期刊文献+

人IL-31的克隆表达及对表皮角化细胞的影响 被引量:10

The effect of IL-31 on normal human epidermal keratinocytes
下载PDF
导出
摘要 目的克隆人IL-31基因,构建真核表达载体,研究人IL-31对表皮角化细胞HaCaT的影响及其作用机制。方法PMA、PHA刺激正常人外周血单个核细胞,提取细胞总RNA,采用RT-PCR克隆人IL-31基因,并将其克隆到真核表达载体pcDNA3.1/myc-His(-)A,进行PCR和双酶切鉴定,并进行序列测定。将重组质粒转染CHO细胞,用RT-PCR和Western blot分析rhIL-31在CHO细胞中的表达。用Ni2+树脂纯化his融合蛋白,用不同剂量rhIL-31刺激HaCaT细胞,利用Transwell穿孔板检测HaCaT细胞培养上清对外周血单个核细胞的趋化作用,荧光定量PCR检测rhIL-31对HaCaT表达趋化因子的影响,Western blot检测STAT3磷酸化。结果成功获得全长人IL-31基因,测序正确,经双酶切、PCR和序列测定鉴定,真核表达质粒构建正确,可在CHO细胞中表达;该目的蛋白刺激人表皮角化细胞HaCaT,细胞培养上清对外周血单个核细胞有趋化作用,HaCaT细胞表达趋化因子MDC、TARC、I-309;细胞STAT3磷酸化增加。结论IL-31作用于正常人表皮角化细胞,细胞培养上清对外周血单个核细胞有趋化作用,HaCaT细胞趋化因子表达增加,细胞STAT3磷酸化增加,提示IL-31可通过STAT3发挥作用。 Objective:To clone the encoding sequence of human IL-31 gene, construct recombinant eukaryotic expression plasmid vector ,and study the effects of rhIL-31 on HaCaT cells and its mechanism for the functions, Methods:Total RNAs were isolated from peripheral blood mononuclear cells after a twelve-hour activation with PMA and PHA. Full-length human IL-31 cDNA was amplified by RT-PCR and cloned into eukaryotic expression plasmid pcDNA3. 1/myc-His( - )A an. sequenced. The expression of rhIL-31 in CHO cells was analyzed by RT-PCR and Western blot. The recombinant protein was purified with Ni^2+ resin. Chemotaxis of the supernatant of HaCaT cells to PBMC was detected through transwell madreporic plate after HaCaT cells were stimulated by different dose of rhIL-31. The promoting effect of the recombinant protein on the production of MDC, TARC and I-309 was detected by real-time RT- PCR. Phosphorylation of STA33 in HaCaT cells was analyzed by Western blot. Results : Obtained full encoding sequence of hIL-31 was identical with that included in Genbank, and the eukaryotic expression vector pcDNA3.1/myc-His( - )A-hIL31 was construct correctly. The recombinant IL-31 could be expressed in CHO cells. It was found that superuatant of stimulated HaCaT cells can chemoattract PBMC. The expression of chemokines and phosphorylated of STAT3 were up-regulated in HaCaT cells, Conclusion: Superuatant of HaCaT cells can chemoattract PBMC after being trigered with rhIL-31 for 6 h. The expression of chemokines was up-regulated in HaCaT cells. HaCaT cells respond to IL-31 stimulation by activating STAT3. [Key words] Human IL-31;Chemokines;Chemotaxis;STAT3
出处 《中国免疫学杂志》 CAS CSCD 北大核心 2007年第4期369-373,共5页 Chinese Journal of Immunology
关键词 人IL-31 表皮角化细胞趋化因子 趋化作用 STAT3 Human IL-31 Chemokines Chemotaxis STAT3
  • 相关文献

参考文献9

  • 1Dillon S R,Sprecher C,Hammond A et al.Interleukin 31,a cytokine produced by actived T cell,induces dermatitis in mice[ J ].Nat Immunol,2004 ;5:752-760. 被引量:1
  • 2Diveu C,Lelievre E,Perret D et al.GPL,a novel cytokine receptor related to GP130 and leukemia inhibitory factor receptor[ J].J Biol Chem,2003 ;278:49850-49859. 被引量:1
  • 3Diven C,Lak-Hal A H,Frofer J et al.Predominant expression of GP130-like(GPL) receptor is required for interkin-31 signaling[ J].Eur Cytokine Netw,2004; 15:291-302. 被引量:1
  • 4Akiko T,Iwao A,Masananori S et al.Expression of IL-31 gene transcripts in NC/Nga mice with atopic dermatitis[ J].European J Pharmacology,2005 ;516:180-181. 被引量:1
  • 5Bilsborough J,Leung D Y,Maurer M et al.IL-31 is associated with cutaneous lymphocyte antigen-positive skin homing T cells in patients with atopic dermatitis[ J].J Allergy Clin Immunol,2006; 117 (2):418-425. 被引量:1
  • 6Sonkoly E,Muller A,Lauerma A I et al.IL-31:a new link between T cells and pruritus in atopic skin inflammation[ J ].J Allergy Clin Immunol,2006 ;117 (2):411-417. 被引量:1
  • 7Activation of phosphoinositide 3-kinases by the CCR4 ligand macrophage-derived chemokine is a dispensable signal for T lymphocyte chemotaxis[ J].J Immunol,2004; 172(12):7761-7770. 被引量:1
  • 8Morita E,Hiragun T,Mihara S et al.Determination of thymus and activation-regulated chemokine (TARC)-contents in scales of atopic dermatitis[J].J Dermatol Sci,2004; 34:237-240. 被引量:1
  • 9Haque N S,Zhang X,French D L et al.CC chemokine I-309 is the principal monocyte chemoattractant induced by apolipoprotein (a) in human vascular endothelial cells[ J].Circulation,2000; 102:786-792. 被引量:1

同被引文献93

引证文献10

二级引证文献18

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