injected into an adenovirus vector,

8、 which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein. Gene therapeutic vectors which transfer a cytotoxic mechanism to tumor cells offer a new route of cancer therapy. （四）基因传递和基因导向技术 inherent advantages (优势): 1 in

9、tegrating (整合) the therapeutic gene into the chromosomal DNA of a target cell,2 deliver the therapeutic gene to large numbers of target cells. 将病毒基因组中与致病相关的基因去掉，保留其携带基因组进入人体细胞的功能，再组装上理想的外源基因，即成为一种病毒载体. 病毒侵染人体细胞时能将自身的基因组携带到细胞内，并利用人体细胞内物质完成自身繁殖，最终导致人类疾病。 腺病毒能感染多种静止期细胞，尤其是呼吸道上皮细胞，其基因携带量大，腺病毒并不插入受体细胞的基因

10、组，以染色体外形式长期存在和表达 目前研究使用的均为缺陷型Ad基因（腺病毒相关病毒）治疗载体，它单独不能复制和增值，但其基因转录可以相当活跃，在适当的结构下，外源基因可以在Ad不繁殖的情况下获得持续高效表达，从而为使用这类载体进行人类基因治疗开辟了新的研究领域。 In the 1990s, late researchers tested a gene therapy treatment that would restore the function of a crucial gene, gamma c, to cells of the immune system. This treatment

11、 appeared very successful, restoring immune function to most of the children who received it.1999,9,17，18岁(8,16)美国青年Jesse Gelsinger:鸟氨酸氨甲酰基转移酶（OTC）(-)遗传性疾病,而在美国宾夕法尼亚州大学人类基因治疗中心接受基因治疗时不幸死亡，成为被报道的首例死于基因治疗中的患者。(3.8X1013)但只有1%的病毒到达靶器官肝脏，而绝大部分的病毒进入其它器官与组织，从而引发了强烈的系统性炎症反应。Procedure of gene therapyGene Ach

12、ievedPCR, Cloned, synthesized, genomic digestionHost cell cultureLymphocyte, endodermis, liver cell, tumor celltransfectionMicroinjection, direct injection electroporationgene gun,Selection identification Marker(neoR), gene defective typeHybridizationexpression expressionIn animalIn cellIn vitroin v

13、ivoIL-1ra gene therapy on DBA/1 mice of collagen-induced arthritis cDNA librariesPCRTTTTTTTTAA+sequencingIL-1ra geneligateIL-1ra: IL-1 receptor antagonistAT clone system (Invitrogen Co.)AT clone vectorpcDI-IL-1ratransfectioncynoviocyteCOS-7in vitroEXPRESSIONIL-1ra proteinELISAmRNApcDI-IL-1rain vivo

14、(gene therapy)muscle injection.DBA/1 micetype II collageninducedDBA/1 mice with arthritis gene gunAdenovirus Disadvantages(缺点)1 免疫原性2 受体的广泛分布卡波西肉瘤叶酸盐受体黑色素瘤( - MHSR)-黑色素细胞刺激激素受体*过度表达抗病毒(anti- Adenovirus) 中和抗体 叶酸盐Fab-S-S- -S-S-卡波西肉瘤叶酸盐受体vector基因导向TNF/HSV-tk抗病毒(anti- Adenovirus) 中和抗体 Fab-S-S- -黑色素细胞刺激激

15、素-S-S-vector( - MHSR)-黑色素细胞刺激激素受体黑色素瘤TNF/HSV-tk一、癌症的基因治疗（一）化学基因疗法化学疗法020406080100化疗出现前后肿瘤治疗水平的变化化疗出现前加化疗后骨肉瘤转移性胚胎睾丸癌弥散性组织细胞淋巴瘤晚期何杰金氏病横纹肌肉瘤蕈样霉菌病急性淋巴性白血病视网膜母细胞瘤巴基特淋巴瘤尤文氏瘤绒癌 肾母细胞瘤有效率 化疗药是肿瘤治疗史上的不朽里程碑0102030405060708090骨肉瘤乳腺癌上颌窦癌肾母细胞瘤几种肿瘤治疗效果的今昔（五年生存率）过去治疗生存率（）现在治疗生存率（）生存率MDR的分子机制肿瘤的MDR（multiple drug re

16、sistance）自杀基因 (suicide gene) : 编码对肿瘤细胞有害的酶类(+相应的原药 )单纯疱疹病毒胸苷激酶（herpes simplex thymidine kinase, HSV-tk）基因TNF:能下调多种耐药性基因（mdr）的表达，从而使肿瘤细胞发生化学致敏作用（chemosensitization）。这一作用作为癌症的基因治疗和化学治疗联合疗法的基础。(1) suicide gene therapy : Enzyme/Pro-drug Therapy: delivery to tumor cells of genes encoding pro-drug convert

17、ing enzymes and then treatment with systemic administration of the respective nontoxic pro-drug.Prodrug (nontoxic) expressiongene. HSV-tk (单纯疱疹病毒胸苷激酶) gene:Coding herpes simplex thymidine kinase the anti-herpetic nucleoside analogues ganciclovir acyclovir bromovinyl-deoxyuridinePhosphorylates磷酸化HSV-

18、tkMonophosphates (一磷酸)ONNNN61OHCH2HOOHHOOHCNH2CCOHHOCH3ganciclovir 9-(1,3-二羟-2-丙氧甲基)鸟苷(ganciclovir,GCV)NH2ONNNNGuanine(G)61herpes simplex thymidine kinase, HSV-tk9-(1,3-二羟-2-丙氧甲基)鸟苷(ganciclovir,GCV)ONNNN61OHCH2HOOH2HOOHCNH2CCOHHOCH3ONNN61OHCH2HOOH2OPOOONH2NOHCCCOHHOCH3OPOOOPOO在细胞内转换成三磷酸形式抑制DNA的合成ONN

19、N61OHCH2HOOH2OPOOONH2NOHCCCOHHOCH3 (dGTP)HOHCH2OOPOOOPOOOPOOOO2NH2NNNNTNF gene :Downregulation(下调) of MDR gene expressionTumor cell chmosensitization As the base: combining gene therapy with chemotherapyTNF/HSV-tkTNF/ HSV-tkExpression of TNF / HSV-tkTumor celltransfectionInjection of GCV（二）免疫基因治疗 对细

20、胞裂解性T细胞(CTL)的直接活化或通过其活化调节物的间接活化是很多基因治疗的研究重心。肿瘤浸润淋巴细胞（TIL）、肿瘤坏死因子（TNF）与白细胞介素-2（IL-2）及其它细胞生长因子。在动物实验中TNF对于小鼠是强力抗肿瘤剂，鼠可耐受400g/kg体重, TNF用于临床，当剂量达到8g/kg体重时即产生明显的副作用。在局部产生高浓度TNF以杀伤肿瘤而不带来严重的副作用。 采用未修饰的TIL细胞治疗肿瘤，TIL细胞有专门攻击肿瘤的特性，导入TNF基因的TIL细胞可望在肿瘤组织中安家，在局部产生高浓度TNF以杀伤肿瘤而不带来严重的副作用。大剂量TNF注射造成恶液质的小鼠模型 在动物实验中TNF对

21、于小鼠是强力抗肿瘤剂，鼠可耐受400g/kg体重, TNF用于临床，当剂量达到8g/kg体重时即产生明显的副作用。TILAttack tumor specific Tumor cellTILTNFtransfectionTNFHoming in tumorTNFTNFProducing Tumor infiltrating lymphocyte（三）血管生成的抑制AngiogenesisVEGF promotes Tumor Angiogenesis肿瘤细胞内皮细胞大鼠和大鼠模型肿瘤系统中已获得成功突变信号传导缺陷性VEGF受体基因转染内皮细胞抑制肿瘤的血管形成二、自身免疫病的基因治疗自身免疫

22、性胰岛损伤I-型糖尿病患者血清中的自身抗体SLE 患者血清中的抗中心粒抗体免疫复合物在 SLE 患者皮下沉积Models of autoimmune diseasesThe experimental models :experimental auto-allergic encephalitis (脑炎), experimental thyroiditis (甲状腺炎) adjuvant induced arthritis (关节炎)Experimental antoimmune encephalomyelitis (脑脊髓膜炎) (EAE)Naturally occurring models

23、:hemolytic anemia in NZB mice systemic lupus erythematosus(狼疮)in NZB/NZW (BW), BXSB and MRL mice diabetes in obese mice AUTOIMMUNE THYROID DISEASE: NEW MODELS OF CELL DEATH IN AUTOIMMUNITYTh1Th2Th0CD4+IFN- 、IL-2TNF IL-4IL-10 促炎症细胞因子 抗炎细胞因子 （一）靶组织基因治疗1 Rheumatoid arthritis (RA)Rheumatoid factor(+)IgM

24、-positive plasma cellsPatients synovial tissueLarge number of cells stained by anti-HLA-DRClass II-positive accessory cell (green)in contact with CD4+T-cellLarge rheumatoid nodule on the forearmGranulomatosis ( Epithelioid cells, Macrophages, Scattered lymphocytes, Plasma cells)Chronic RA (Classical

25、 swan-neck deformities)Cytokines emerge from B and T cells initiating tissue destruction in rheumatoid arthritis.IL-1, TNF-, IL-6, GM-CSF抗炎cytokines : IL-10, TGF-, IL-1ra and TNF-R. 抗炎机制不能有效地下调类风湿关节炎的炎症过程，类风湿关节炎是细胞因子间平衡失调的结果。治疗策略1 soluble receptors2 anti-TNF antibody3 anti-receptor antibody4 recepto

26、r antagonistpcDI-IL-1rain vivo (gene therapy)muscle injection.DBA/1 micetype II collageninducedDBA/1 mice with arthritis gene gunIL-1ra gene therapy on DBA/1 mice of collagen-induced arthritis 22. Diabetes The NOD/Ba colonyThe NOD/Ba colony was established in 1987. There is a stable cumulative incid

27、ence of diabetes in the colony of approximately 55% in females and 15% in males at 30 weeks of age. Destruction of pancreatic islet -cells by infiltrating T-cells in the Nonobese diabetic (NOD) mouseInsulin stained by rhodamine-conjugated antibodiesT cells by fluoresceinated anti-CD3Transgenic NOD m

28、ouse (IL-4)胰岛细胞表达IL-4不发生胰岛炎和以后发生的糖尿病（二）T细胞介导基因治疗MS: multiple sclerosis MS 患者中枢神经系统的实质性损伤 MS 中枢神经系统的组织损伤病灶Experimental antoimmune encephalomyelitis (EAE), a demyelinating model for multiple sclerosisEarly lesion: infiltration of lymphocytes and monocytes Large demyelinating lesionsLarge demyelinated

29、plaquesAcute EAE in cat with optic nerve involvement. Experimental antoimmune encephalomyelitis (EAE), a demyelinating model for multiple sclerosis induced by immunizatin with brain antigens in complete freunds adjuvant (CFA)Early lesion of EAE in the rat at 9 days after immununization with rat spin

30、al cord homogenate in CFA. The lesion in brain white matter, which is probably a few hours old, shows perivenous infiltration of lymphocytes and monocytes (a pure mononuclear inflammation) with cells invading the nervous parenchyma. Myelin is not stained.Lumbar spinal cord of rat with chromic EAE af

31、ter immunization with myelin proteolipid protein. Large demyelinating lesions in dorsal columns, in both left (large) and right (small) columns, as well as on lower left. Also gray matter involved with ongoing inflammation, in particular affecting left dorsal horn. Normal myelin is stained brown.Chr

32、onic relapsing EAE in guinea-pig. Large demyelinated plaques in brain white matter (arrows) closely similar to plaques of multiple sclerosis. Acute EAE in cat with optic nerve involvement. EAETh1MS: multiple sclerosisTh2注入Th2细胞 EAETh1IL-10TransfectionTh1PLP抑制EAE T细胞基因修饰 第二节 抗体的免疫学治疗一、单克隆抗体二、双向特异性抗体的免疫治疗作用TUMORCELL-S-S-Fab免疫毒素( 植物和