摘要
目的 探讨乏氧对甘氨酸-N-甲基转移酶(Glycine-N- methyltransferase,GNMT)表达的影响,以及GNMT与缺氧诱导因子1α (hypoxia inducible factor 1 alpha,HIF1α)表达之间的关系。方法 HepG2,293T,H460 3株细胞常规培养并设立常氧组和乏氧组,待细胞生长至50%~60%融合后,将乏氧组细胞放入1%氧气乏氧箱继续培养24 h,常氧组继续常规培养24 h后,运用半定量PCR,实时荧光定量PCR方法分析HepG2,293T,H460 3株细胞乏氧后相对于常氧GNMT基因的表达变化;通过小RNA干扰方法敲低HepG2细胞中HIF1α基因和HIF2A基因,运用实时荧光定量技术检测干扰效率,并检测GNMT基因的表达变化。结果 乏氧能够下调HepG2,293T,H460 3株细胞中GNMT基因的表达,HepG2细胞中抑制最明显;3株细胞的GNMT基因表达抑制率分别为68%,17%,21%;在HepG2细胞中敲低HIF1α基因能够引起GNMT表达明显上调,而敲低HIF2A后GNMT表达无明显变化。结论 在HepG2细胞中乏氧可能通过HIF1α下调GNMT的表达。
Objective To explore the changing of glycine-N-methyltransferase (GNMT) gene expression during hypoxia,and the relationship between GNMT and hypoxia inducible factor 1 alpha (HIF1α). Methods The HepG2,293T,H460 cell lines were cultured and divided into two groups:hpoxia goup and normal group.After grown to 50% to 60% confluence,the hyoxia group cells were incubated at 1%O2 for 24 hours,and the nomal group cells were cultrued nomally for 24 hours.Then,the alteration of the hypoxia group′s GNMT gene expression compared with the nomal group were observed by RT-PCR and realtime PCR;HIF1A gene and HIF2A gene were knock down by small interfering RNA (siRNA) in HepG2 cells,transfection efficiency and GNMT gene expression were detected by realtime PCR. Results Hypoxia down regulate GNMT gene expression in three cell lines,especially in HepG2 cells.The inhibitory rate of HepG2,293T,H460 cells was 68%,17%,21%.HIF1A knockdown led to significant increased expression of GNMT gene in HepG2 cells,howeverHIF2A knockdown can′t. Conclusions Hpoxia down regulate GNMT gene expression in a HIF1α dependent way in HepG2 cells.
出处
《复旦学报(医学版)》
CAS
CSCD
北大核心
2013年第5期511-515,共5页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金项目(81071180)~~