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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。
Objective To explore the role of myeloid cell trigger receptor 1 in the secretion of tumor necrosis factor-α and interleukin-1β by endotoxin-stimulated murine macrophage cell line RAW264.7 using small interfering RNA. Methods Small interfering RNA with high interference rate was designed and synthesized. Plasmid pLKO1.1-myeloid cell-triggered receptor 1 was constructed by using pLKO1.1 as a vector. The murine macrophage cell line RAW264.7 was divided into 4 groups: blank group; endotoxin group; empty plasmid group (pLKO1.1 group): pL-KO1.1 transfected cells by liposome method; Small interfering RNA group): transfected cells with pLKO1.1-myeloid cell trigger receptor 1, 24 h after endotoxin stimulation real-time quantitative PCR were detected myeloid cell trigger receptor 1, tumor necrosis factor alpha and interleukin 1β mRNA levels were detected by ELISA were detected in the supernatant of tumor necrosis factor α, interleukin 1β content. Results Compared with the endotoxin group, the mRNA level of tumor necrosis factor alpha and interleukin 1 beta in the small interfering RNA-RNA interference group was significantly decreased (P <0.01). The tumor necrosis in the cell culture supernatant Factor α, interleukin 1β content was significantly lower (P <0.01). Conclusions Small interfering RNA (siRNA) may decrease the secretion of TNF - α and IL - 1β by endotoxin - induced mouse macrophage RAW264.7 by inhibiting the expression of receptor 1 gene by myeloid cells.