TREM-1siRNA对内毒素刺激巨噬细胞杉RAW264．7分泌炎性因子影响

目的探讨靶向小鼠髓样细胞触发受体1（triggering receptor expressed on myeloid cells-1，TREM-1）的小分子干扰RNA（small inter-feting RNA，siRNA）在体外对内毒素刺激小鼠巨噬细胞株RAW264．7分泌肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）、白纽胞介素1p（interleukin1B，IL-1β）的抑制作用。方法合成1条靶向小鼠TREM-1分子的siRNA序列，以增强型绿色荧光蛋白（EGFP）作为报告因子，荧光显微镜观察EGFP的荧光强度，验证siRNA序列的干扰率。以pLKO1-1为载体构建针对TREM-1的pLKO1．1-TREM1-shRNA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（LPS组）；空质粒组（pLKO1．1组）：采用脂质体法将pLKO1．1转染细胞；干扰组（siRNA组）：将pLKO1．1-TREM1-shRNA转染细胞；转染72h后用内毒素刺激24h，并以实时定量PCR分别检测TREM．1、TNF-α与IL-1β的表达；以ELISA分别检测上清液中TNF-α、IL-1β含量。结果与LPS组比较，siRNA组中TREM．1、TNF—α、IL-1β的mRNA显著下降（P〈0．01），上清液中TNF-α、IL-1β含量明显降低（P〈0．01）。结论小分子干扰RNA可能通过抑制TREM-1基因的表达而减少内毒素诱导小鼠巨噬细胞264．7中TNF-α、IL-1β的分泌。

Effects of TREM-1 siRNA on Lpopolysaecharide-induced Inflammatory Factor Secretion from Mice Macrophage Cell Lines RAW264.7

Objective To investigate the role of TREM-1 in tumor necrosis factor-α （TNF-α） and interleukin-1β （IL- 1β） secretion from lipopolysaccharide induced mice macrophage cell lines RAW264.7. Methods The small interfering RNA （siRNA） that targeted on TREM-1 of mice was synthesized, and the enhanced green fluorescent protein was used as report factor,the cells were observed under inverted fluorescen microscoper to detect the suppression effect of siRNA on EGFP expression. Then pLKO1.1-puro-TREM-1 that targeted on TREM-1 was constructed with the pLKO1.1 was used as carrier. The mice macrophage cell lines RAW264. 7 were divided into 4 groups： control group （control）; lipopolysaccharide group （LPS） ; empty plasmid group （pLKO1.1） （just transfected with pLKO1.1 ） ; interference group （siRNA） （transfected with pLKO1. 1-puro-TREM1.24 h after stimulated with LPS）. The 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-1β were decreased sinificantlvC P 〈0.01）; moreover, the concentrations of TNF-α and IL-1β were lower than other groups significantly （P 〈 0. 01 ）. Conclusion Small interfering RNA may reduced TNF-α, IL-1β secretion in LPS-induced macrophage 264. 7 through inhibiting the expression of TREM-1 gene.