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浙江大学学报（医学版）
浙江大学学报（医学版）  2013, Vol. 42 Issue (5): 498-503    DOI: 10.3785/j.issn.1008-9292.2013.05.004
论著     
CCL21-CD40L融合蛋白的肿瘤免疫实验研究
宫婷1，李鸿立2，巴一2
1.天津医科大学总医院肿瘤科，天津 300052； 2.天津市肿瘤医院消化内科，天津 300060
CCL21-CD40L fusion gene induce augmented antitumor activity in colon cancer
GONG Ting1, LI Hong-Li2, BA Yi2
1.Department of Oncology,Tianjin General Hospital,Tianjin 300052,China;2.Department of gastrointestinal Oncology,Tianjin Cancer Hospital,Tianjin 300060,China
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摘要：

目的：探讨CCL21-exCD40L融合基因的抗肿瘤作用。
方法：构建CCL21-exCD40L真核表达载体，Western blot法检测CCL21-exCD40L融合蛋白的表达，Transwell法检测CCL21-exCD40L融合基因趋化活性，体外转染CCL21-exCD40L融合基因，观察其在小鼠结肠癌肿瘤模型中的抗肿瘤作用。
结果：成功构建真核表达载体pcDNA3.1+/CCL21-exCD40L，Transwell法验证融合蛋白对树突细胞具有较强趋化功能，其每高倍镜视野穿膜细胞数是空载体对照组的14.95倍。将融合基因原位转染肿瘤局部，荷瘤小鼠全部存活，肿瘤体积缩小。
结论：CCL21-exCD40L融合基因能通过真核细胞正常表达，CCL21-exCD40L融合蛋白能够趋化树突细胞，在体内发挥抗肿瘤作用。
关键词： 趋化因子CCL21;  CD40配体;  重组融合蛋白质类;  基因疗法;  结肠肿瘤;  疾病模型, 动物    
Abstract:

Objective： Toinvestigate the anti-tumor activity of CCL21-exCD40Leukaryotic expression vector.
Methods： CCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4 ，and then was cloned into expression vector pcDNA3.1+.pcDNA3.1+/CCL21 and pcDNA3.1+/exCD were constructed as negative control.Wsestern blot was used to identify the fusion protein.CHO cells was transfected with pcDNA3.1+/CCL21-exCD，pcDNA3.1+/CCL21 and pcDNA3.1＋，respectively.The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection.
Results： Gene sequencing and restrictive digestion proved the successful construction of pcDNA3.1+/CCL21-exCD40L，and its expression was conformed by western blot.The transfectant supernantes of pcDNA3.1+/CCL21-exCD40 group had a significant chmotactic function to DCs，of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field.After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse，the tumor mass reduced remarkablely，and all the mouse in fusion gene group survived after 4 weeks.
Conclusion： CCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time，which is more effective than all controlgroup.
Key words: Chemokine CCL21    CD40 ligand    Recombinant fusion proteins    Gene therapy    Colonic neoplasms    Disease models, Animal
收稿日期: 2012-11-15 出版日期: 2013-09-25
:  R 730.51  
基金资助:

天津市教委自然科学基金(20060111).
通讯作者: 巴一（1969-），女，博士，教授，博士生导师，主要从事消化道肿瘤的临床诊治及基础研究;E-mail:olingdar@hotmail.com   
作者简介: 宫婷（1984-），女，硕士，主要从事肿瘤治疗的临床及科研工作;E-mail:gt2096@gmail.com
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引用本文:

宫婷, 李鸿立, 巴一. CCL21-CD40L融合蛋白的肿瘤免疫实验研究[J]. 浙江大学学报（医学版）, 2013, 42(5): 498-503.

GONG Ting, LI Hong-Li, BA Yi. CCL21-CD40L fusion gene induce augmented antitumor activity in colon cancer. Journal of ZheJiang University(Medical Science), 2013, 42(5): 498-503.

链接本文:

https://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2013.05.004    或    https://www.zjujournals.com/med/CN/Y2013/V42/I5/498

[1] HENDON S E,DIPALMA J A.U.S.practices for colon cancer screening [J].Keio J Med,2005,54(4):179-183.
[2] HU Taishan,CHEN Haixu,YANG Yungui,et al(胡泰山,陈海旭,杨运桂，等).Construction and expression of ScFv against antibacterial peptide Magainin II [J].J Cell Mol Immunol(细胞与分子免疫学杂志),2000,16(4):317-320（in Chinese）.
[3] STEINMAN R M,POPE M.Exploiting dendritic cells to improve vaccine efficacy [J].J Clin Invest,2002,109:1519-1526.
[4] HALLIDAY G M,LE S.Transforming growth factor-beta produced by progressor tumors inhibits,while IL-10 produced by regressor tumors enhances,Langerhans cell migration from skin [J].Int Immunol,2001,13(9):1147-1154.
[5] OHM J E,CARBONE D P.VEGF as a mediator of tumor-associated immunodeficiency [J].Immunol Res,2001,23(2-3):263-272.
[6] HEDRICK J A,ZLOTNIK A,et al.Identification and characterization of a novel beta chemokine containing six conserved cysteines [J].J Immunol,1997,159(4):1589-1593.
[7] CHEN S C,VASSILEVA G,KINSLEY D,et al.Ectopic expression of the murine chemokines CCL21a and CCL21b induces the formation of lymph node-like structures in pancreas [J].J Immunol,2002,168(3):1001-1008.
[8] SHARMA S,STOLINA M,ZHU L,et al.Secondary lymphoid organ chemokine reduces pulmonary tumor burden in spontaneous murine bronchoalveolar cell carcinoma [J].Cancer Res,2001,61(17):6406-6412.
[9] ARMITAGE R J, FANSLOW W C, STROCKBINE L,et al.Molecular and biological characterization of a murine ligand for CD40 [J].Nature,1992,357(6373):80-82.
[10] CLAYTON A,COURT J,NAVABI H,et al.Analysis of antigen presenting cell derived exosomes,based on immuno-magnetic isolation and flow cytometry [J].J Immunol Methods,2001,247(1-2):163-174.
[11] LIU Y,QURESHI M,XIANG J.Antitumor immune responses derived from transgenic expression of CD40 ligand in myeloma cells [J].Cancer Biother Radiopharm,2002,17(1):11-18.
[12] HAASE C, MICHELSEN B K,JORGENSEN T N.CD40 is necessary for activation of naive T cells by a dendritic cell line in vivo but not in vitro [J].Scand J Immunol,2004,59(3):237-245.
[13] MAZZEI G J,EDGERTON M D.Herpes simplex virus (HSV) amplicon-mediated codelivery of secondary lymphoid tissue chemokine and CD40L results in augmented antitumor activity [J].Cancer Res,2002,62(22):6545-6551.
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