人CCL25基因慢病毒表达载体的构建及其对TEC黏附分子表达的影响
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摘要
通过构建人趋化因子CCL25基因过表达慢病毒载体并转染人原代胸腺上皮细胞(TEC),探讨过表达CCL25对人TEC黏附分子表达的影响,为研究CCL25在重症肌无力患者胸腺异常表达的作用奠定基础。PCR和DNA测序鉴定携带CCL25基因的慢病毒构建成功,病毒滴度约为4×108 Tu/ml,慢病毒对TEC感染效率达80%。Western blotting结果显示感染组细胞CCL25蛋白表达量显著增高。RT-PCR结果显示,与对照组相比,感染组HLA-A、HLA-DR表达无显著变化,P-selectin、VCAM-1和ICAM-1表达显著增高,提示我们人胸腺上皮细胞CCL25基因的过表达可一定程度影响TEC的功能特性,可能以通过增加P-selectin、VCAM-1和ICAM-1的表达量来影响淋巴祖细胞的胸腺归巢或胸腺内胸腺细胞的阳性选择过程,进而引起胸腺细胞发育异常,细胞亚群比例失调。
Our aims are to construct a lentiviral vector over-expressing human CCL25 gene,and to examine its ability to infect human thymus epithelial cell(TEC)in vitro.The primers for PCR amplification of CCL25 gene fragments were designed.The pCD513B-1vectors were digested by XbaI and EcoRI enzyme,followed by ligase mediated ligation to CCL25 fragments.Correctly connected cloning pCD513B-1-CCL25 was identified by PCR and DNA sequencing and was co-transfected into HEK 293 T cells with plasmid pMD2.0Gand plasmid psPAX2.The supernatant containing lentivirus lenti-CCL25 gene was harvested,and the titer value of lentiviral Vector was about 4×108 Tu/ml.The recombinant lentivirus were transfected into the original generation of human TEC(TEC obtained from thymic tissure of patients with congenital heart diseases).Expression rate of GFP was observed about 80%.Western blotting showed that the infected TEC had significantly higher expression of CCL25 protein.RT-PCR showed that the infected TEC had significantly higher expression of P-selectin、VCAM-1and ICAM-1 mRNA,but there was no difference in HLA-A and HLA-DR expression.
Our aims are to construct a lentiviral vector over-expressing human CCL25 gene,and to examine its ability to infect human thymus epithelial cell(TEC)in vitro.The primers for PCR amplification of CCL25 gene fragments were designed.The pCD513B-1vectors were digested by XbaI and EcoRI enzyme,followed by ligase mediated ligation to CCL25 fragments.Correctly connected cloning pCD513B-1-CCL25 was identified by PCR and DNA sequencing and was co-transfected into HEK 293 T cells with plasmid pMD2.0Gand plasmid psPAX2.The supernatant containing lentivirus lenti-CCL25 gene was harvested,and the titer value of lentiviral Vector was about 4×108 Tu/ml.The recombinant lentivirus were transfected into the original generation of human TEC(TEC obtained from thymic tissure of patients with congenital heart diseases).Expression rate of GFP was observed about 80%.Western blotting showed that the infected TEC had significantly higher expression of CCL25 protein.RT-PCR showed that the infected TEC had significantly higher expression of P-selectin、VCAM-1and ICAM-1 mRNA,but there was no difference in HLA-A and HLA-DR expression.
引文
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[11]Williams KM,Lucas PJ,Bare CV,et al.CCL25increases thymopoiesis after androgen withdrawal[J].Blood,2008,112:3255-3263.
[12]Carramolino L,Zaballos A,Kremer L,et al.Expression of CCR9beta-chemokine receptor is modulated in thymocyte differentiation and selectively maintained in CD8+T cells from secondary lymphoid organs[J].Blood,2001,97:850-857.
[13]Youn BS,Kim YJ,Mantel C,et al.Blocking of c-FLEPLindependent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction[J].Blood,2001,98:925-933.
[14]Lutz CP,Satwinder KS,Rosette JF,et al.Vascular cell adhesion molecule-1(VCAM-1)and intercellular adhesion molecule-1(ICAM-1)provide co-stimulation in positive selection along with survival of selected thymocytes[J].Mol Immunol,2008,45:42-48.
[15]Dzenetdina D,Zúniga-Pflücker JC.Positive selection of T cells,an in vitro view[J].Semin in Immunol,2010,22:276-286.
[16]张琳,郝选明,邓树勋,等.P-选择素/P-选择素糖蛋白配体-1与骨髓祖细胞胸腺归巢[J].天津师范大学学报(自然科学版),2011,31:77-81.
[2]Zaballos A,Gutiérrez J,Varona R,et al.Cutting edge:identification of the orphan chemokine receptor GPR-9-6as CCR9,the receptor for the chemokine TECK[J].J Immunol,1999,162:5671-5675.
[3]Meurens F,Whale J,Brownlie R,et al.Expression of mucosal chemokines TECK/CCL25and MEC/CCL28during fetal development of the ovine mucosal immune system[J].Immunology,2007,120:544-555.
[4]Hill ME,Shiono H,Newsom-Davis J,et al.The myasthenia gravis thymus:a rare source of human autoantibody-secreting plasma cells for testing potential therapeutics[J].J Neuroimmunol,2008,15:50-56.
[5]苏艳宾,李倩如,高峰,等.趋化因子及其受体在重症肌无力患者胸腺组织的异常表达[J].现代免疫学,2011,31:248-252.
[6]蒋觉安,蒋建华,薛群,等.重症肌无力患者外周血CD28-T细胞亚群变化及临床意义[J].现代免疫学,2012,32:458-462.
[7]Wang W,Malani M,Ostlie N,et al.C57BL/6mice genetically deficient in IL-12/IL-23and IFN-gamma are susceptible to experimental autoimmune myasthenia gravis,suggesting a pathogenic role of non-Th1cells[J].J Immunol,2007,178:7072-7080.
[8]Panse RL,Clairac GR,Bismuth J,et al.Microarrays reveal distinct gene signatures in the thymus of seropositive and seronegative myasthenia gravis patients and the role of CC chemokine ligand 21in thymic hyperplasia[J].J Immunol,2006,177:7868-7879.
[9]Amel M,Geraldine CC,Rozen LP,et al.The chemokine CXCL13is a key molecule in autoimmune myasthenia gravis[J].Blood,2006,108:432-439.
[10]Gossens K,Naus S,Corbel SY,et al.Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25[J].J Exp Med,2009,206:761-778.
[11]Williams KM,Lucas PJ,Bare CV,et al.CCL25increases thymopoiesis after androgen withdrawal[J].Blood,2008,112:3255-3263.
[12]Carramolino L,Zaballos A,Kremer L,et al.Expression of CCR9beta-chemokine receptor is modulated in thymocyte differentiation and selectively maintained in CD8+T cells from secondary lymphoid organs[J].Blood,2001,97:850-857.
[13]Youn BS,Kim YJ,Mantel C,et al.Blocking of c-FLEPLindependent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction[J].Blood,2001,98:925-933.
[14]Lutz CP,Satwinder KS,Rosette JF,et al.Vascular cell adhesion molecule-1(VCAM-1)and intercellular adhesion molecule-1(ICAM-1)provide co-stimulation in positive selection along with survival of selected thymocytes[J].Mol Immunol,2008,45:42-48.
[15]Dzenetdina D,Zúniga-Pflücker JC.Positive selection of T cells,an in vitro view[J].Semin in Immunol,2010,22:276-286.
[16]张琳,郝选明,邓树勋,等.P-选择素/P-选择素糖蛋白配体-1与骨髓祖细胞胸腺归巢[J].天津师范大学学报(自然科学版),2011,31:77-81.