论文部分内容阅读
目的探讨葡萄糖转运载体(GLUT)-4是否参与骨髓间充质干细胞(MSCs)的葡萄糖转运以及Akt基因转染提高MSCs耐缺氧能力是否与GLUT-4易位和表达有关。方法将经Akt基因转染和未转染的MSCs均行常氧(5%CO2)和缺氧(94%N2、1%O2和5%CO2)37℃孵育8 h。放射同位素法检测氚标-脱氧葡萄糖(3H-G)的摄取量,免疫细胞化学染色、Western blot和RT-PCR分别检测GLUT-4的蛋白质和mRNA表达。结果①缺氧转染组的3H-G摄取量是缺氧非转染组的(1.39±0.13)倍(P<0.05),但仍低于常氧非转染组(P<0.05)。②MSCs在常氧或缺氧、Akt基因转染或无转染的条件下均可表达GLUT-4蛋白。与常氧非转染组比较,缺氧非转染组GLUT-4 mRNA和蛋白的表达水平明显降低(P<0.05)。③与缺氧非转染组比较,缺氧转染组的GLUT-4 mRNA〔(1.756±0.152)倍〕和蛋白〔细胞总GLUT-4蛋白(1.653±0.312)倍,细胞膜GLUT-4蛋白(2.041±0.258)倍〕的表达水平明显提高(P<0.05),且GLUT-4蛋白易位明显;但与常氧非转染组比较,其GLUT-4 mRNA和蛋白的表达水平仍较低(P<0.05)。④MSCs的3H-G摄取量与细胞膜中GLUT-4蛋白的表达呈正相关(r=0.415,P=0.001)。结论GLUT-4可能参与MSCs的葡萄糖转运,Akt基因提高MSCs耐缺氧能力可能与提高GLUT-4的表达和易位有关。
Objective To investigate whether glucose transporter (GLUT) -4 is involved in glucose transport in bone marrow mesenchymal stem cells (MSCs) and whether Akt gene transfection enhances hypoxia tolerance in MSCs is related to GLUT-4 translocation and expression. Methods Both MSCs transfected with Akt gene and untransfected MSCs were incubated with normoxia (5% CO2) and hypoxia (94% N2, 1% O2 and 5% CO2) for 8 h at 37 ℃. Radioimmunoassay was used to detect the uptake of tritiated-deoxyglucose (3H-G). Immunocytochemistry, Western blot and RT-PCR were used to detect the protein and mRNA expression of GLUT-4. Results ① The uptake of 3H-G in hypoxia group was (1.39 ± 0.13) times higher than that in hypoxia group (P <0.05), but still lower than that in non-hypoxia group (P <0.05). ②MSCs can express GLUT-4 protein under normoxia or hypoxia, Akt gene transfection or without transfection. Compared with normoxia non-transfection group, the expression of GLUT-4 mRNA and protein in hypoxia non-transfection group was significantly decreased (P <0.05). ③Compared with hypoxic group, GLUT-4 mRNA 〔(1.756 ± 0.152) fold〕 and protein 〔total cell GLUT-4 protein (1.653 ± 0.312)〕, cell membrane GLUT-4 protein (P <0.05), and the translocation of GLUT-4 protein was obvious; however, the expression level of GLUT-4 mRNA and protein was still low compared with normoxia non-transfection group (P <0.05) P <0.05). The 3H-G uptake of MSCs was positively correlated with the expression of GLUT-4 in the cell membrane (r = 0.415, P = 0.001). Conclusion GLUT-4 may be involved in the glucose transport of MSCs, and Akt gene may be related to the increase of GLUT-4 expression and translocation.