对TGF-β不敏感肿瘤特异性细胞毒性T淋巴细胞的培养及其在前列腺癌免疫治疗中的应用
|
摘要
目的
肿瘤特异性细胞毒性T淋巴细胞(CTL)是体内抗肿瘤免疫最为有效的效应细胞,但其激活需要抗原呈递细胞(APC)处理并呈递相应的抗原。树突状细胞(DC)是目前已知功能最强的APC,可以激活初始T细胞和记忆性T细胞。DC可提供T细胞活化所需的共刺激信号,在体内外诱导CTL的生成。肿瘤组织匀浆、细胞裂解物包含全部抗原信息,用其致敏DC细胞,体外可以激发T细胞大量增殖,产生肿瘤特异性CTL。转化生长因子β(transforming growth factor-β,TGF-β)是肿瘤细胞分泌的功能最强的免疫抑制因子。TGF-β具有通过抑制肿瘤抗原呈递、淋巴细胞的杀伤作用等多种途径,负性调节机体对肿瘤的免疫应答和促进肿瘤生长的作用。我们通过用携带显性负相的TGF-βⅡ型受体基因的慢病毒载体感染人CTL,使其对TGF-β不敏感,为晚期前列腺癌的治疗探索一条新的治疗途径。
方法
我们用同一患者来源的DC、肿瘤组织匀浆、T淋巴细胞培养出肿瘤特异性CTL,然后以慢病毒为载体将TβRⅡ-DNglytk基因转染到肿瘤特异性CTL,从而去除了TGF-β对肿瘤特异性CTL的抑制作用。我们先用PC-3肿瘤细胞裂解物致敏健康人来源DC,体外刺激T淋巴细胞,获得对TGF-β不敏感肿瘤特异性CTL,并且通过流式细胞术,荧光抗体染色,Western—blot,MTT等方法对其进行了分析、鉴定。在此基础之上,我们将前列腺根治性切除术后标本移植到裸鼠身上,进行扩增,获得足量的肿瘤组织,用肿瘤组织匀浆作用于同一患者来源DC,然后刺激T淋巴细胞,成功获得了对TGF-β不敏感肿瘤特异性CTL,并经体内实验证实了对TGF-β不敏感肿瘤特异性CTL的有效性。
结果
本实验采用人重组IL-4、人重组GM-CSF、PC-3肿瘤细胞裂解物体外诱导PBMC成功培养出负载肿瘤抗原的DC细胞,以此致敏T淋巴细胞,获得肿瘤特异性细胞毒性T淋巴细胞;成功将TβRⅡDNglytk基因转入肿瘤特异性CTL细胞,并获得鉴定;转染对细胞状态、活性影响很小;体内、外实验验证转染后一定程度上去除了TGF-β对CTL杀伤肿瘤细胞的抑制。
结论
对TGF-β不敏感肿瘤特异性CTL培养成功及其体内、外实验研究表明:这一方法在肿瘤免疫治疗中具有潜在的应用前景,为肿瘤的过继免疫治疗提供了又一新的思路。
Objectives
Cytotoxic T lymphocytes(CTL) are the most important effect cell in anti-tumor immune response in vivo. CTL must be activated through the presentation of appropriate antigen by antigen-present cells(APC). Dendritic cells(DC) are the potentest APC. As we known, they can activate not only naive T cells but memory T cell. Cytotoxic T lymphocytes(CTL) can be induced by DCs which can provide co-stimulating signal that is indispensable to activation of T cells. Cancer cells produce large amounts of TGF-β, which is a potent immunosuppressant . The high levels of TGF-βproduced by cancer cells have a negative effect on surrounding cells, including the host immune cells. As a result, cancer cells are able to escape the host's immune surveillance program, leading to tumor progression and metastasis .
Material and methods
The present study involved isolation and generation of dendritic cells, CD4+ T cells, and CD8+ T cells from patients with prostate cancer. Next, We harvested tumor cells from patients with advanced prostate cancer undergoing radical prostatectomy to generate the primary cancer cell cultures but failed to subculture the cells. Because of the limited quantity of prostate tumor, we primed the CTL with PC3 cell lysates and dendritic cells. Tumor reactive CTL will be generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of PC-3 cells and IL-2,IL-4,GM-CSF. Then, we will render these cells insensitive to TGF-βby transfection with the TβRⅡDN-tk construct. The control CTL will be transfected with the TRANSglytk vector. Then we performed adoptive transfer of autologous tumor-reactive TGF-βinsensitive CTL into these immuno-deficient mice bearing human prostate tumors. Finally, the effectiveness of tumor-reactive TGF-βinsensitive CTL in vivo were determined using FACS, immunofluorescence, western-blot, MTT and so on.
Results
The mixed culture of lymphocytes and DCs made the autologous tumor-reacive CTL cells. Establish the efficacy of transfection using the lentivirus-based gene transfer method to generate TGF-βinsensitive CTL cells: Tumor reactive CTL were infected with the lentiviral particle containing TβRⅡDNglytk and TRANSglytk. Infection efficiency were assessed for V5-epitope using anti-V5-FITC. The results showed that adoptive transfer of tumor-reactive TGF-β-insensitive CTL in immunodeficient mice bearing xenograft tumor from human Prostate carcinoma can significantly suppress the growth of the xenograft tumor.
Conclusion
Our experiment made a foundation for further application of tumor-reactive TGF-βinsensitive CTL in adoptive immunotherapy of prostate tumors.
肿瘤特异性细胞毒性T淋巴细胞(CTL)是体内抗肿瘤免疫最为有效的效应细胞,但其激活需要抗原呈递细胞(APC)处理并呈递相应的抗原。树突状细胞(DC)是目前已知功能最强的APC,可以激活初始T细胞和记忆性T细胞。DC可提供T细胞活化所需的共刺激信号,在体内外诱导CTL的生成。肿瘤组织匀浆、细胞裂解物包含全部抗原信息,用其致敏DC细胞,体外可以激发T细胞大量增殖,产生肿瘤特异性CTL。转化生长因子β(transforming growth factor-β,TGF-β)是肿瘤细胞分泌的功能最强的免疫抑制因子。TGF-β具有通过抑制肿瘤抗原呈递、淋巴细胞的杀伤作用等多种途径,负性调节机体对肿瘤的免疫应答和促进肿瘤生长的作用。我们通过用携带显性负相的TGF-βⅡ型受体基因的慢病毒载体感染人CTL,使其对TGF-β不敏感,为晚期前列腺癌的治疗探索一条新的治疗途径。
方法
我们用同一患者来源的DC、肿瘤组织匀浆、T淋巴细胞培养出肿瘤特异性CTL,然后以慢病毒为载体将TβRⅡ-DNglytk基因转染到肿瘤特异性CTL,从而去除了TGF-β对肿瘤特异性CTL的抑制作用。我们先用PC-3肿瘤细胞裂解物致敏健康人来源DC,体外刺激T淋巴细胞,获得对TGF-β不敏感肿瘤特异性CTL,并且通过流式细胞术,荧光抗体染色,Western—blot,MTT等方法对其进行了分析、鉴定。在此基础之上,我们将前列腺根治性切除术后标本移植到裸鼠身上,进行扩增,获得足量的肿瘤组织,用肿瘤组织匀浆作用于同一患者来源DC,然后刺激T淋巴细胞,成功获得了对TGF-β不敏感肿瘤特异性CTL,并经体内实验证实了对TGF-β不敏感肿瘤特异性CTL的有效性。
结果
本实验采用人重组IL-4、人重组GM-CSF、PC-3肿瘤细胞裂解物体外诱导PBMC成功培养出负载肿瘤抗原的DC细胞,以此致敏T淋巴细胞,获得肿瘤特异性细胞毒性T淋巴细胞;成功将TβRⅡDNglytk基因转入肿瘤特异性CTL细胞,并获得鉴定;转染对细胞状态、活性影响很小;体内、外实验验证转染后一定程度上去除了TGF-β对CTL杀伤肿瘤细胞的抑制。
结论
对TGF-β不敏感肿瘤特异性CTL培养成功及其体内、外实验研究表明:这一方法在肿瘤免疫治疗中具有潜在的应用前景,为肿瘤的过继免疫治疗提供了又一新的思路。
Objectives
Cytotoxic T lymphocytes(CTL) are the most important effect cell in anti-tumor immune response in vivo. CTL must be activated through the presentation of appropriate antigen by antigen-present cells(APC). Dendritic cells(DC) are the potentest APC. As we known, they can activate not only naive T cells but memory T cell. Cytotoxic T lymphocytes(CTL) can be induced by DCs which can provide co-stimulating signal that is indispensable to activation of T cells. Cancer cells produce large amounts of TGF-β, which is a potent immunosuppressant . The high levels of TGF-βproduced by cancer cells have a negative effect on surrounding cells, including the host immune cells. As a result, cancer cells are able to escape the host's immune surveillance program, leading to tumor progression and metastasis .
Material and methods
The present study involved isolation and generation of dendritic cells, CD4+ T cells, and CD8+ T cells from patients with prostate cancer. Next, We harvested tumor cells from patients with advanced prostate cancer undergoing radical prostatectomy to generate the primary cancer cell cultures but failed to subculture the cells. Because of the limited quantity of prostate tumor, we primed the CTL with PC3 cell lysates and dendritic cells. Tumor reactive CTL will be generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of PC-3 cells and IL-2,IL-4,GM-CSF. Then, we will render these cells insensitive to TGF-βby transfection with the TβRⅡDN-tk construct. The control CTL will be transfected with the TRANSglytk vector. Then we performed adoptive transfer of autologous tumor-reactive TGF-βinsensitive CTL into these immuno-deficient mice bearing human prostate tumors. Finally, the effectiveness of tumor-reactive TGF-βinsensitive CTL in vivo were determined using FACS, immunofluorescence, western-blot, MTT and so on.
Results
The mixed culture of lymphocytes and DCs made the autologous tumor-reacive CTL cells. Establish the efficacy of transfection using the lentivirus-based gene transfer method to generate TGF-βinsensitive CTL cells: Tumor reactive CTL were infected with the lentiviral particle containing TβRⅡDNglytk and TRANSglytk. Infection efficiency were assessed for V5-epitope using anti-V5-FITC. The results showed that adoptive transfer of tumor-reactive TGF-β-insensitive CTL in immunodeficient mice bearing xenograft tumor from human Prostate carcinoma can significantly suppress the growth of the xenograft tumor.
Conclusion
Our experiment made a foundation for further application of tumor-reactive TGF-βinsensitive CTL in adoptive immunotherapy of prostate tumors.
引文
1 Jemal A,Siegel R,et al.Cancer statistics,2007.CA Cancer J Clin.2007Jan-Feb;57(1):43-66.
2 Gao HW,Li YL,et al.Mass screening of prostate cancer in a Chinese population:the relationship between pathological features of prostate cancer and serum prostate specific antigen.Asian J Androl.2005 Jun;7(2):159-63.
3 杨全成 徐勇 李凯 张晓光 牛远杰.263例前列腺癌患者诊断治疗现状.2006,33:339-341.
4 叶定伟,李长岭.前列腺癌发病趋势的回顾和展望.中国癌症杂志,2007,17:177-180.
5 De La Taille A,Vacherot F,Salomon L,et al.Hormone-refractory prostate cancer:a multi-step and multi-event process.Prostate Cancer Prostatic Dis,2001,4:204-212.
6 吕晨,钟朝晖.前列腺癌的内分泌治疗现状及研究进展.临床泌尿外科杂志,2007,22:872-875.
7 叶定伟,孙燕.激素抵抗性前列腺癌的治疗进展.中国癌症杂志,2007,17:220-224.
8 宁波.肿瘤免疫与肿瘤免疫逃逸机制.中国实用妇科与产科杂志,2000,16:339-340
9 Gorelik L,Constant S,Flavell RA.Mechanism of transforming growth factor beta-induced inhibition of T helper type 1 differentiation.J Exp Med.2002;195(11):1499-1505.
10 Gorelik L,Fields PE,Flavell RA.Cutting edge:TGF-beta inhibits Th type 2development through inhibition of GATA-3 expression.J Immunol.2000;165(9):4773-4777.
11 Mosmann TR,Moore KW.The role of IL-10 in crossregulation of TH1 and TH2responses.Immunol Today.1991;12(3):A49-53
12 Strobl H,Knapp W.TGF-betal regulation of dendritic cells.Microbes Infect.1999;1(15):1283-1290.
13 Morelli AE,Zahorchak AF,Larregina AT,Colvin BL,Logar AJ,Takayama T,Falo LD,Thomson AW.Cytokine production by mouse myeloid dendritic cells in relation todifferentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation.Blood.2001;98(5):1512-1523.
14 李振莉,畅继武.肿瘤的热休克蛋白90一肽复合物诱导产生特异性细胞毒性T 淋巴细胞的体外研究.中华医学杂志,2004:vol84,No.20:1701-1704.
15 吴玉章.基础免疫学.北京:科学出版社,2003:1364
16 Edington H,Agarwala S,Kirkwood JM.Biologic Therapy[J].lClin Plast Surg,2000,27:643.
17 Rosenberg A.Progress in human tumor immunology and immunotherapy[J].Nature,2001,30:170.
18 Biatolah MK,Thomas KE.Current concepts in cancer vaccine strategies[J].Bio Tech,2001,30:170.
19 You Z.Hesler J,Rollins L,el al.A retrogen strategy for presentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potem antitumor T helper and CTL responses[J],cancer Res,2001.61(1):197205
20 Caux C,Massacri C,Vanbervliet B,et al.CD34+ Hematopoietic Progen-itors FromHuman Cord Blood Differentiate AlongTwo Independent Den-dritic Cell Pathways in Response to Granulocyte-Macrophage Colony-Stimulating Factor Plus Tumor Necrosis Factor:Ⅱ.Functional Analysis.J Blood,1997,90(4):1458-1470
21 Anita Reddy,Mark Sapp,MaryFeldman,et al.A Monocyte Conditioned Medium Is More Effective Than Defined Cytokines in Mediating the Ter-minal Maturation of Human Dendritic cells.J Blood,1997,90(9):3641-3646.
22 吴松泉.细胞免疫中共刺激分子研究进展.国外医学·免疫学分册,2000,23(1):16-18.
23 Bou rgeios C,Roeha B.Tanchot C.A role for CD40 expression on CD8+T cell in the generation of CD8+T cell memory[J].Science,2002.297(5589):2060-2063.
24 Jansscn EM.Lemtncns EE,Woife T.CD4+T cells are required for secondary expansion and memory in CD8+T lymphoeytes [J],Nature,2003,421(6925):852-856.
25 Cho Y,Miyamoto M,Kato K,et al.CD4+ and CD8+ T cells cooperate to improve prognosis of patients with esophageal squamous cell careinoma[J].Cancer Res,2003,63(7):1555-1559.
26 Rifkin DB,Kojima S,Abe M,Harpel JG.TGF-beta:structure,function,and formation.Thromb Haemost.1993 Jul 1;70(1):177-9.
27 李颖,段世刚,陈平.TGF-β/Smads信号转导途径与肿瘤发生发展的关系消化外科,2006,5:487-490.
28 Carrington LM,Albon J,Anderson I,et al.Differential regulation of key stages in early corneal wound healing by TGF-beta isoforms and their inhibitors.Invest Ophthalmol Vis Sci,2006,47:1886-94.
29 Ferguson MW,O'Kane S.Scar-free healing:from embryonic mechanisms to adult therapeutic intervention.Philos Trans R Soc Lond B Biol Sci,2004,359:839-50.
30 赵志德,寇伯君,李胜文等,肾癌患者TGFβ1表达和肿瘤微血管密度检测的临床意义.中华泌尿外科杂志,1999:20:332-4.
31 Murray PA,Barret RJ,Travers M,et al.The prognostic significance of transforming growth factors in human breast carcinoma.Br J Cancer 1993;67:1408-13.
32 Friess H,Yamanaka Y,Buchler M,et al.Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival.Gastroenterology.1993;105:1846-56.
33 Chin D,Boyle GM,Parsons PG,et al.What is transforming growth factor-beta (TGF-beta)?.Br J Plast Surg,2004,57:215-21.
34 Bottner M,Krieglstein K,Unsicker K.The transforming growth factor-betas:structure,signaling,and roles in nervous system development and functions.J Neurochem,2000,75:2227-40.
35 Itoh S,ItohF,Goumans MJ,Ten Dijke P.Signaling of transforming growth factor-beta family members through Smad proteins.Eur J Biochem.2000Dec;267(24):6954-6967.
36 Itoh S,Itoh F,Goumans MJ,TenDijke P.Signaling of transforming growth factor-beta family members through Smad proteins.Eur J Biochem.2000 Dec;267(24):6954-6967.
37 Stroschein SL,Wang W,Zhou S,Zhou Q,Luo K.Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.Science.l999;286(5440):771-774.
38 Annunziato F, Cosmi L, Liotta F, et al. Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes. J Exp Med, 2002,196:379-87.
39 Azuma T, Takahashi T, Kunisato A, et al. Human CD4+ CD25+ regulatory T cells suppress NKT cell functions. Cancer Res, 2003,63:4516-20.
40 Witham TF, Villa L, Yang T, et al. Expression of a soluble transforming growth factor-beta (TGFbeta) receptor reduces tumorigenicity by regulating natural killer (NK) cell activity against 9L gliosarcoma in vivo. J Neurooncol, 2003,64:63-9.
41 Hsiao YW, Liao KW, Hung SW, et al. Tumor-infiltrating lymphocyte secretion of IL-6 antagonizes tumor-derived TGF-beta 1 and restores the lymphokine-activated killing activity. J Immunol, 2004,172:1508-14.
42 Bacchetta R, Sartirana C, Levings MK, et al. Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines. Eur J Immunol, 2002,32:2237-45.
43 Mitra R, Khar A. Suppression of macrophage function in AK-5 tumor transplanted animals: role of TGF-betal. Immunol Lett, 2004,91:189-95.
44 Castriconi R, Cantorii C, Delia Chiesa M, et al. Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells. Proc Natl Acad Sci U S A, 2003,100:4120-5.
45 Cox DA. Transforming growth factor-beta 3. Cell Biol Int, 1995,19:357-71.
46 Wu L, Siddiqui A, Morris DE, et al. Transforming growth factor beta 3 (TGF beta 3) accelerates wound healing without alteration of scar prominence. Histologic and competitive reverse-transcription-polymerase chain reaction studies. Arch Surg, 1997,132:753-60.
47 Lin HY, Moustakas A. TGF-beta receptors: structure and function. Cell Mol Biol (Noisy-le-grand), 1994,40:337-49.
48 Denicourt C, Dowdy SF. Another twist in the transforming growth factor beta-induced cell-cycle arrest chronicle. Proc Natl Acad Sci U S A, 2003,100:15290-1.
49 Rich J, Borton A, Wang X. Transforming growth factor-beta signaling in cancer. Microsc Res Tech, 2001,52:363-73.
50 Laouar Y, Sutterwala FS, Gorelik L, et al. Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma. Nat Immunol, 2005,6:600-7.
51 Song C, Jun SY, Hong JH, et al. Transforming growth factor-beta downregulates interleukin-2-induced phosphorylation of signal transducer and activator of transcription 5 in human renal cell carcinoma. J Cancer Res Clin Oncol, 2007,133:487-92.
52 Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene, 2005,24:5764-74.
53 Castanares C, Redondo-Horcajo M, Magan-Marchal N, et al. Signaling by ALK5 mediates TGF-beta-induced ET-1 expression in endothelial cells: a role for migration and proliferation. J Cell Sci, 2007,120:1256-66.
54 Kominsky SL, Doucet M, Brady K, et al. TGF-beta promotes the establishment of renal cell carcinoma bone metastasis. J Bone Miner Res, 2007,22:37-44.
55 Seth P, Wang ZG, Pister A, et al. Development of oncolytic adenovirus armed with a fusion of soluble transforming growth factor-beta receptor II and human immunoglobulin Fc for breast cancer therapy. Hum Gene Ther, 2006,17:1152-60.
56 Swallow CJ, Partridge EA, Macmillan JC, et al. alpha2HS-glycoprotein, an antagonist of transforming growth factor beta in vivo, inhibits intestinal tumor progression. Cancer Res, 2004,64:6402-9.
57 Haider SK, Beauchamp RD, Datta PK. A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers. Neoplasia, 2005,7:509-21.
58 Ge R,Rajeev V,Ray P,et al.Inhibition of growth and metastasis of mouse mammary carcinoma by selective inhibitor of transforming growth factor-beta type Ⅰ receptor kinase in vivo.Clin Cancer Res,2006,12:4315-30.
59 Biswas S,Guix M,Rinehart C,et al.Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression.J Clin Invest,2007,117:1305-13.
60 Ye L,Zhang H,Zhang L,et al.Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells.Int J Oncol,2006,29:1149-57.
61 Chen Y,Bhushan A,Vale W.Smad8 mediates the signaling of the ALK-2[corrected]receptor serine kinase.Proc Natl Acad Sci U S A,1997,94:12938-43.
62 Lawler S,Feng XH,Chen RH,et al.The type Ⅱ transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues.J Biol Chem,1997,272:14850-9.
63 Franzen P,ten Dijke P,Ichijo H,et al.Cloning of a TGF beta type Ⅰ receptor that forms a heteromeric complex with the TGF beta type Ⅱ receptor.Cell,1993,75:681-92.
64 Robe PA,Nguyen-Khac MT,Lambert F,et al.Sulfasalazine unveils a contact-independent HSV-TK/ganciclovir gene therapy bystander effect in malignant gliomas.Int J Oncol,2007,30:283-90.
65 单纯疱疹病毒胸苷激酶基因联合更昔洛韦治疗肿瘤的机制.食品与药品,2007,9:52-55.
[1] Jemal A, Siegel R, et al. Cancer statistics, 2007. CA Cancer J Clin. 2007 Jan-Feb;57(1):43-66. PMID: 17237035
[2] Gao HW, Li YL,et al. Mass screening of prostate cancer in a Chinese population: the relationship between pathological features of prostate cancer and serum prostate specific antigen. Asian J Androl. 2005 Jun;7(2):159-63. PMID: 15897972
[3] Galvin JP, Spaeny-Dekking LH, Wang B, et al. Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo J Immunol. 1999,162(9):5345-5350. PMID: 10228010
[4] Andersen MH, Schrama D, Thor Straten P, et al. Cytotoxic T cells. Invest Dermatol, 2006, 126: 32-41. PMID: 16417215
[5] Shafer-Weaver K, Anderson M, et al. T cell tolerance to tumors and cancer immunotherapy. Adv Exp Med Biol. 2007;601:357-68. PMID: 17713024
[6] Yeh YA, Wang JW , Fan CY, et al. Expression of Fas ligand in metastatic prostatic carcinoma :suggestive of possible clonal expansion of subpopulation with metastatic potential. Diagn ,Mol Pathol, 2001, 10(4), 236～41. PMID: 11763314
[7] Laouar Y,Sutterwala FS,Gorelik L,et al. Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma. Nat Immunol. 2005 Jun;6(6):600-7. Epub 2005 Apr 24. PMID: 15852008
[8] Song C,Jun SY,Hong JH,et al.Transforming growth factor-beta downregulates interleukin-2-induced phosphorylation of signal transducer and activator of transcription 5 in human renal cell carcinoma.J Cancer Res Clin Oncol. 2007 Feb 6.PMID: 17279417
[9] Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 2005; 24: 5764-5774. PMID: 16123809
[10] Castanares C, Redondo-Horcajo M, Magan-Marchal N,et al.Signaling by ALK5 mediates TGF-beta-induced ET-1 expression in endothelial cells: a role for migration and proliferation. J Cell Sci. 2007 Apr 1;120(Pt 7):1256-1266 .PMID: 17376964
[11]Dreicer R. See W. A, Klein E. A. Phase II trial of GM—CSF in advanced prostate cancer. Invest. New Drugs, 2001, 19: 261—265 .PMID: 11561685
[12] Rini BI,Weinberg V, Bok R, el al.Prostate specific anligen kinetics as a measure of granulocyte—macrophage colony—stimulating factor with pr0gre8sion of prostate cancer. J Clin Oncol, 2003, 21: 99—105. PMID: 12506177
[13] Angelov GS, Tomkowiak M, Mareais A, et al. Flt3 ligand—generated murine plasmacytoid and conventional dendritic cells differ in theircapacity to prime najve CD8+ T cells and to generate memory cells invivo. J Immunol, 2005,175: 189—195. PMID: 15972647
[14] Ciavarra RP, Somer KD, Brown RR. et al. Flt3-ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer. Cancer Res, 2000, 60: 2081—2084. PMID: 10786663
[15] Higano CS, Vogelzang NJ, Sosman J.A, et al. Safety and biologicalactivity of repeated doses of recombinant human Flt3 ligand in patients with bone scan-negative hormone-refractory prostate cancer. Clin Cancer Res. 2004 Feb 15;10(4):1219-25. PMID: 14977818
[16] Heiser A, Coleman D, Dannull J, et al. Autologous dendritic cels transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors[J]. J Clin Invest, 2002, 109(3): 409-417. PMID: 11828001
[17] Li N, Qin H, Li X, et al. Potent systemic antitumor immunity induced by vaccination with chemotactic-prostate tumor associated antigen gene-modified tumor cell and blockade of B7-H1. J Clin Immunol. 2007 Jan;27(1):117-30. Epub 2006 Dec 16. PMID: 17180470
[18] Qin H, Zhou C, Wang D, et al .Enhancement of antitumour immunity by a novel chemotactic antigen DNA vaccine encoding chemokines and multiepitopes of prostate-tumour-associated antigens. Immunology. 2006 Mar;117(3):419-30.PMID: 16476062
[19] Dannull J, DienerPA,Prikler L, et al. Protate stem cell antigen is apromising candidate for immunotherapy of advanced protate cancer Cancer Res , 2000,60, 5522-5528. PMID: 11034097
[20] Saffran DC, Raitano AB,Hubert RS, et al. Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival Of mice bearing human protate cancer xenografts.Proc Natl Acad sci USA, 2001. 98. 2658～ 2663 .PMID: 11226295
[21] May KF Jr, Roychowdhury S,BhttD,et al. Anti-human CTLA-4 monoclonal antibody promotes T-cell expansion and immunity in a hu-PBL-SCID model: a new method for preclinical screening of Costimulatory monoclonal antibodies. Blood,2005 , 105(3): 1114-1120. PMID: 15486062
[22] Lute KD, May KF Jr, Lu P,et al. Human CTLA4 knock-in mice unravel the quantitative link between tumor immunity and autoimmunity induced by anti-CTLA-4 antibodies. Blood, 2005,106(9): 3127-3133. PMID: 16037385
2 Gao HW,Li YL,et al.Mass screening of prostate cancer in a Chinese population:the relationship between pathological features of prostate cancer and serum prostate specific antigen.Asian J Androl.2005 Jun;7(2):159-63.
3 杨全成 徐勇 李凯 张晓光 牛远杰.263例前列腺癌患者诊断治疗现状.2006,33:339-341.
4 叶定伟,李长岭.前列腺癌发病趋势的回顾和展望.中国癌症杂志,2007,17:177-180.
5 De La Taille A,Vacherot F,Salomon L,et al.Hormone-refractory prostate cancer:a multi-step and multi-event process.Prostate Cancer Prostatic Dis,2001,4:204-212.
6 吕晨,钟朝晖.前列腺癌的内分泌治疗现状及研究进展.临床泌尿外科杂志,2007,22:872-875.
7 叶定伟,孙燕.激素抵抗性前列腺癌的治疗进展.中国癌症杂志,2007,17:220-224.
8 宁波.肿瘤免疫与肿瘤免疫逃逸机制.中国实用妇科与产科杂志,2000,16:339-340
9 Gorelik L,Constant S,Flavell RA.Mechanism of transforming growth factor beta-induced inhibition of T helper type 1 differentiation.J Exp Med.2002;195(11):1499-1505.
10 Gorelik L,Fields PE,Flavell RA.Cutting edge:TGF-beta inhibits Th type 2development through inhibition of GATA-3 expression.J Immunol.2000;165(9):4773-4777.
11 Mosmann TR,Moore KW.The role of IL-10 in crossregulation of TH1 and TH2responses.Immunol Today.1991;12(3):A49-53
12 Strobl H,Knapp W.TGF-betal regulation of dendritic cells.Microbes Infect.1999;1(15):1283-1290.
13 Morelli AE,Zahorchak AF,Larregina AT,Colvin BL,Logar AJ,Takayama T,Falo LD,Thomson AW.Cytokine production by mouse myeloid dendritic cells in relation todifferentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation.Blood.2001;98(5):1512-1523.
14 李振莉,畅继武.肿瘤的热休克蛋白90一肽复合物诱导产生特异性细胞毒性T 淋巴细胞的体外研究.中华医学杂志,2004:vol84,No.20:1701-1704.
15 吴玉章.基础免疫学.北京:科学出版社,2003:1364
16 Edington H,Agarwala S,Kirkwood JM.Biologic Therapy[J].lClin Plast Surg,2000,27:643.
17 Rosenberg A.Progress in human tumor immunology and immunotherapy[J].Nature,2001,30:170.
18 Biatolah MK,Thomas KE.Current concepts in cancer vaccine strategies[J].Bio Tech,2001,30:170.
19 You Z.Hesler J,Rollins L,el al.A retrogen strategy for presentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potem antitumor T helper and CTL responses[J],cancer Res,2001.61(1):197205
20 Caux C,Massacri C,Vanbervliet B,et al.CD34+ Hematopoietic Progen-itors FromHuman Cord Blood Differentiate AlongTwo Independent Den-dritic Cell Pathways in Response to Granulocyte-Macrophage Colony-Stimulating Factor Plus Tumor Necrosis Factor:Ⅱ.Functional Analysis.J Blood,1997,90(4):1458-1470
21 Anita Reddy,Mark Sapp,MaryFeldman,et al.A Monocyte Conditioned Medium Is More Effective Than Defined Cytokines in Mediating the Ter-minal Maturation of Human Dendritic cells.J Blood,1997,90(9):3641-3646.
22 吴松泉.细胞免疫中共刺激分子研究进展.国外医学·免疫学分册,2000,23(1):16-18.
23 Bou rgeios C,Roeha B.Tanchot C.A role for CD40 expression on CD8+T cell in the generation of CD8+T cell memory[J].Science,2002.297(5589):2060-2063.
24 Jansscn EM.Lemtncns EE,Woife T.CD4+T cells are required for secondary expansion and memory in CD8+T lymphoeytes [J],Nature,2003,421(6925):852-856.
25 Cho Y,Miyamoto M,Kato K,et al.CD4+ and CD8+ T cells cooperate to improve prognosis of patients with esophageal squamous cell careinoma[J].Cancer Res,2003,63(7):1555-1559.
26 Rifkin DB,Kojima S,Abe M,Harpel JG.TGF-beta:structure,function,and formation.Thromb Haemost.1993 Jul 1;70(1):177-9.
27 李颖,段世刚,陈平.TGF-β/Smads信号转导途径与肿瘤发生发展的关系消化外科,2006,5:487-490.
28 Carrington LM,Albon J,Anderson I,et al.Differential regulation of key stages in early corneal wound healing by TGF-beta isoforms and their inhibitors.Invest Ophthalmol Vis Sci,2006,47:1886-94.
29 Ferguson MW,O'Kane S.Scar-free healing:from embryonic mechanisms to adult therapeutic intervention.Philos Trans R Soc Lond B Biol Sci,2004,359:839-50.
30 赵志德,寇伯君,李胜文等,肾癌患者TGFβ1表达和肿瘤微血管密度检测的临床意义.中华泌尿外科杂志,1999:20:332-4.
31 Murray PA,Barret RJ,Travers M,et al.The prognostic significance of transforming growth factors in human breast carcinoma.Br J Cancer 1993;67:1408-13.
32 Friess H,Yamanaka Y,Buchler M,et al.Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival.Gastroenterology.1993;105:1846-56.
33 Chin D,Boyle GM,Parsons PG,et al.What is transforming growth factor-beta (TGF-beta)?.Br J Plast Surg,2004,57:215-21.
34 Bottner M,Krieglstein K,Unsicker K.The transforming growth factor-betas:structure,signaling,and roles in nervous system development and functions.J Neurochem,2000,75:2227-40.
35 Itoh S,ItohF,Goumans MJ,Ten Dijke P.Signaling of transforming growth factor-beta family members through Smad proteins.Eur J Biochem.2000Dec;267(24):6954-6967.
36 Itoh S,Itoh F,Goumans MJ,TenDijke P.Signaling of transforming growth factor-beta family members through Smad proteins.Eur J Biochem.2000 Dec;267(24):6954-6967.
37 Stroschein SL,Wang W,Zhou S,Zhou Q,Luo K.Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.Science.l999;286(5440):771-774.
38 Annunziato F, Cosmi L, Liotta F, et al. Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes. J Exp Med, 2002,196:379-87.
39 Azuma T, Takahashi T, Kunisato A, et al. Human CD4+ CD25+ regulatory T cells suppress NKT cell functions. Cancer Res, 2003,63:4516-20.
40 Witham TF, Villa L, Yang T, et al. Expression of a soluble transforming growth factor-beta (TGFbeta) receptor reduces tumorigenicity by regulating natural killer (NK) cell activity against 9L gliosarcoma in vivo. J Neurooncol, 2003,64:63-9.
41 Hsiao YW, Liao KW, Hung SW, et al. Tumor-infiltrating lymphocyte secretion of IL-6 antagonizes tumor-derived TGF-beta 1 and restores the lymphokine-activated killing activity. J Immunol, 2004,172:1508-14.
42 Bacchetta R, Sartirana C, Levings MK, et al. Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines. Eur J Immunol, 2002,32:2237-45.
43 Mitra R, Khar A. Suppression of macrophage function in AK-5 tumor transplanted animals: role of TGF-betal. Immunol Lett, 2004,91:189-95.
44 Castriconi R, Cantorii C, Delia Chiesa M, et al. Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells. Proc Natl Acad Sci U S A, 2003,100:4120-5.
45 Cox DA. Transforming growth factor-beta 3. Cell Biol Int, 1995,19:357-71.
46 Wu L, Siddiqui A, Morris DE, et al. Transforming growth factor beta 3 (TGF beta 3) accelerates wound healing without alteration of scar prominence. Histologic and competitive reverse-transcription-polymerase chain reaction studies. Arch Surg, 1997,132:753-60.
47 Lin HY, Moustakas A. TGF-beta receptors: structure and function. Cell Mol Biol (Noisy-le-grand), 1994,40:337-49.
48 Denicourt C, Dowdy SF. Another twist in the transforming growth factor beta-induced cell-cycle arrest chronicle. Proc Natl Acad Sci U S A, 2003,100:15290-1.
49 Rich J, Borton A, Wang X. Transforming growth factor-beta signaling in cancer. Microsc Res Tech, 2001,52:363-73.
50 Laouar Y, Sutterwala FS, Gorelik L, et al. Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma. Nat Immunol, 2005,6:600-7.
51 Song C, Jun SY, Hong JH, et al. Transforming growth factor-beta downregulates interleukin-2-induced phosphorylation of signal transducer and activator of transcription 5 in human renal cell carcinoma. J Cancer Res Clin Oncol, 2007,133:487-92.
52 Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene, 2005,24:5764-74.
53 Castanares C, Redondo-Horcajo M, Magan-Marchal N, et al. Signaling by ALK5 mediates TGF-beta-induced ET-1 expression in endothelial cells: a role for migration and proliferation. J Cell Sci, 2007,120:1256-66.
54 Kominsky SL, Doucet M, Brady K, et al. TGF-beta promotes the establishment of renal cell carcinoma bone metastasis. J Bone Miner Res, 2007,22:37-44.
55 Seth P, Wang ZG, Pister A, et al. Development of oncolytic adenovirus armed with a fusion of soluble transforming growth factor-beta receptor II and human immunoglobulin Fc for breast cancer therapy. Hum Gene Ther, 2006,17:1152-60.
56 Swallow CJ, Partridge EA, Macmillan JC, et al. alpha2HS-glycoprotein, an antagonist of transforming growth factor beta in vivo, inhibits intestinal tumor progression. Cancer Res, 2004,64:6402-9.
57 Haider SK, Beauchamp RD, Datta PK. A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers. Neoplasia, 2005,7:509-21.
58 Ge R,Rajeev V,Ray P,et al.Inhibition of growth and metastasis of mouse mammary carcinoma by selective inhibitor of transforming growth factor-beta type Ⅰ receptor kinase in vivo.Clin Cancer Res,2006,12:4315-30.
59 Biswas S,Guix M,Rinehart C,et al.Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression.J Clin Invest,2007,117:1305-13.
60 Ye L,Zhang H,Zhang L,et al.Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells.Int J Oncol,2006,29:1149-57.
61 Chen Y,Bhushan A,Vale W.Smad8 mediates the signaling of the ALK-2[corrected]receptor serine kinase.Proc Natl Acad Sci U S A,1997,94:12938-43.
62 Lawler S,Feng XH,Chen RH,et al.The type Ⅱ transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues.J Biol Chem,1997,272:14850-9.
63 Franzen P,ten Dijke P,Ichijo H,et al.Cloning of a TGF beta type Ⅰ receptor that forms a heteromeric complex with the TGF beta type Ⅱ receptor.Cell,1993,75:681-92.
64 Robe PA,Nguyen-Khac MT,Lambert F,et al.Sulfasalazine unveils a contact-independent HSV-TK/ganciclovir gene therapy bystander effect in malignant gliomas.Int J Oncol,2007,30:283-90.
65 单纯疱疹病毒胸苷激酶基因联合更昔洛韦治疗肿瘤的机制.食品与药品,2007,9:52-55.
[1] Jemal A, Siegel R, et al. Cancer statistics, 2007. CA Cancer J Clin. 2007 Jan-Feb;57(1):43-66. PMID: 17237035
[2] Gao HW, Li YL,et al. Mass screening of prostate cancer in a Chinese population: the relationship between pathological features of prostate cancer and serum prostate specific antigen. Asian J Androl. 2005 Jun;7(2):159-63. PMID: 15897972
[3] Galvin JP, Spaeny-Dekking LH, Wang B, et al. Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo J Immunol. 1999,162(9):5345-5350. PMID: 10228010
[4] Andersen MH, Schrama D, Thor Straten P, et al. Cytotoxic T cells. Invest Dermatol, 2006, 126: 32-41. PMID: 16417215
[5] Shafer-Weaver K, Anderson M, et al. T cell tolerance to tumors and cancer immunotherapy. Adv Exp Med Biol. 2007;601:357-68. PMID: 17713024
[6] Yeh YA, Wang JW , Fan CY, et al. Expression of Fas ligand in metastatic prostatic carcinoma :suggestive of possible clonal expansion of subpopulation with metastatic potential. Diagn ,Mol Pathol, 2001, 10(4), 236～41. PMID: 11763314
[7] Laouar Y,Sutterwala FS,Gorelik L,et al. Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma. Nat Immunol. 2005 Jun;6(6):600-7. Epub 2005 Apr 24. PMID: 15852008
[8] Song C,Jun SY,Hong JH,et al.Transforming growth factor-beta downregulates interleukin-2-induced phosphorylation of signal transducer and activator of transcription 5 in human renal cell carcinoma.J Cancer Res Clin Oncol. 2007 Feb 6.PMID: 17279417
[9] Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 2005; 24: 5764-5774. PMID: 16123809
[10] Castanares C, Redondo-Horcajo M, Magan-Marchal N,et al.Signaling by ALK5 mediates TGF-beta-induced ET-1 expression in endothelial cells: a role for migration and proliferation. J Cell Sci. 2007 Apr 1;120(Pt 7):1256-1266 .PMID: 17376964
[11]Dreicer R. See W. A, Klein E. A. Phase II trial of GM—CSF in advanced prostate cancer. Invest. New Drugs, 2001, 19: 261—265 .PMID: 11561685
[12] Rini BI,Weinberg V, Bok R, el al.Prostate specific anligen kinetics as a measure of granulocyte—macrophage colony—stimulating factor with pr0gre8sion of prostate cancer. J Clin Oncol, 2003, 21: 99—105. PMID: 12506177
[13] Angelov GS, Tomkowiak M, Mareais A, et al. Flt3 ligand—generated murine plasmacytoid and conventional dendritic cells differ in theircapacity to prime najve CD8+ T cells and to generate memory cells invivo. J Immunol, 2005,175: 189—195. PMID: 15972647
[14] Ciavarra RP, Somer KD, Brown RR. et al. Flt3-ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer. Cancer Res, 2000, 60: 2081—2084. PMID: 10786663
[15] Higano CS, Vogelzang NJ, Sosman J.A, et al. Safety and biologicalactivity of repeated doses of recombinant human Flt3 ligand in patients with bone scan-negative hormone-refractory prostate cancer. Clin Cancer Res. 2004 Feb 15;10(4):1219-25. PMID: 14977818
[16] Heiser A, Coleman D, Dannull J, et al. Autologous dendritic cels transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors[J]. J Clin Invest, 2002, 109(3): 409-417. PMID: 11828001
[17] Li N, Qin H, Li X, et al. Potent systemic antitumor immunity induced by vaccination with chemotactic-prostate tumor associated antigen gene-modified tumor cell and blockade of B7-H1. J Clin Immunol. 2007 Jan;27(1):117-30. Epub 2006 Dec 16. PMID: 17180470
[18] Qin H, Zhou C, Wang D, et al .Enhancement of antitumour immunity by a novel chemotactic antigen DNA vaccine encoding chemokines and multiepitopes of prostate-tumour-associated antigens. Immunology. 2006 Mar;117(3):419-30.PMID: 16476062
[19] Dannull J, DienerPA,Prikler L, et al. Protate stem cell antigen is apromising candidate for immunotherapy of advanced protate cancer Cancer Res , 2000,60, 5522-5528. PMID: 11034097
[20] Saffran DC, Raitano AB,Hubert RS, et al. Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival Of mice bearing human protate cancer xenografts.Proc Natl Acad sci USA, 2001. 98. 2658～ 2663 .PMID: 11226295
[21] May KF Jr, Roychowdhury S,BhttD,et al. Anti-human CTLA-4 monoclonal antibody promotes T-cell expansion and immunity in a hu-PBL-SCID model: a new method for preclinical screening of Costimulatory monoclonal antibodies. Blood,2005 , 105(3): 1114-1120. PMID: 15486062
[22] Lute KD, May KF Jr, Lu P,et al. Human CTLA4 knock-in mice unravel the quantitative link between tumor immunity and autoimmunity induced by anti-CTLA-4 antibodies. Blood, 2005,106(9): 3127-3133. PMID: 16037385