The c-Myc expression and apoptosis of mouse thymocytes treated with dexamethasone

AIM: To study c-Myc expression and its relationship with caspase-3 in a dexamethasone (DEX)- induced mouse thymocyte apoptosis model, and discuss the role of c-Myc in cell apoptosis. METHODS: Mouse thymocyte apoptosis was induced by 1 μmol/L DEX, the apoptotic and necrosis cells were measured by Annexin V-FITC/PI double staining flowcytometry at 30 min, 3 h, 6 h and 9 h . Electron microscopy observation was carried out at 6 h, and c-Myc and caspase-3 contents were tested by Western blot at 0, 30, 60, 180 min. RESULTS: By 1μmol/L DEX treatment, the apoptosis rates of thymocytes at 30 min, 3 h, 6 h, 9 h were (5.70±0.46)%, (35.79±1.13)%, (50.61±2.15)% and (35.52±1.66)%,respectively; in control group, they were (5.97±0.25)%, (10.20±0.71)%, (12.10±0.66)% and (15.45±0.51)% (P0.01). At same time intervals, the necrosis rates of thymocytes in DEX group were (4.58±0.51)%, (4.66±0.67)%, (25.36±1.64)% and (46.99±2.67)%; in control group, they were (4.38±0.39)%, (4.19±0.73)%, (9.63±1.25)% and (13.38±0.72)%. Typical apoptotic cells were observed at 6 h in DEX group by electron microscopy. Obvious expression of c-Myc was detected at 0 min in control group, then c-Myc content increased at 30 min and reduced at 1 h and 3 h, in DEX group, c-Myc expression was higher than that in control group, and got a peak expression at 30 min, then significantly reduced at 1h and 3 h. Caspase-3 increased following culture time lapse in control group, while its content was more in DEX group at 0 min, peak content was detected at 30 min, and significant reduction at 1 h and 3 h. CONCLUSION: These results implied a c-Myc mediated cell apoptosis pattern in the DEX- induced mouse thymocyte apoptosis model. c-Myc and caspase-3 signals may have feedback inhibitory regulation in this process.     

CD137 signaling molecules promote proliferation of pulmonary artery endothelial cells by aerobic glycolysis

AIM:To investigate whether CD137 signaling molecules promote the proliferation of pulmonary artery endothelial cells (PAECs) by aerobic glycolysis. METHODS:The experiments of mouse PAECs were performed as follows. (1) Stimulating factors TNF-α (10 μg/L), ET-1 (10 mmol/L) and 5-HT (1 μmol/L) were used to stimulate the cells for 24 h. (2) After stimulation with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group (treatment with 5 mg/L CD137L recombinant protein to activate CD137-CD137L signaling), c-Myc inhibitor group (pretreatment with 10 μmol/L c-Myc inhibitor 10074-G5, dissolved in DMSO, for 30 min, followed by treatment with 5 mg/L CD137L recombinant protein) and DMSO group (pretreated with DMSO at the same volume to c-Myc inhibitor group for 30 min followed by CD137L recombinant protein treatment). (3) After stimulated with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group and 2-deoxyglucose (2-DG) group (pretreatment with 10 mmol/L glycolysis inhibitor 2-DG for 30 min followed by CD137L recombinant protein treatment).The expression of membrane protein and total protein of CD137 in the PAECs was detected by flow cytometry and Western blot, respectively. The protein levels of glycolytic enzymes such as hexokinase (HK2), 6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 3 (PFKFB3) and c-Myc were measured by Western blot. The enzyme activity of HK2 and PFKFB3 was detected by HK2 kit and PFK kit, respectively. Glucose oxidase method was used to measure the glucose uptake rate, and lactate colorimetric assay was conducted for analyzing lactic acid production. CCK-8 assay and EdU staining were used to detect proliferation of the PAECs. RESULTS:Compared with control group, TNF-α, ET-1 and 5-HT significantly increased the expression of CD137 membrane protein and total protein in the PAECs (P<0.05). The protein levels and enzyme activity of HK2 and PFKFB3 protein in CD137 agonist group were significantly higher than those in control group (P<0.05). Compared with control group, the lactic acid production and glucose consumption in CD137 agonist group were significantly increased. The protein level of c-Myc was significantly higher than that in control group after stimulation with CD137L recombinant protein, while c-Myc inhibitor 10074-G5 significantly inhibited the promoting effect of CD137L recombinant protein on glycolysis (P<0.05). The results of CCK-8 assay and EdU staining showed that the cell proliferation in CD137 agonist group was significantly increased compared with control group, while glycolysis inhibitor 2-DG significantly inhibited the proliferation-enhancing effect of CD137 signaling activation on the cells (P<0.05). CONCLUSION:CD137 signaling molecules may modulate the aerobic glycolysis by up-regulating c-Myc, thus promoting the proliferation of mouse PAECs.     

牛c-Myc/TAT/9R融合表达载体构建及其原核表达研究

原癌基因c-Myc虽然在许多肿瘤组织中特异性高表达,但它也是机体正常细胞生长与增殖所必需的因子,然而目前家畜诱导多潜能干细胞（iPS）研究中关于多功能转录因子c-Myc的报道很少.为此,研究拟利用蛋白转导区域（PTD）的跨膜作用,构建TAT-bc-Myc-9R融合表达载体,并对其进行原核表达,旨在为转录因子蛋白重编程牛体细胞奠定基础.试验以牛c-Myc基因编码区序列为参考,设计并合成含酶切位点、跨膜转导肽TAT和9R的引物,利用PCR等基因工程方法获得包括牛c-Myc、TAT和9R的重组原核表达载体（pTAT-HA-bc-Myc-9R）;酶切鉴定和DNA测序结果表明,试验成功获得bc-Myc-9R重组序列,成功构建重组质粒pTAT-bc-Myc-9R;不同IPTG浓度和诱导时间条件对该重组载体的诱导表达和SDS-PAGE分析表明,TAT-bc-Myc-9R在0.6 mmol/L IPTG和37℃诱导6h条件下表达约57KDa的目的蛋白.这些结果为PTD介导的转录因子蛋白直接重编程牛体细胞以获得iPS研究积累了科学资料.