人B7-2瘤苗与DC瘤苗体外联合诱导抗食管癌的作用
路静,赵军,宋谦,崔自由,赵国强,黄幼田,杨洪艳,赵继敏,董子明,郑州大学基础医学院病理生理教研室 河南省郑州市 450052
路静,女, 1974-04-01生, 河南省洛阳人,汉族.1996年河南医科大学本科毕业,现为郑州大学基础医学院病理生理学专业在读硕士,主要从事肿瘤的生物治疗的研究.
河南省科技厅攻关项目,No. 0324410034
河南省科技创新人才工程,No. 20008402
通讯作者:董子明,450052, 河南省郑州市,郑州大学基础医学院病理生理教研室. dongzm@zzu.edu.cn
电话:0371-66658157
, 百拇医药
收稿日期:2005-03-06 接受日期: 2005-04-08
Combination effect of human B7-2 and dentric cells vaccines in anti-esophageal cancer in vitro
Jing Lu, Jun Zhao, Qian Song, Zi-You Cui, Guo-Qiang Zhao, Hong-Yan Yang,You-Tian Huang, Ji-Min Zhao, Zi-Ming Dong
Jing Lu, Jun Zhao, Qian Song, Zi-You Cui, Guo-Qiang Zhao, You-TianHuang, Hong-Yan Yang, Ji-Min Zhao, Zi-Ming Dong, Department ofPathophysiology, School of Basic Medical Sciences, Zhengzhou University,Zhengzhou 450052, Henan Province, China
, 百拇医药
Supported by the Key Project of Science and Technology Departmentof Henan Province, No.0324410034 and the Project of Science and TechnologyInnovation Talent of Henan Provience, No.20008402
Correspondence to: Dr. Zi-Ming Dong, Department of Pathophysiology,School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450052,Henan Province, China. dongzm@zzu.edu.cn
Received: 2005-03-06 Accepted: 2005-04-08
, 百拇医药
Abstract
AIM: To investigate the roles of human B7-2 combined with dentriccell (DC) vaccine in inducing anti-tumor immunity against esophagealcancer in vitro.
METHODS: Human esophageal cancer cell line EC9706 was transfectedwith the vector of pEGFP-N3-B7-2 by lipofectamine method. The mononuclearcells (MNCs) were separated from cord blood by density gradientcentrifugation (Ficall-Hypaque) and then were induced to differentiate bycell factors. On the 3rdday, EC9706 cytolysis antigen was added into the medium, in which DCsgradually matured and expressed special tumor antigen. After the maturedDCs were co-cultured with autologous T cells derived from cord blood for 3days, the T cells were activated to become tumor specific cytotoxic Tlymphocytes (CTL). The inhibition of CTL on the transfected anduntransfected EC9706 cells was detected by methyl thiazolyl tetrazolium (MTT)assay.
, 百拇医药
RESULTS: The EGFP-B7-2 fusion gene was expressed mainly on themembrane of EC9706 cells, which proved the transfection was successful.Mature DCs with tumor cytolysis antigene was able to activate naive Tcells to become tumor specialized CTL, and the CTL had significantinhibitory effect or killing response on EC9706 cells transfected withpEGFP-N3-B7-2(F = 21.672,P = 0.000).
CONCLUSION: The human B7-2 combined with DC vaccine can inducesignificant killing response on esophageal cancer cell.
, http://www.100md.com
Key Words: Human B7-2; Vaccine; Cord blood; Dendritic cell;Esophageal cancer; Cytotoxic T lymphocyte; Green fluorescent protein
Lu J, Zhao J, Song Q, Cui ZY, Zhao GQ, Huang YT, Yang HY, Zhao JM, Dong ZM.Combination effect of human B7-2 and dentric cells vaccines inanti-esophageal cancer in vitro. Shijie Huaren Xiaohua Zazhi 2005;13(12):1382-1385
摘要
, 百拇医药
目的: 研究人B7-2瘤苗和负载了肿瘤细胞冻融抗原的DC瘤苗体外联合诱导抗食管癌的免疫作用.
方法: 应用脂质体转染技术,将融合基因表达载体pEGFP-N3-B7-2转染人食管癌细胞株EC9706.采用密度梯度离心法从脐血中分离单个核细胞(MNC)后,获得单核细胞(Mo).在细胞因子作用下诱导分化，第3d加入EC9706的冻融抗原，共培养4d后获得负载肿瘤抗原的成熟树突状细胞(MDC).将致敏DC与从脐血中分离的T淋巴细胞共培养3d，获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝(MTT)法检测CTL对转染和未转染的EC9706的细胞毒作用.
结果: 融合基因在食管癌细胞株EC9706的胞膜上定位表达.脐血来源的DC可负载并递呈肿瘤抗原，激活自体T淋巴细胞，诱导肿瘤特异性CTL产生，对转染pEGFP-N3-B7-2的EC9706细胞有显著杀伤作用(F= 21.672，P= 0.000).
, 百拇医药
结论: 人B7-2瘤苗和DC瘤苗体外联合应用,诱导出明显的杀伤食管癌细胞的免疫效应.
关键词: 人B7-2;疫苗;脐血;树突状细胞;食管癌;细胞毒T淋巴细胞;绿色荧光蛋白
路静,赵军,宋谦,崔自由,赵国强,黄幼田,杨洪艳,赵继敏,董子明.人B7-2瘤苗与DC瘤苗体外联合诱导抗食管癌的作用.世界华人消化杂志 2005;13(12):1382-1385:空质粒组; B:重组质粒组.
图2 培养的脐血单个核细胞(IF×400). A:培养3 d; B:培养7 d.
, 百拇医药
3 讨论
当前，如何能使机体内肿瘤细胞成为好的抗原递呈细胞，进而激活抗肿瘤免疫成为至关重要的问题.就肿瘤细胞活化细胞而言，B7分子提供共刺激信号以顺式共刺激方式最为有效[4]，B7基因修饰的瘤细胞能诱导有效的免疫反应[5].抗原特异性CD8+T细胞介导的抗肿瘤免疫效应中，要求肿瘤细胞本身表达MHCI类分子并提呈抗原.对于MHCI类分子阴性的低免疫原性肿瘤来说，即使导入B7基因也难以诱导CTL免疫应答，但是B7分子能通过增强NK细胞活性杀伤低免疫原性肿瘤[6-7].Wilson et al[7]报道，鼠黑色素瘤细胞B16.F1转染人B7-1或B7-2增加了人NK细胞介导的肿瘤杀伤作用.用特异性抗体封闭B7-1或B7-2分子后，该杀伤作用显著降低.
, http://www.100md.com
肿瘤患者的DC存在表型和功能上的缺陷，不能引发有效的抗肿瘤免疫反应[8].反复大量抽取肿瘤患者的外周血去诱导DC，使大多数肿瘤患者不能耐受.我们采用脐血单个核细胞诱生DC是基于:(1)纯化CD34+干细胞费用高昂，不利于将来用于临床;(2)虽然CD34+干细胞是DC的主要前体细胞但并不是唯一的，脐血中的单核细胞也能在细胞因子的诱导下生成DC;(3)CD34+细胞来源的DC形成晚，分化成熟需12-14 d，单个核细胞诱生DC周期短，仅需7-9 d，更方便于应用，并且两种方法获得的DC的形态、CDla表面抗原的表达没有显著的差异，二者均具有刺激同种T细胞增殖的能力[9].DC在抗肿瘤免疫中发挥重要作用，有关运用DC治疗恶性肿瘤已成为热点.由于肿瘤免疫逃逸的机制之一是其阻止了DC的成熟，而非直接抑制DC的功能[10]，因此在体外将DC扩增或同时用肿瘤抗原致敏DC，使其分化成熟，然后将这种功能正常且携带相应的肿瘤抗原的DC回输体内，可有效地诱导机体产生肿瘤特异性CTL反应[11].
, 百拇医药
食管癌组织B7分子表达缺陷，不能引起有效的免疫应答[4].我们应用脂质体转染技术将pEGFP-N3-B7-2转染人食管癌细胞株EC9706，制备B7-2瘤苗.其抗瘤免疫机制是将T细胞活化所需要共刺激分子B7-2导入食管癌细胞EC9706，通过B7-CD28途径将瘤细胞与T细胞联系在一起，诱导出不依赖于APC的直接的T细胞激活;但有文献报道非免疫系肿瘤缺乏第一信号，即使提供共刺激信号也不能有效激活T细胞[7].故我们考虑联合应用DC瘤苗，利用DC高效的加工提呈抗原能力，尤其是已负载了抗原的DC-Pr瘤苗能更快速地直接将抗原提呈给T细胞，使之识别而活化.食管癌具有异质性大、抗原性弱，缺乏特异性肿瘤抗原的特点，和多数肿瘤一样不能激发有效的免疫反应[12].我们将上述两种途径有机结合在一起，在体外诱导出了有效的抗食管癌细胞效应，为食管癌的生物治疗提供一定的实验依据.如前所述，B7分子在体内还能通过增强NK细胞活性杀伤低免疫原性肿瘤，这为B7-2肿瘤疫苗的应用提供了进一步的理论依据.
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4 参考文献
1 Boussiotis VA, Freeman GJ, Gribben JG, Nadler LM. The role ofB7-1/B7-2: CD28/CLTA-4 pathways in the prevention
of anergy, induction of productive immunityand down杛egulation of the immuneresponse. Immunol Rev
1996;153:5-26
2 晏梅, 高冬玲, 文小岗, 付淑莉, 张云汉. 树突状细胞在食管癌T细胞抗肿瘤免疫中的作用. 中国肿瘤临床 2000;27:663
, 百拇医药
3 路静, 杨洪艳, 赵军, 黄幼田, 赵国强, 董子明. 人B7-2和增强型绿色荧光蛋白融合基因表达载体的构建. 山东医药
2005;45:19-20
4 Chen L. Manipulation of T cell response to tumors by targeting oncostimulatory pathway. Leukemia 1997;
11(Suppl 3):567-569
5 Pizzoferrato E. B7-2 expression above a threshold elicitsanti-tumor immunity as effective as interleukin-12 and
prolongs survival in murine B-celllymphoma. Int J Cancer 2004;110:61-69
, 百拇医药
6 Yoon SK, Kim TG, Cho HI, Lee BS, Cho SH, Han NI, Lee YS, Jang JW,Chung KW, Sun HS, Kim BS. Suppression of
tumor formation and induction of naturalkiller cell activity in BALB/c nude mice by human B7-1 (CD80) gene
transfer subcutaneously injected with humanhepatocellular carcinoma cells (Huh-7). Taehan Kan Hakhoe
Chi 2003;9:124134
7 Wilson JL, Charo J, Martin-Fontecha A, Dellabona P, Casorati G,Chambers BJ, Kiessling R, Bejarano MT, Ljunggren HG.
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NK cell triggering by the humancostimulatory molecules CD80 and CD86. J Immunol 1999;163:4207-4212
8 Steinbrink K, Wolfl M, Jonuleit H, Knop J, Enk AH. Induction oftolerance by IL-10-treated dendritic cells. J
Immunol 1997;159:4772-4780
9 裴莉, 陈洁平, 梁后杰. 人脐血不同前体细胞体外诱导分化为树突状细胞的研究. 重庆医学 2004;33:835-837
10 Steinman RM, Turley S, Mellman I, Inaba K. The induction oftolerance by dendritic cells that have captured
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apoptotic cells. J Exp Med 2000;191:411-416
11 Geiger JD, Hutchinson RJ, Hohenkirk LF, McKenna EA, Yanik GA,Levine JE, Chang AE, Braun TM, Mule JJ. Vaccination
of pediatric solid tumor patientswith tumor lysate-pulsed dendritic cells can expand specific T cells andmediate
tumor regression. Cancer Res 2001;61:8513-8519
12 Chen LP. Immunological ignorance of silent antigens as anexplanation of tumor evasion. Immunol Today
1998;19:27-30
编辑 潘伯荣 审读 张海宁, http://www.100md.com( 路 静, 赵 军, 宋 谦, 崔自由, 赵国强, 黄幼田, 杨洪艳, 赵继敏, 董子明)
路静,女, 1974-04-01生, 河南省洛阳人,汉族.1996年河南医科大学本科毕业,现为郑州大学基础医学院病理生理学专业在读硕士,主要从事肿瘤的生物治疗的研究.
河南省科技厅攻关项目,No. 0324410034
河南省科技创新人才工程,No. 20008402
通讯作者:董子明,450052, 河南省郑州市,郑州大学基础医学院病理生理教研室. dongzm@zzu.edu.cn
电话:0371-66658157
, 百拇医药
收稿日期:2005-03-06 接受日期: 2005-04-08
Combination effect of human B7-2 and dentric cells vaccines in anti-esophageal cancer in vitro
Jing Lu, Jun Zhao, Qian Song, Zi-You Cui, Guo-Qiang Zhao, Hong-Yan Yang,You-Tian Huang, Ji-Min Zhao, Zi-Ming Dong
Jing Lu, Jun Zhao, Qian Song, Zi-You Cui, Guo-Qiang Zhao, You-TianHuang, Hong-Yan Yang, Ji-Min Zhao, Zi-Ming Dong, Department ofPathophysiology, School of Basic Medical Sciences, Zhengzhou University,Zhengzhou 450052, Henan Province, China
, 百拇医药
Supported by the Key Project of Science and Technology Departmentof Henan Province, No.0324410034 and the Project of Science and TechnologyInnovation Talent of Henan Provience, No.20008402
Correspondence to: Dr. Zi-Ming Dong, Department of Pathophysiology,School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450052,Henan Province, China. dongzm@zzu.edu.cn
Received: 2005-03-06 Accepted: 2005-04-08
, 百拇医药
Abstract
AIM: To investigate the roles of human B7-2 combined with dentriccell (DC) vaccine in inducing anti-tumor immunity against esophagealcancer in vitro.
METHODS: Human esophageal cancer cell line EC9706 was transfectedwith the vector of pEGFP-N3-B7-2 by lipofectamine method. The mononuclearcells (MNCs) were separated from cord blood by density gradientcentrifugation (Ficall-Hypaque) and then were induced to differentiate bycell factors. On the 3rdday, EC9706 cytolysis antigen was added into the medium, in which DCsgradually matured and expressed special tumor antigen. After the maturedDCs were co-cultured with autologous T cells derived from cord blood for 3days, the T cells were activated to become tumor specific cytotoxic Tlymphocytes (CTL). The inhibition of CTL on the transfected anduntransfected EC9706 cells was detected by methyl thiazolyl tetrazolium (MTT)assay.
, 百拇医药
RESULTS: The EGFP-B7-2 fusion gene was expressed mainly on themembrane of EC9706 cells, which proved the transfection was successful.Mature DCs with tumor cytolysis antigene was able to activate naive Tcells to become tumor specialized CTL, and the CTL had significantinhibitory effect or killing response on EC9706 cells transfected withpEGFP-N3-B7-2(F = 21.672,P = 0.000).
CONCLUSION: The human B7-2 combined with DC vaccine can inducesignificant killing response on esophageal cancer cell.
, http://www.100md.com
Key Words: Human B7-2; Vaccine; Cord blood; Dendritic cell;Esophageal cancer; Cytotoxic T lymphocyte; Green fluorescent protein
Lu J, Zhao J, Song Q, Cui ZY, Zhao GQ, Huang YT, Yang HY, Zhao JM, Dong ZM.Combination effect of human B7-2 and dentric cells vaccines inanti-esophageal cancer in vitro. Shijie Huaren Xiaohua Zazhi 2005;13(12):1382-1385
摘要
, 百拇医药
目的: 研究人B7-2瘤苗和负载了肿瘤细胞冻融抗原的DC瘤苗体外联合诱导抗食管癌的免疫作用.
方法: 应用脂质体转染技术,将融合基因表达载体pEGFP-N3-B7-2转染人食管癌细胞株EC9706.采用密度梯度离心法从脐血中分离单个核细胞(MNC)后,获得单核细胞(Mo).在细胞因子作用下诱导分化，第3d加入EC9706的冻融抗原，共培养4d后获得负载肿瘤抗原的成熟树突状细胞(MDC).将致敏DC与从脐血中分离的T淋巴细胞共培养3d，获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝(MTT)法检测CTL对转染和未转染的EC9706的细胞毒作用.
结果: 融合基因在食管癌细胞株EC9706的胞膜上定位表达.脐血来源的DC可负载并递呈肿瘤抗原，激活自体T淋巴细胞，诱导肿瘤特异性CTL产生，对转染pEGFP-N3-B7-2的EC9706细胞有显著杀伤作用(F= 21.672，P= 0.000).
, 百拇医药
结论: 人B7-2瘤苗和DC瘤苗体外联合应用,诱导出明显的杀伤食管癌细胞的免疫效应.
关键词: 人B7-2;疫苗;脐血;树突状细胞;食管癌;细胞毒T淋巴细胞;绿色荧光蛋白
路静,赵军,宋谦,崔自由,赵国强,黄幼田,杨洪艳,赵继敏,董子明.人B7-2瘤苗与DC瘤苗体外联合诱导抗食管癌的作用.世界华人消化杂志 2005;13(12):1382-1385:空质粒组; B:重组质粒组.
图2 培养的脐血单个核细胞(IF×400). A:培养3 d; B:培养7 d.
, 百拇医药
3 讨论
当前，如何能使机体内肿瘤细胞成为好的抗原递呈细胞，进而激活抗肿瘤免疫成为至关重要的问题.就肿瘤细胞活化细胞而言，B7分子提供共刺激信号以顺式共刺激方式最为有效[4]，B7基因修饰的瘤细胞能诱导有效的免疫反应[5].抗原特异性CD8+T细胞介导的抗肿瘤免疫效应中，要求肿瘤细胞本身表达MHCI类分子并提呈抗原.对于MHCI类分子阴性的低免疫原性肿瘤来说，即使导入B7基因也难以诱导CTL免疫应答，但是B7分子能通过增强NK细胞活性杀伤低免疫原性肿瘤[6-7].Wilson et al[7]报道，鼠黑色素瘤细胞B16.F1转染人B7-1或B7-2增加了人NK细胞介导的肿瘤杀伤作用.用特异性抗体封闭B7-1或B7-2分子后，该杀伤作用显著降低.
, http://www.100md.com
肿瘤患者的DC存在表型和功能上的缺陷，不能引发有效的抗肿瘤免疫反应[8].反复大量抽取肿瘤患者的外周血去诱导DC，使大多数肿瘤患者不能耐受.我们采用脐血单个核细胞诱生DC是基于:(1)纯化CD34+干细胞费用高昂，不利于将来用于临床;(2)虽然CD34+干细胞是DC的主要前体细胞但并不是唯一的，脐血中的单核细胞也能在细胞因子的诱导下生成DC;(3)CD34+细胞来源的DC形成晚，分化成熟需12-14 d，单个核细胞诱生DC周期短，仅需7-9 d，更方便于应用，并且两种方法获得的DC的形态、CDla表面抗原的表达没有显著的差异，二者均具有刺激同种T细胞增殖的能力[9].DC在抗肿瘤免疫中发挥重要作用，有关运用DC治疗恶性肿瘤已成为热点.由于肿瘤免疫逃逸的机制之一是其阻止了DC的成熟，而非直接抑制DC的功能[10]，因此在体外将DC扩增或同时用肿瘤抗原致敏DC，使其分化成熟，然后将这种功能正常且携带相应的肿瘤抗原的DC回输体内，可有效地诱导机体产生肿瘤特异性CTL反应[11].
, 百拇医药
食管癌组织B7分子表达缺陷，不能引起有效的免疫应答[4].我们应用脂质体转染技术将pEGFP-N3-B7-2转染人食管癌细胞株EC9706，制备B7-2瘤苗.其抗瘤免疫机制是将T细胞活化所需要共刺激分子B7-2导入食管癌细胞EC9706，通过B7-CD28途径将瘤细胞与T细胞联系在一起，诱导出不依赖于APC的直接的T细胞激活;但有文献报道非免疫系肿瘤缺乏第一信号，即使提供共刺激信号也不能有效激活T细胞[7].故我们考虑联合应用DC瘤苗，利用DC高效的加工提呈抗原能力，尤其是已负载了抗原的DC-Pr瘤苗能更快速地直接将抗原提呈给T细胞，使之识别而活化.食管癌具有异质性大、抗原性弱，缺乏特异性肿瘤抗原的特点，和多数肿瘤一样不能激发有效的免疫反应[12].我们将上述两种途径有机结合在一起，在体外诱导出了有效的抗食管癌细胞效应，为食管癌的生物治疗提供一定的实验依据.如前所述，B7分子在体内还能通过增强NK细胞活性杀伤低免疫原性肿瘤，这为B7-2肿瘤疫苗的应用提供了进一步的理论依据.
, 百拇医药
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编辑 潘伯荣 审读 张海宁, http://www.100md.com( 路 静, 赵 军, 宋 谦, 崔自由, 赵国强, 黄幼田, 杨洪艳, 赵继敏, 董子明)
