体外Akt基因转染对骨髓间充质干细胞葡萄糖转运载体-4表达和易位的影响

Translocation and Expression of GLUT-4 in Bone Marrow Mesenchymal Stem Cells Transfected with Akt Gene of Rat Ex Vivo

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 elucidate whether glucose transporters-4（GLUT-4） takes part in glucose uptake of mesenchymal stem cells（MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo.Methods MSCs,transfected by Akt gene and no,were cultured with normoxia（5% CO2） or hypoxia（94%N2,1%O2 and 5% CO2） at 37 ℃ for 8 h.Glucose uptake was assayed by using radiation isotope 2——deoxy-D-glucose（3H-G） and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry,Western blot and RT-PCR,respectively.Results ①3H-G intake of MSCs was significantly increased in hypoxia-transfection group than that in hypoxia-non-transfection（（1.39±0.13） fold,P0.05）,but which was lower than that in normoxia-non-transfection group,P0.05.②GLUT-4 was expressed by MSCs under any conditions.Compared with normoxia-non-transfection group,hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly（P0.05）.③Compared with hypoxia-non-transfection group,the expression of GLUT-4（mRNA（1.756±0.152） fold,total protein in cell（1.653±0.312） fold,protein in plasma membrane（2.041±0.258） fold） was increased in hypoxia-transfection group significantly（P0.05）,but which was lower than that in normoxia-non-transfection group（P0.05）.④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane（r=0.415,P=0.001）.Conclusion GLUT-4 may take part in glucose uptake of MSCs,and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.