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目的观察脂多糖(1ipopolyssac-chafide,LPS)及促炎症细胞因子IL-1β诱导人肾小管上皮细胞株(HK-2)β防御素-2(hBD-2)的表达及抗菌活性,探索肾脏的先天性防御病原体机制。方法给予不同质量浓度的LPS(0.1、1、10μg/ml)及IL-1β(0.1、1、10 ng/ml)刺激HK-2细胞12 h,采用实时荧光定量PCR检测HK-2细胞hBD-2 mRNA的表达,Western印迹及ELISA法检测hBD-2蛋白的表达。菌落计数法检测细胞上清对尿路致病性大肠杆菌(UTI89)和克雷白杆菌(TOP52)临床分离株的抗菌效果。结果1)HK-2细胞有微量hBD-2 mRNA表达,不同质量浓度的LPS、IL-1β刺激HK-2细胞后,hBD-2 mRNA表达呈剂量依赖性,与对照组比较差异具有统计学意义(P<0.01);2)不同质量浓度LPS及IL-1β刺激细胞后,均可诱导HK-2细胞分泌hBD-2,并在该实验质量浓度范围内呈剂量依赖性,与对照组比较差异具有统计学意义(P<0.05);3)1μg/ml的LPS及1 ng/ml的IL-1β刺激HK-2细胞12 h后,细胞浆中均可见hBD-2蛋白表达;4)LPS(1μg/ml)及IL-1β(1 ng/ml)刺激HK-2细胞24 h后的细胞上清对UTI89和TOP52有杀菌作用。结论一定剂量的LPS及前炎症细胞因子可诱导HK-2细胞hBD-2表达,hBD-2的表达可能是肾小管最初防御反应,起着发挥防御病原微生物及免疫重要作用。
Objective To observe the expression and antibacterial activity of hBD-2 induced by 1ipopolyssac-chafide (LPS) and pro-inflammatory cytokine IL-1β in human renal tubular epithelial cells (HK-2) Congenital defense against pathogens. Methods HK-2 cells were stimulated with different concentrations of LPS (0.1,1,10μg / ml) and IL-1β (0.1,1,10 ng / ml) for 12 hours. Real-time fluorescent quantitative PCR was used to detect hBD- 2 mRNA expression, Western blot and ELISA assay of hBD-2 protein expression. The colony counting method was used to detect the antibacterial effect of cell supernatant on the clinical isolates of UTI89 and TOP52. Results 1) The expression of hBD-2 mRNA was down-regulated in HK-2 cells. The expression of hBD-2 mRNA in HK-2 cells was dose-dependently different concentrations of LPS and IL-1β stimulated HK-2 cells compared with the control group (P <0.01). 2) HK-2 cells induced hBD-2 secretion after stimulated with different concentrations of LPS and IL-1β in a dose-dependent manner, (P <0.05) .3) The expression of hBD-2 protein in the cytoplasm of HK-2 cells stimulated by 1μg / ml LPS and 1ng / ml IL-1βfor 12 hours.4) The expression of LPS 1μg / ml) and IL-1β (1ng / ml) stimulated HK-2 cells 24 hours after the cell supernatant of UTI89 and TOP52 have a bactericidal effect. Conclusions A certain dose of LPS and pro-inflammatory cytokines can induce the expression of hBD-2 in HK-2 cells. The expression of hBD-2 may be the primary defensive response of tubules, which plays an important role in defense against pathogenic microorganisms and immunity.