期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

人CXCR3基因转染细胞株的构建、鉴定及初步功能研究 被引量：5

Establishment of human CXCR3 gene-transfected cell line and study on its biological function
暂未订购
导出
摘要 目的:构建含有人CXCR3基因的重组逆转录病毒载体,获得稳定表达人CXCR3分子的基因转染细胞株L929-CXCR3,研究CXCR3与其配体Mig、IP-10及I-TAC相互作用引起的L929-CXCR3的迁移效应。方法:TRIzol一步法从经PHA和IL-2活化的人PBMC中抽提总RNA,RT-PCR扩增人CXCR3全长基因,装入逆转录病毒载体pEGZ-term,与两辅助病毒载体脂质体法共转染包装细胞293T,收集培养上清感染L929细胞,500μg/mlzeocin加压筛选获得稳定表达人CXCR3分子的基因转染细胞株L929-CXCR3。采用流式细胞术和RT-PCR分别从蛋白及基因水平对CXCR3的表达进行鉴定。Transwell分析L929-CXCR3细胞在Mig、IP-10及I-TAC作用下的迁移率。结果:构建了含人CXCR3基因的重组逆转录病毒载体,建立了人CXCR3基因转染细胞株L929-CXCR3,其膜表面CXCR3分子的阳性表达率为98.4%;趋化L929-CXCR3发生迁移的最小配体浓度分别为Mig10ng/ml、IP-105ng/ml及I-TAC1ng/ml,相应迁移率分别为2.46%、2.34%及2.24%。结论:L929-CXCR3细胞株的建立为研究CXCR3信号转导与生物学功能及制备相应的单克隆抗体奠定了基础。 Objective：To construct recombinant human CXCR3 gene retroviral vector and obtain L929-CXCR3 gene transfected cell line for stably expressing human CXCR3.Further conduct study on L929-CXCR3 migratory effect resulted from interaction by CXCR3 and its ligands Mig,IP-10 and I-TAC.Methods：Total RNA was extracted from activated human peripheral blood mononuclear cells by PHA and IL-2 with TRIzol.Human CXCR3 gene of full length was amplified by RT-PCR,and then it was inserted into retrovirus vector pEGZ-term.The recombinant vector together with its helper virus vector were co-transfected into package cell 293T with LipofectamineTM 2000.The supernatant of 293T was collected for infecting L929 cells,and cell clones stably expressing human CXCR3 molecule were screened by zeocin（500 μg/ml）.Used FCM and RT-PCR to verify expression of CXCR3 at protein level and gene level,respectively.Migratory ability of L929-CXCR3 interacted with its ligands was studied by transwell system.Results：Had constructed recombinant human CXCR3 gene retroviral vector and obtained L929-CXCR3 gene-transfected cell line.Positive expression rate of CXCR3 on membrane was 98.4%,and the cells could directly migrate when induced by its ligands.For 10 ng/ml Mig,5 ng/ml IP-10 and 1 ng/ml I-TAC,the migration rates was 2.46%,2.34% and 2.24% respectively.Conclusion：Construction of L929-CXCR3 cell line has laid a good foundation on research of biologic characteristics of CXCR3 signal path and preparation of CXCR3 monoclonal antibody.
作者 孙杰 刘玉华 陈永井 郭静雅 陈昌友 胡玲玲 陈明心 杜阳阳 徐耀瑜 邱玉华
机构地区 苏州大学医学部免疫学系 杭州师范大学附属医院检验科 南京医科大学附属第二医院检验科
出处 《中国免疫学杂志》 CAS CSCD 北大核心 2010年第5期387-391,共5页 Chinese Journal of Immunology
基金 科技部科技重大专项资助(No.2009ZX09103-705)
关键词 CXCR3 趋化因子 基因转染 逆转录病毒 稳定表达 CXCR3 Chemokine Gene transfection Retrovirus Stably expression
分类号 R392.11 [医药卫生—免疫学]
  • 相关文献

参考文献17

  • 1Liping L,Melissa K C,De R H et al.Chemokine receptor CXCR3:an unexpected enigma[J].Curr Top Dev Biol,2005;68:149-181.
  • 2Marcel L,Pius L,Nicole B et al.Lymphocyte-specific chemokine receptor CXCR3:regulation,chemokine,binding and gene localization[J].Eur J Immunol,1998;28(14):3696-3705.
  • 3Laura L,Michela F,Francesco A et al.An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10,Mig,and I-TAC,and acts as functional receptor for platelet factor 4[J].J Exp Med,2003;197(11):1537-1549.
  • 4杨秀利,储以微,邓富刚,熊思东.IP10对机体抗肿瘤免疫应答的增强作用及其机制[J].中国免疫学杂志,2006,22(2):99-103. 被引量：8
  • 5Robert M S,Marie D B,Javier M et al.Cancer CXC chemokine networks and tumour angiogenesis[J].Eur J Cancer,2006;42(6):768-778.
  • 6Marcel L,Basil G,Pius L et al.Chemokine receptor specific for IP-10 and Mig:structure,function,and expression in activated T-lymphocytes[J].J Exp Med,1996;184:963-969.
  • 7Romagnani P,Lasagni L,Annunziato F et al.CXC chemokines:the regulatory link between inflammation and angiogenesis[J].Trends Immunol,2004;25(4):201-209.
  • 8Katherine E C,Christine A S,Timothy J P et al.Interferon inducible T cell alpha chemoattractant (I-TAC):a novel Non-ELR CXC chemokine with potent activity on activated T cells through selective high afflnity Binding to CXCR3[J].J Exp Med,1998;187(12):2009-2021.
  • 9Tonya C W,Salah R,Xinrong M et al.Antagonism of CXCR3 inhibits Lung metastasis in a murine model of metastatic breast cancer[J].Cancer Res,2006;66:(15):7701-7707.
  • 10康辉,王亚男,范霞,姜拥军,刁莹莹,代娣,尚红.趋化因子及受体与中国HCV/HIV合并感染相关性的研究[J].中国免疫学杂志,2006,22(6):569-572. 被引量：1

二级参考文献29

  • 1Ochsenbein A F,Sierro S,Odermatt B et al.Roles of tumor localization,second signals and cross priming in cytotoxic T-cell induction[J].Nature,2001;411(6841):1058-1064.
  • 2Mohan K,Ding Z,Hanly J et al.IFN-gamma-inducible T cell alpha chemoattractant is a potent stimulator of normal human blood T lymphocyte transendothelial migration:differential regulation by IFN-gamma and TNF-alpha[J].J Immunol,2002;168(15):6420-6428.
  • 3Ogasawara K,Hida S,Weng Y et al.Requirement of the IFN-alpha/beta-induced CXCR3 chemokine signalling for CD8^+T cell activation[J].Genes Cells,2002;7(3):309-320.
  • 4Yu P,Liu W,Chin R K et al.Priming of Naive T cells inside tumors leadsto eradication of established tumors[J].Nature Immunology,2004;5(2):141-149.
  • 5Hensbergen P J,Wijnands P G J T B,Schreurs W J et al.The CXCR3 targeting chemokine CXCL11 has potent antitumor activity in vivo involving attraction of CD8^+T lymphocytes but not inhibition of angiogensis[J].J Immunother,2005;28(4):343-351.
  • 6Dufour J H,Dziejman M,Liu M T et al.IFN-gamma-inducible protein 10(IP-10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking[J].J Immunol,2002;168(7):3195-3204.
  • 7Dorsey R,Kundu N,Yang Q et al.Immunotherapy with interleukin-10 depends on the CXC chemokines inducible protein-10 and monokine induced by IFN-gamma[J].Cancer Res,2002;62(9):2606-2610.
  • 8Pertl U,Luster A D,Varki N M et al.IFN-gamma-inducible protein-10 is essential for the generation of a protective tumor-specific CD8+T cell response induced by single-chain IL-12 gene therapy[J].J Immunol,2001;166(11):6944-6951.
  • 9Luster A D,Leder P.IP10,a CXC-chemokine,elicits a potent thymus-dependent antitumor response in vivo[J].J Exp med,1993;178(3):1057-1065.
  • 10Regulier E,Paul S,Marigliano M et al.Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach[J].Cancer Gene Ther,2001;8(1):45-54.

共引文献7

同被引文献33

  • 1高金明 ,LUBao .趋化因子CXC受体3在博莱霉素诱导的肺纤维化中的作用[J].中华结核和呼吸杂志,2005,28(1):28-32. 被引量：8
  • 2周京国,郭晓兰,唐中,袁国华,魏锦,杨明辉,何兰,蒋红.可溶性尿激酶型纤溶酶原激活物受体在RA患者血浆和滑液的水平及其临床意义[J].中华风湿病学杂志,2006,10(12):713-716. 被引量：5
  • 3李文珠,陶惠然,庄国洪,胡庆中,王生育,陈彩霞.抗死亡受体DR5抗体对大鼠佐剂型关节炎的作用机理探讨[J].中华微生物学和免疫学杂志,2007,27(7):651-655. 被引量：2
  • 4Laura L,Michela F,Francesco A et al.An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth Induced by IP-10,Mig,and I-TAC,and acts as functional receptor for platelet Factor 4[J].J Exp Med,2003;197(11):1537-1549.
  • 5Wang D,Dubois R N,Richmond A.The role of chemokines in intestinal inflammation and cancer[J].Curr Opin Pharmacol,2009;9(6):688-696.
  • 6Walser T C,Rifat S,Ma X et al.Antagonism of CXCR3 inhibits lung metastasis in a murine model of metastatic breast cancer[J].Cancer Res,2006;66(15):7701-7707.
  • 7Pradelli E,Karimdjee-Soilihi B,Michiels J F et al.Antogonism of chemokine receptor CXCR3 inhibits osteosarcoma metastasis to lungs[J].Int J Cancer,2009;125(11):2586-2594.
  • 8Slettenaar V I,Wilson J L.The chemokine network:a target in cancer biology?[J].Adv Drug Deliv Rev,2006;58(8):962-974.
  • 9Robledo M M,Bartolome R A,Longo N et al.Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells[J].J Biol Chem,2001;276(48):45098-45105.
  • 10Kawada K,Sonoshita M,Sakashita H et al.Pivotal role of CXCR3 in melanoma cell metastasis to lymph nodes[J].Cancer Res,2004;64(11):4010-4017.

引证文献5

二级引证文献14

中国免疫学杂志
2010年 第5期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部