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

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.

The effect of neuropeptides on proliferation of rat bone marrow mesenchymal stem cells

Objective To investigate the effects and mechanism of calcitonin gene-related peptide(CGRP)and substance P (SP) on proliferation of rat bone marrow mesenchymal stem cells.Methods The rBMSCs were isolated using whole bone marrow

SD大鼠间充质干细胞的分离培养及生物学特性初探

目的:探讨体外分离培养、纯化SD大鼠骨髓间充质干细胞(mesenchymalstemcells,MSCs)的方法,明确增殖及生长特征。方法:从骨髓中提取间充质干细胞,加入淋巴分离液后用密度梯度离心培养法和静止贴壁培养法,分离纯化大鼠骨髓MSCs,对其进行传代扩增和形态学观察,细胞计数,绘制生长曲线。结果:(1)通过离体培养,可使体内环境下低丰度的骨髓间充质干细胞实现数目扩增;(2)体外单层培养条件下贴壁生长的骨髓间充质干细胞大多数具有成纤维细胞生长特性。结论:对比两种分离方法可知淋巴分离法更有利于骨髓间充质干细胞的纯化,体外培养成功为今后进一步利用体内间充质干细胞研究细胞衰老,并建立衰老模型奠定了基础。