IL-17A对山羊乳腺上皮细胞分泌免疫防御因子功能的影响

Effect of IL-17A on the secretory of immune defense factors in goat mammary epithelial cells

白细胞介素17(interleukin-17,IL-17)是Th17细胞的特征性细胞因子,被认为是一种关键的炎性细胞因子,在宿主抵抗胞外菌、真菌感染,以及炎症、超敏反应、自身免疫性疾病等多种疾病的病程中起到了重要的作用。为研究IL-17A对山羊乳腺上皮细胞免疫防御功能的影响,本研究根据GenBank公布的奶山羊IL-17A完全CDS区序列设计了真核表达引物,从Con A活化的奶山羊外周血单个核细胞(PBMCs)中提取总RNA,反转录PCR法扩增目的序列。扩增出的目的片段连接于真核表达载体pEGFP-N1,并转入大肠杆菌Trans5α感受态细胞保存,将鉴定正确的重组质粒命名为IL-17A-pEGFP-N1。将重组质粒转染入HEK293T细胞进行瞬时表达,收取真核表达上清作为刺激物。选取成年健康奶山羊,通过组织块贴壁法制备原代山羊乳腺上皮细胞(GMEC)。用IL-17A真核表达上清(终质量浓度:15 mg/L)刺激山羊乳腺上皮细胞,通过Real-time PCR方法检测GMEC分泌的各种促炎性细胞因子、趋化因子以及防御性蛋白表达水平的变化。结果表明,IL-17A可以促进山羊乳腺上皮原代细胞分泌多种促炎性细胞因子、趋化因子和防御性蛋白,暗示IL-17A可能参与了奶山羊乳腺炎的发生,并在山羊乳腺免疫防御中起到了一定的调节作用。

Interleukin-17(IL-17),the characteristic cytokine of Th17 cells,is considered to be a key inflammatory cytokine in host defense against extracellular bacteria,fungal infections,and also plays a pivotal role in various diseases such as inflammation,hypersensitivity reactions and autoimmune diseases.In the present study,the eukaryotic expression primers were designed according to the complete CDS region of the goat IL-17 A published on GenBank,and total RNA was extracted from Con A stimulated peripheral blood mononuclear cells(PBMCs)from dairy goats.The target fragment was amplified and ligated into the eukaryotic expression vector pEGFP-N1 and then the recombinant vector was transformed into E.coli Trans5αcompetent cells for storage,and the recombinant plasmid was named IL-17 A-pEGFP-N1.The recombinant plasmid was transfected into HEK293 T cells for transient expression and the supernatant was collected as stimulant.An adult healthy dairy goat in the active lactation period were chosen to sample the mammary tissue and the goat mammary epithelial primary cells(GMECs)were prepared by the tissue block adherence method.The supernatant(final concentration of 15 mg/L)of the IL-17 A-pEGFP-N1-HEK293 T cells was used to stimulate goat mammary epithelial cells,and the expression levels of various pro-inflammatory cytokines,chemokines and defensive proteins secreted by GMEC were detected by Real-time PCR.The results showed that IL-17 Acould promote the secretion of various pro-inflammatory cytokines,chemokines and defensive proteins from goat mammary epithelial primary cells,which suggested that IL-17 A may be involved in the development of mastitis in dairy goats and play a regulatory role in goat mammary gland immune defense.