The effect of IL-31 on normal human epidermal keratinocytes

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 and sequenced. The expression of rhIL-31 in CHO cells was analyzed by RT-PCR and Western blot. The recombinant protein was purified with Ni2+ 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 STAT3 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 supernatant of stimulated HaCaT cells can chemoattract PBMC. The expression of chemokines and phosphorylated of STAT3 were up-regulated in HaCaT cells.Conclusion:Supernatant 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.