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

Abstract

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 94%N2,1%O 2, 5%CO 2 at 37±. At different hypoxia time points, 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 immunocytochemistry 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 < 0.01) and tended to descend slowly with hypoxia time duration, for which there was no statistical significance( 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 <0.01)and DR(0.04 ± 1.79%,0.93 ± 1.85%,3.11 ± 2.14%,4.09 ± 2.36%,4.72 ± 2.05% and 4.91 ± 3.72% at 0,0.5,1,4 and 8 h, respectively, P < 0.05) at different hypoxia time points significantly increased compared to those time in normoxia; The AR further went up with time ( P < 0.05), however there was no statistical significance( 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 < 0.01) and degraded with time (in an hypoxic environment - P < 0.01). IOD of p-Akt protein of MSCs at different hypoxia time points significantly increased(0.367 ± 0.031,0.556 ± 0.023,0.579 ± 0.013, 0.660 ± 0.024, 0.685 ± 0.039 and 0.685 ± 0.011, respectively) compared to their equivalents in normoxia( P < 0.05), however, there was no statistical significance( P > 0.05) for different hypoxia time points. Hypoxia may result in ultramicrostructure changes, such as defluvium of Microvilli, apoptotic body, “argination” 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.