摘要
通过对组蛋白乙酰化和去乙酰化的研究,探索NO信号激活LO2细胞中凝血因子FⅧ（coagulation factorⅧ,FⅧ）重表达的分子机制。建立L-精氨酸激活人肝细胞LO2内源凝血因子FⅧ重表达的分子细胞模型。取对数生长期人类永生化肝细胞LO2随机分为：正常组和L-精氨酸组、组蛋白乙酰化抑制剂组和组蛋白去乙酰化抑制剂组。分别培养0、12 h、24 h、36 h、48 h、60 h。用RT-PCR方法检测各组中人FⅧ基因的转录水平。构建核转录因子（nuclear factor k B,NF-k B1）flag标签质粒,转染LO2细胞,用染色质免疫共沉淀（Ch IP）检测FⅧ基因启动子区域组蛋白乙酰化水平。实验显示L-精氨酸组和组蛋白去乙酰化抑制剂组中有人FⅧ基因m RNA的转录,正常组和组蛋白乙酰化抑制剂组没有出现FⅧ基因m RNA的转录。Ch IP检测NF-k B1与FⅧ基因启动子区域结合时,FⅧ基因启动子区域组蛋白乙酰化水平提高。上述研究表明组蛋白乙酰化抑制剂能取消LO2细胞中内源人FⅧ基因的上调,而组蛋白去乙酰化抑制剂能协同LO2细胞中内源人FⅧ基因的上调。NO信号在LO2细胞中通过调节FⅧ基因启动子区域组蛋白乙酰化,招募转录因子NF-k B1激活人FⅧ基因的重表达。
Through the research on the acetylation and deacetylation of histone, to explore the molecular mechanism of NO signaling to activate the expression of coagulation factor Ⅷ （FⅧ） in LO2 cells. To build L-arginine to the culture medium of human liver cells LO2 in vitro, to make LO2 re-express FⅧ. The LO2 cells at logarithm growth phase were selected and randomly divided into blank control group and L-arginine group, the C646 group and the SAHA group respectively, culture 0 h, 12 h, 24 h, 36 h, 48 h, 60 h. RT-PCR method was used to detect the transcription level of FⅧ gene in each group. （NF-kB1） flag plasmid was transfected into LO2 cells. The levels of histone acetylation in the promoter region of FⅧ gene were detected by immunoprecipitation （CHIP）. Results RT-PCR show that L-arginine group and the SAHA group has the transcription of human FⅧ mRNA in LO2 cells, The blank control group and the C646 group had no change. ChIP detection NF-kB1 and FⅧ gene promoter region binding, FⅧ gene promoter region histone acetylation level increased. The histone acetylation inhibitor can abrogate the re-expression of human FⅧ gene. Histone deacetylation inhibitors enhance the re-expression of human FⅧ gene. NO signaling in LO2 cells by regulating the FⅧ gene promoter region histone acetylation,recruiting transcription factor NF-kB 1 activated the expression of the human FⅧ gene.
作者
代晓璐
刘桂岑
苏小超
张军
Dai Xiaolu;Liu Guicen;Su Xiaochao;Zhang Jun(Department of Cell Biology and Genetics, Basic Medical College, Chongqing Medical University, Chongqing, 400016)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2018年第7期3232-3237,共6页
Genomics and Applied Biology
基金
重庆市教委科研项目(KJ08030)资助
