异丙酚对脂多糖诱导肺泡巨噬细胞高迁移率族蛋白B1启动子转录活性的影响  

Effect of propofol on the transcriptional activity of HMGB1 promoter in alveolar macrophages induced by lipolmlysaccharide

作　　者：丁宁[1] 肖慧[2] 许立新[1] 佘守章[1] 

机构地区：[1]广州市第一人民医院麻醉科,广州510180 [2]广州市第一人民医院门诊部,广州510180

出　　处：《中华急诊医学杂志》2010年第2期156-160,共5页Chinese Journal of Emergency Medicine

摘　　要：目的探讨异丙酚对脂多糖（lipopolysaccharide，LPS）诱导肺泡巨噬细胞（alveolar macrophages，AM）高迁移率族蛋白B1（high mobility group box 1 protein，HMGBI）启动子转录激活的影响。方法以基因重组技术将HMGBI启动子克隆人荧光素酶报告基因表达载体pGL3-Basic，得到重组质粒pGt3-HMGB1P，采用脂质体介导的转染技术将质粒转染AM细胞。根据转染质粒和施加刺激的不同分为以下各组：未转染组；转染pGL3-Basic组；转染pGL3-HMGB1P组；转染pGL3-HMGB1P＋IPS（100mg／L）刺激组；转染pG13-HMGB1P＋IPS（100mg／L）＋异丙酚（5mg／L）处理组。通过检测荧光素酶活性来观察启动子活性变化，分别采用Western blot和RT-PCR方法检测细胞HMGB1蛋白和mRNA的表达。应用单因素方差分析进行不同组别间的比较。结果酶切和测序结果证实重组体pGt3-HMGB1P构建正确，荧光素酶活性检测显示pGL3-HMGB1P在AM细胞中有效表达。与转染pGL3-HMGB1P组比较，转染pGL3-HMGB1P＋LPS刺激组HMGB1启动子的转录活性（423±27），HMGB1蛋白（0．49±0．03）和mRNA（0．48±0．04）的表达均显著增加（P〈0．05）；与转染pGL3．HMGB1P＋LPS刺激组比较，转染pGL3．HMGB1P＋LPS＋异丙酚处理组HMGB1动子的转录活性（207±13），HMGB1蛋白（0．17±0．02）和mRNA（0．13±0．02）的表达均显著降低（P〈0．05）。结论异丙酚在转录水平通过抑制LPS诱导HMGB1启动子的转录激活而影响HMGB1的表达。Objective To investigate the effects of propofol on the transcriptional activity of high mobility group box 1 protein （HMGB1） promoter in alveolar macrophages （AMs） induced by lipopolysaccharide （LPS）. Method HMGBI promoter sequence was sub-cloned into the Sac Ⅰ/Hind Ⅲ sites of the firefly luciferase reporter gene vector, pGL3-Basic, to obtain the recombinant plasmid pGL3-HMGB1P. AMs were divided into follow- ing groups： untransfeetion group, pGL3-Basic transfection group, pGL3-HMGB1P transfection group, pGL3- HMGB1P＋ LPS （100 mg/L） group and pGL3-HMGB1P ＋ LPS（100 mg/L） ＋ propofol （5 mg/L） group. The rel- ative activities were determined in the cell lysates to evaluate the activity of HMGBI promoter. The expression of HMGB1 mRNA and HMGB! protein level were detected by using RT-PCR and Western blot analysis, respectively. Differences among groups were analyzed by using One-way ANOVA. Results Double restriction enzyme digestion and sequencing both confirmed the successful construction of the recombinant plasmid pGL3-HMGB1P, which could be effectively expressed in AMs. Compared with pGL3-HMGB1P transfection group, the luciferase activity （423±27）, and the expression of HMGB1 mRNA （0.48 ± 0.04） and HMGB1 protein level （0.49 ± 0.03） in pGL3-HMGB1P ＋ LPS group were obviously increased （ P 〈 0.05）. Compared with pGL3-HMGB1P ＋ LPS group, the luciferase activity （207 ± 13）, and expression of HMGB1 mRNA （0.13 ±0.02） and HMGB1 protein level （0.17± 0.02） in pGL3-HMGBIP ＋ LPS ＋ propofol group were obviously decreased （P 〈 0.05）. Conclusions Propofol inhibits the activity of HMGB1 promoter and affects the expression of HMGB1 induced by LPS at the transcriptional level.

关 键 词：异丙酚 脂多糖 高迁移率族蛋白B1 肺泡巨噬细胞 转录活性 启动子 炎症反应 细胞因子 

分 类 号：R686[医药卫生—骨科学]