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pLKO1.1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24h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-1β (IL-1β) secretion from lipopolysaccharide-induced mice macrophage cell lines RAW264.7. METHODS Designing and synthesizing small interfering RNA (siRNA) with high intangerference ratio,then constructing pLKO1.1-puro-TREM-1 The mice macrophage cell lines RAW264.7 were divided into four 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 stimulation 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-1β were decreased significantly (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 reduce TNF-α, IL-1β secretion in LPS-induced macrophage 264.7 through inhibiting the expression of TREM-1 gene.
关键词
髓样细胞触发受体1 /
小分子干扰RNA /
内毒素 /
小鼠巨噬细胞 /
肿瘤坏死因子α /
白细胞介素1βdoi：10.11669/cpj.2013.09.009
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Key words
striggering receptor expressed on myeloid cells-1 (TREM-1) /
small interfering RNA (siRNA) /
lipopolysaccharide (LPS) /
mice macrophage /
tumor necrosis factor α (TNF-α) /
interleukin 1β (IL-1β)
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中图分类号：
R965
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参考文献
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脚注
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基金
广东省社会发展重点引导项目（2009B030801335）
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