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目的筛选靶向晚期炎症因子高迁移率族蛋白1(High mobility group box 1 protein,HMGB1)基因的高效RNA干扰(siRNA)序列,并检测其体外生物学效应。方法选择针对HMGB1基因mRNA的3条不同序列,用T7体外转录系统合成3条siRNA,转染RAW264.7细胞,6 h后,加入500 ng/ml脂多糖(Lipopolysaccharide,LPS)分别刺激24、48 h,另设单纯LPS刺激组和只加培养液的对照组。采用RT-PCR检测各组细胞中HMGB1基因mRNA的转录水平,ELISA检测细胞培养上清液中HMGB1的含量。结果体外合成的HMGB1 siRNA1～3经琼脂糖凝胶电泳分析,均可见21 bp的RNA片段;经LPS刺激24和48 h,RAW264.7细胞中HMGB1基因mRMA的转录水平和细胞培养上清液中HMGB1的含量与对照组相比均显著升高,3条HMGB1 siRNA均能抑制细胞中HMGB1基因mRNA的转录水平,并减少细胞培养上清液中HMGB1的含量,其中以HMGB1siRNA1的效果最为明显。结论 LPS可刺激RAW264.7细胞释放大量HMGB1,同时增加细胞中mRNA的转录水平;HMGB1siRNA能有效降低HMGB1蛋白和mRNA水平,有望成为控制晚期炎症发展的新的治疗手段。
Objective To screen high-performance RNA interference (siRNA) sequences targeting the high mobility group box 1 protein (HMGB1) gene and test its biological effects in vitro. METHODS: Three siRNAs targeting HMGB1 gene were synthesized and transfected into RAW264.7 cells by using T7 in vitro transcription system. After 6 h, 500 ng / ml lipopolysaccharide (LPS) was added to stimulate 24,48 h, another set of pure LPS stimulation group and only add culture medium control group. The transcription level of HMGB1 mRNA in each group was detected by RT-PCR, and the content of HMGB1 in the cell culture supernatant was detected by ELISA. Results The mRNA of HMGB1 synthesized in vitro was analyzed by agarose gel electrophoresis and the 21 bp RNA fragment was observed. The mRNA level of mRMA of HMGB1 gene in RAW264.7 cells stimulated by LPS for 24 h and 48 h was significantly higher than that of the cell culture supernatant HMGB1 significantly increased compared with that of the control group. All the three HMGB1 siRNAs could inhibit the transcription of HMGB1 mRNA and decrease the content of HMGB1 in the cell culture supernatant, especially HMGB1siRNA1. Conclusion LPS stimulates the release of a large number of HMGB1 in RAW264.7 cells and increases the mRNA transcription level in cells. HMGB1 siRNA can effectively decrease the level of HMGB1 protein and mRNA, which is expected to be a new therapeutic strategy to control the development of advanced inflammation.