小分子干扰RNA沉默髓样细胞触发受体1对内毒素诱导小鼠巨噬细胞株RAW264.7分泌炎性因子的影响

Effects of TREM-1 Gene Silencing on Lipopolysaccharide-induced Inflammatory Factor Secretion from Mice Macrophage Cell Lines RAW264.7

目的利用小分子干扰RNA探讨髓样细胞触发受体1在内毒素刺激小鼠巨噬细胞株RAW264.7分泌肿瘤坏死因子α、白细胞介素1β中的作用。方法设计并合成干扰率高的小分子干扰RNA,以pLKO1.1为载体构建pLKO1.1-髓样细胞触发受体1干扰质粒。将小鼠巨噬细胞株RAW264.7分为4组:空白组;内毒素组;空质粒组(pLKO1.1组):采用脂质体法将pL-KO1.1转染细胞;干扰组(小分子干扰RNA组):将pLKO1.1-髓样细胞触发受体1转染细胞,内毒素刺激24 h后实时定量PCR分别检测髓样细胞触发受体1、肿瘤坏死因子α与白细胞介素1β的mRNA水平;以ELISA法分别检测细胞上清液中肿瘤坏死因子α、白细胞介素1β含量。结果与内毒素组比较,小分子干扰RNA组细胞中髓样细胞触发受体1、肿瘤坏死因子α、白细胞介素1β的mRNA含量显著下降(P<0.01);细胞培养上清液中肿瘤坏死因子α、白细胞介素1β含量明显降低(P<0.01)。结论小分子干扰RNA可能通过抑制髓样细胞触发受体1基因的表达而减少内毒素诱导的小鼠巨噬细胞RAW264.7中肿瘤坏死因子α、白细胞介素1β的分泌。

ABSTRACT：OBJECTIVE To investigate the role of TREM-1 in tumor necrosis factor-α （TNF-α） and interleukin-1 β （IL-1 β） se- cretion from lipopolysaccharide-induced mice macrophage cell lines RAW264. 7. METHODS Designing and synthesizing small inter-fering RNA （siRNA） with high intangerference ratio, then constructing pLKO1.1-puro-TREM-I The mice maerophage cell lines RAW264. 7 were divided into four groups： control group （control）; lipopolysaecharide group （LPS）; empty plasmid group （pLKO1.1）--just transfected with pLKO1. 1 ; interference group （siRNA）--transfected with pLKO1.1-puro-TREM1.24 h after stimu- lation with LPS, real-time PCR was used to detect the mRNA levels of TREM-1, TNF-α and IL-1β respectively. The concentrations of TNF-α and IL-1β were assayed by ELISA. RESULTS In siRNA group,the mRNA levels of TREM-1 ,TNF-α and IL-1 β were de- creased significantly （P 〈 0. 01 ） ; moreover, the concentrations of TNF-α and IL-115 were lower than other groups significantly （P 〈 0. 01 ）. CONCLUSION Small interfering RNA may reduce TNF-α, IL-1 β secretion in LPS-induced macrophage 264. 7 through in- hibiting the expression of TREM-1 gene.