人IL-31的克隆表达及对表皮角化细胞的影响

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Ni^2＋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。

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.