Cell multiplication, apoptosis and p-Akt protein expression of bone mesenchymal stem cells of rat under hypoxia environment

摘要

Objective:To elucidate whether cell multiplication, apoptosis, glucose intake and p-Akt protein expression of bone Mesenchymal Stem Cells(MSCs) of rats is influenced by a hypoxic environment ex vivo. Methods:Passage 3 of bone marrow MSCs taken from Wistar rats,were cultured in a culturing chamber with 94N_2,1O_2,5CO_2 at 37℃. At different hypoxia time pbints,0,0.5, 1,4 and 8 h, glucose uptake was assayed by using radiation isotope ~3H-G, Apoptotic Rate(AR) and dead rate(DR) were analyzed by flow cytometry(FCM) after Annexin V/PI staining, cell multiplication (by MTT methods) and p-Akt protein by immunocyto-chemistry and western blot. Results:Assay for CD29~+,CD44~+,CD71~+,CD34~-, Tn T~+(after 5-azacytidine agent inducing) and ALP~+(after bone differentiation agent inducing) suggested these bone-derived cells were MSCs. The ~3H-G intaking ratio (CPM/ flask value: 157 ± 11,110 ± 11,107 ± 13,103 ± 10,100 ± 9 and 98 ± 10) of MSCs at different hypoxia time points, significantly decreased compared to that of normoxia (P 0.05). The AR(0.09 ± 2.03, 12.9 ± 1.72, 13.7 ± 2.26, 13.8 ± 3.01, 14.1 ± 2.78 and 14.7 ± 4.01 at 0,0.5,1,4 and 8 h,respectively,p 0.05) for the DR. Optical absorption value of MTT methods at different hypoxia time points significantly decreased compared to those with a corresponding normoxia time (P 0.05) for different hypoxia time points. Hypoxia may result in ultramicrostructure changes, such as defluvium of Microvilli, apoptotic body, "margination" and so on and are further aggravated with hypoxia time stretching. Conclusion:Hypoxia may lead to a depression of MSCs intaking glucose, creep of cell multiplication, upregulation of p-Akt protein and apoptosis of MSCs ex vivo.