HIF—1α在小鼠ACTH垂体腺瘤细胞AtT-20的抗凋亡作用

Antiapoptotic role of HIF-1α in mouse ACTH pituitary adenoma cells AtT-20 in hypoxia

目的观察缺氧诱导因子（HIF）-1α在小鼠垂体促肾上腺皮质激素腺瘤细胞ART-20生长增殖中的作用，验证HIF-1α能否保护AtT-20细胞免于低氧诱导的细胞凋亡。方法不同浓度的CoCl2诱导AtT-20细胞发生低氧，MTT方法观察CoCl2促进细胞生长增殖的作用，实时定量PCR和Western印迹检测常氧和低氧环境下HIF-1α mRNA和蛋白水平的改变；细胞转染HIF-1α-siRNA后，CoCl2低氧诱导，实时定量PCR和Western印迹验证siRNA下调HIF-1α的效率，凋亡检测系统Annexin V—FITC和TUNEL法检验细胞凋亡情况。结果在一定浓度（≤100μmol／L）和时间（≤48h）范围内，CoCl2可以促进AtT-20细胞的生长增殖，并呈浓度和时间正相关性；HIF-1α -siRNA转染后，CoCl2低氧诱导处理的细胞发生凋亡的比率大幅度上升（P〈0．05）。结论HIF-1α 在AtT-20细胞生长增殖中发挥抗凋亡的作用。

Objective To explore the role of hypoxia inducible factor （HIF）-1α in AtT-20 cells proliferation and to investigate whether HIF-1α could protect AtT-20 cells from hypoxia-induced apoptosis. Methods AtT-20 cells were treated with various concentrations of CoCl2 to induce hypoxia. MTT was applied to detect whether COCl2 could stimulate the growth of pituitary adenoma cells. Western blotting and real time PCR were used to evaluate changes in HIF-1α protein and mRNA levels. AtT-20 cells were transfected with siRNA targeting HIF-1α mRNA sequences or mock siRNA, followed by treatment with different concentrations of COCl2 for 24 h,and compared with normoxia groups. The efficacy of RNAi was assessed via real-time PCR and western blotting. Apoptosis was determined by Annexin V-FITC PI staining and Tdt-mediated dUTP nick end-labeling （TUNEL） assay. Results CoCl2 triggered the proliferation of AtT-20 ceils in a concentration and time dependent manner. However,the viability of the ceils when transfected with HIF-1α-siRNA and then treated with COCl2 was decreased greatly. Annexin V-FITC PI staining and TUNEL analysis showed that a significantly greater proportion of AtT-20 cells transfected with specific siRNA duplexes and subsequently exposed to hypoxia demonstrated apoptosis to a large extent when compared with nontransfected cells. Conclusions These findings strongly suggest that HIF-1α triggers the growth of AtT-20 cells and plays an antiapoptotic role in AtT-20 ceils in hypoxia.