摘要
目的研究髓样分化蛋白2(MD-2)在脂多糖(LPS)诱导的急性肺损伤(ALI)大鼠肺组织中的表达及其作用。方法 20只健康雄性SD大鼠随机分为LPS组和对照组,通过支气管肺泡灌洗分离肺泡巨噬细胞,采用RT-PCR、Western blot和免疫组化方法分别检测MD-2 mRNA和蛋白的表达,ELISA方法测定血清肿瘤坏死因子α(TNF-α)水平。将MD-2 siRNA转染大鼠肺泡巨噬细胞系NR8383,以终浓度1μg/mL的LPS刺激细胞,采用RT-PCR和Western blot方法分别测定细胞MD-2mRNA和蛋白的表达,ELISA方法测定细胞培养上清液TNF-α水平。结果 LPS组大鼠肺泡巨噬细胞和肺组织标本中MD-2 mRNA和蛋白的表达均显著高于对照组(P<0.01),血清TNF-α水平明显升高(P<0.01)。在LPS刺激下,NR8383细胞MD-2 mRNA和蛋白的表达显著增加(P<0.01),细胞培养上清液中TNF-α水平升高(P<0.01)。经MD-2 siRNA处理后,NR8383细胞在LPS刺激下MD-2mRNA和蛋白的表达未见明显增加(P>0.05),细胞培养上清液中TNF-α水平未见明显升高(P>0.05)。结论 MD-2在LPS所致ALI大鼠肺组织中表达显著上调,沉默肺泡巨噬细胞MD-2基因可抑制LPS刺激引起的细胞因子分泌增加,提示MD-2在LPS所致大鼠ALI中起重要作用。
Objective To explore the expression of myeloid differentiation protein 2（MD-2） in rat lung and its role in acute lung injury（ALI） induced by lipopolysaccharide（LPS）.Methods Twenty male SD rats were randomly divided into a LPS group and a control group.The wet/dry ratios of lung tissues were measured and the histological changes of lung tissues were observed under microscope.Alveolar macrophages were collected from bronchial alveolar lavage fluid（BALF）.The MD-2 mRNA and protein expressions were detected by RT-PCR,Western blot,and immunohistochemistry respectively.The MD2-siRNA oligo were transfected into NR8383 cells and 1 μg/mL LPS was used to stimulate the cells.The expressions of MD-2 mRNA and protein were detected by RT-PCR and Western blot.The levels of TNF-α in rat serum and cell culture supernatant were detected by ELISA.Results Compared with the control group,the expressions of MD-2 mRNA and protein in alveolar macrophages and lung tissue were elevated（P0.01）,as well as the level of TNF-α in rat serum.The expressions of MD-2 mRNA and protein in NR8383 cell and the level of TNF-α in supernatant increased obviously after LPS stimulation （P0.01）.There were no changes of MD-2 mRNA and protein expressions and TNF-α of NR8383 cells treated by MD-2 siRNA with or without LPS stimulation（P0.05）.Conclusions The expression of MD-2 in lung increases obviously after challenged by LPS.Knockdown MD-2 gene of NR8383 cell by MD-2 siRNA can inhibit TNF-α secretion induced by LPS stimulation.MD-2 may play an important role in rat ALI induced by LPS.
出处
《中国呼吸与危重监护杂志》
CAS
2010年第6期614-618,共5页
Chinese Journal of Respiratory and Critical Care Medicine
基金
复旦大学附属中山医院青年基金项目(编号:2008027)
关键词
髓样分化蛋白2
急性肺损伤
脂多糖
肺泡巨噬细胞
Myeloid differentiation protein 2 Acute lung injury Lipopolysaccharide Alveolar macrophages
