Objective ：To elucidate whether cell multiplication, apoptosis, glucose intake and p-Akt protein expression of bone Mesenchyreal Stem Cells（MSCs） of rats is influenced by a hypoxic environment ex vivo. Methods ：Pa...Objective ：To elucidate whether cell multiplication, apoptosis, glucose intake and p-Akt protein expression of bone Mesenchyreal 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%O2,5%CO2 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 MTr 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 MTr 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, ＂margination＂ and so on and are further aggravated with hypoxia time stretching. Conclusion： Hypoxia may lead to a depression of MSCs intaldng glucose, creep of cell multiplication, upregulation of p-Akt protein and apoptosis of MSCs ex vivo.展开更多
文摘Objective ：To elucidate whether cell multiplication, apoptosis, glucose intake and p-Akt protein expression of bone Mesenchyreal 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%O2,5%CO2 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 MTr 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 MTr 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, ＂margination＂ and so on and are further aggravated with hypoxia time stretching. Conclusion： Hypoxia may lead to a depression of MSCs intaldng glucose, creep of cell multiplication, upregulation of p-Akt protein and apoptosis of MSCs ex vivo.
