免疫治療PPT精品課件

1、免疫治療ImmunotherapyGene therapy can be broadly defined as the transfer of genetic material into a cell to transiently (短暫) or permanently(永久) alter the cellular phenotype(表型). 第一節(jié) 基因治療 (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生殖細胞治療Somatic Gene Therapy體細胞治療Introduction of a nucleic acid or target gene (transgene) directly into cells is referred to as transfection(轉(zhuǎn)染). transduction (轉(zhuǎn)導(dǎo)) 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 (修飾的細胞) 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(腺苷脫氨酶）基因突變（常染色體隱性遺傳）引起免疫缺陷AMPIMP肌苷腺苷ADA腺苷 脫氧腺苷dAMPdADPdATP抑制核苷酸還原酶，阻礙DNA合成T、B 細胞增殖障礙腺苷脫氨酶 (adenosine deaminase ) ADA 重度聯(lián)合免疫缺陷癥（Severe Combined ImmunoDeficiency ） The first case for gene the

6、rapy in the world is SCIDOKT3抗體,IL-2ADAADAADAmonocyteT cell從1990年轉(zhuǎn)移ADA基因到現(xiàn)在的大部分基因治療臨床試驗都是先從病人體內(nèi)獲得某種細胞（例如T淋巴細胞），進行培養(yǎng)，在體外完成基因轉(zhuǎn)移后，篩選成功轉(zhuǎn)移的細胞擴增培養(yǎng)，然后重新輸入患者體內(nèi)。這種效果較為可靠，稱其為體外（ex vivo）基因治療。 受治患兒為4歲女孩，于1990年9月14日開始接受白細胞透入，用梯度分離患兒離體血細胞得到單核細胞，培養(yǎng)這些細胞并刺激T淋巴細胞分化，與帶正常ADA基因的載體共培養(yǎng)數(shù)日，然后將T細胞輸回患兒體內(nèi)。受治患兒在其后的10個半月中共接受7次自體

7、細胞輸注，患兒免疫功能增強，臨床癥狀改善。ADA基因校正的T細胞相當(dāng)于正常人的20-25，其后六個半月未接受基因治療，然后三至五個月接受一次基因治療。1991年1月起第二例患兒（9歲女孩）接受同樣基因治療并取得類似療效。兩例患兒在接受基因治療奏效后較少感染，從密閉環(huán)境轉(zhuǎn)人正常人生活，并均已入學(xué)。 1994年美國科學(xué)家利用經(jīng)過修飾的腺病毒為載體，成功地將治療遺傳性囊性纖維化病的正?；騝fdr 轉(zhuǎn)入患者肺組織中。這種直接往人體組織細胞中轉(zhuǎn)移基因的治病方法叫做體內(nèi)（in vivo）基因治療。 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）基因傳遞和基因?qū)蚣夹g(shù) inherent advantages (優(yōu)勢): 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. 將病毒基因組中與致病相關(guān)的基因去掉，保留其攜帶基因組進入人體細胞的功能，再組裝上理想的外源基因，即成為一種病毒載體. 病毒侵染人體細胞時能將自身的基因組攜帶到細胞內(nèi)，并利用人體細胞內(nèi)物質(zhì)完成自身繁殖，最終導(dǎo)致人類疾病。 腺病毒能感染多種靜止期細胞，尤其是呼吸道上皮細胞，其基因攜帶量大，腺病毒并不插入受體細胞的基因

10、組，以染色體外形式長期存在和表達 目前研究使用的均為缺陷型Ad基因（腺病毒相關(guān)病毒）治療載體，它單獨不能復(fù)制和增值，但其基因轉(zhuǎn)錄可以相當(dāng)活躍，在適當(dāng)?shù)慕Y(jié)構(gòu)下，外源基因可以在Ad不繁殖的情況下獲得持續(xù)高效表達，從而為使用這類載體進行人類基因治療開辟了新的研究領(lǐng)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歲(8,16)美國青年Jesse Gelsinger:鳥氨酸氨甲酰基轉(zhuǎn)移酶（OTC）(-)遺傳性疾病,而在美國賓夕法尼亞州大學(xué)人類基因治療中心接受基因治療時不幸死亡，成為被報道的首例死于基因治療中的患者。(3.8X1013)但只有1%的病毒到達靶器官肝臟，而絕大部分的病毒進入其它器官與組織，從而引發(fā)了強烈的系統(tǒng)性炎癥反應(yīng)。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(缺點)1 免疫原性2 受體的廣泛分布卡波西肉瘤葉酸鹽受體黑色素瘤( - MHSR)-黑色素細胞刺激激素受體*過度表達抗病毒(anti- Adenovirus) 中和抗體 葉酸鹽Fab-S-S- -S-S-卡波西肉瘤葉酸鹽受體vector基因?qū)騎NF/HSV-tk抗病毒(anti- Adenovirus) 中和抗體 Fab-S-S- -黑色素細胞刺激激

15、素-S-S-vector( - MHSR)-黑色素細胞刺激激素受體黑色素瘤TNF/HSV-tk一、癌癥的基因治療（一）化學(xué)基因療法化學(xué)療法020406080100化療出現(xiàn)前后腫瘤治療水平的變化化療出現(xiàn)前加化療后骨肉瘤轉(zhuǎn)移性胚胎睪丸癌彌散性組織細胞淋巴瘤晚期何杰金氏病橫紋肌肉瘤蕈樣霉菌病急性淋巴性白血病視網(wǎng)膜母細胞瘤巴基特淋巴瘤尤文氏瘤絨癌 腎母細胞瘤有效率 化療藥是腫瘤治療史上的不朽里程碑0102030405060708090骨肉瘤乳腺癌上頜竇癌腎母細胞瘤幾種腫瘤治療效果的今昔（五年生存率）過去治療生存率（）現(xiàn)在治療生存率（）生存率MDR的分子機制腫瘤的MDR（multiple drug re

16、sistance）自殺基因 (suicide gene) : 編碼對腫瘤細胞有害的酶類(+相應(yīng)的原藥 )單純皰疹病毒胸苷激酶（herpes simplex thymidine kinase, HSV-tk）基因TNF:能下調(diào)多種耐藥性基因（mdr）的表達，從而使腫瘤細胞發(fā)生化學(xué)致敏作用（chemosensitization）。這一作用作為癌癥的基因治療和化學(xué)治療聯(lián)合療法的基礎(chǔ)。(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 (單純皰疹病毒胸苷激酶) 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-(1,3-二羥-2-丙氧甲基)鳥苷(ganciclovir,GCV)NH2ONNNNGuanine(G)61herpes simplex thymidine kinase, HSV-tk9-(1,3-二羥-2-丙氧甲基)鳥苷(ganciclovir,GCV)ONNNN61OHCH2HOOH2HOOHCNH2CCOHHOCH3ONNN61OHCH2HOOH2OPOOONH2NOHCCCOHHOCH3OPOOOPOO在細胞內(nèi)轉(zhuǎn)換成三磷酸形式抑制DNA的合成ONN

19、N61OHCH2HOOH2OPOOONH2NOHCCCOHHOCH3 (dGTP)HOHCH2OOPOOOPOOOPOOOO2NH2NNNNTNF gene :Downregulation(下調(diào)) 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）免疫基因治療 對細

20、胞裂解性T細胞(CTL)的直接活化或通過其活化調(diào)節(jié)物的間接活化是很多基因治療的研究重心。腫瘤浸潤淋巴細胞（TIL）、腫瘤壞死因子（TNF）與白細胞介素-2（IL-2）及其它細胞生長因子。在動物實驗中TNF對于小鼠是強力抗腫瘤劑，鼠可耐受400g/kg體重, TNF用于臨床，當(dāng)劑量達到8g/kg體重時即產(chǎn)生明顯的副作用。在局部產(chǎn)生高濃度TNF以殺傷腫瘤而不帶來嚴重的副作用。 采用未修飾的TIL細胞治療腫瘤，TIL細胞有專門攻擊腫瘤的特性，導(dǎo)入TNF基因的TIL細胞可望在腫瘤組織中安家，在局部產(chǎn)生高濃度TNF以殺傷腫瘤而不帶來嚴重的副作用。大劑量TNF注射造成惡液質(zhì)的小鼠模型 在動物實驗中TNF對

21、于小鼠是強力抗腫瘤劑，鼠可耐受400g/kg體重, TNF用于臨床，當(dāng)劑量達到8g/kg體重時即產(chǎn)生明顯的副作用。TILAttack tumor specific Tumor cellTILTNFtransfectionTNFHoming in tumorTNFTNFProducing Tumor infiltrating lymphocyte（三）血管生成的抑制AngiogenesisVEGF promotes Tumor Angiogenesis腫瘤細胞內(nèi)皮細胞大鼠和大鼠模型腫瘤系統(tǒng)中已獲得成功突變信號傳導(dǎo)缺陷性VEGF受體基因轉(zhuǎn)染內(nèi)皮細胞抑制腫瘤的血管形成二、自身免疫病的基因治療自身免疫

22、性胰島損傷I-型糖尿病患者血清中的自身抗體SLE 患者血清中的抗中心?？贵w免疫復(fù)合物在 SLE 患者皮下沉積Models of autoimmune diseasesThe experimental models :experimental auto-allergic encephalitis (腦炎), experimental thyroiditis (甲狀腺炎) adjuvant induced arthritis (關(guān)節(jié)炎)Experimental antoimmune encephalomyelitis (腦脊髓膜炎) (EAE)Naturally occurring models

23、:hemolytic anemia in NZB mice systemic lupus erythematosus(狼瘡)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 促炎癥細胞因子 抗炎細胞因子 （一）靶組織基因治療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. 抗炎機制不能有效地下調(diào)類風(fēng)濕關(guān)節(jié)炎的炎癥過程，類風(fēng)濕關(guān)節(jié)炎是細胞因子間平衡失調(diào)的結(jié)果。治療策略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)胰島細胞表達IL-4不發(fā)生胰島炎和以后發(fā)生的糖尿?。ǘ㏕細胞介導(dǎo)基因治療MS: multiple sclerosis MS 患者中樞神經(jīng)系統(tǒng)的實質(zhì)性損傷 MS 中樞神經(jīng)系統(tǒng)的組織損傷病灶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細胞 EAETh1IL-10TransfectionTh1PLP抑制EAE T細胞基因修飾 第二節(jié) 抗體的免疫學(xué)治療一、單克隆抗體二、雙向特異性抗體的免疫治療作用TUMORCELL-S-S-Fab免疫毒素( 植物和