Department of Orthopedic Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, China.
J Surg Res. 2013 Oct;184(2):873-9. doi: 10.1016/j.jss.2013.03.082. Epub 2013 Apr 17.
Bone marrow-derived mesenchymal stem cells (BMSCs) undergo hypoxia-induced apoptosis when cells are transplanted from a normoxic to a hypoxic microenvironment in vivo. The effect of the osteogenic microenvironment on BMSCs under hypoxic conditions has not yet been revealed.
In the current study, we investigated the effects on BMSCs of hypoxia and osteogenic induction (OI) individually and in combination. We isolated BMSCs from rat bone marrow and confirmed them by recognition of surface antigens using cytometry. After passaging the BMSCs to the third generation, we treated them with the following conditions: 1% oxygen and OI, normoxia and OI, and 1% oxygen without OI; normoxia without OI was the control condition. On days 3, 7, 14, and 21, we detected the expression levels of hypoxia inducible factor-1α and alkaline phosphate via Western blotting. Cellular apoptosis was detected by Hoechst staining and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick end labeling; caspase activity was also detected.
The expression of hypoxia inducible factor-1α was induced and up-regulated when BMSCs were grown under 1% oxygen. The incidence of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine, 5'-triphosphatenick end labeling-positive cells in the hypoxia plus OI group was much lower than that in the hypoxia group without OI. Caspase activity increased on days 3, 7, 14, and 21. The absolute value of caspase was statistically higher in the BMSC hypoxia group than in the other three groups, whose values were similar to each other.
Osteogenic induction could protect BMSCs against hypoxia-induced apoptosis. Bone marrow-derived mesenchymal stem cells may be appropriate candidate cells for cytotherapy for skeletal diseases.
骨髓间充质干细胞（BMSCs）在体内从常氧环境移植到低氧微环境时会发生缺氧诱导的凋亡。低氧条件下成骨微环境对 BMSCs 的影响尚未揭示。
在本研究中，我们分别研究了低氧和成骨诱导（OI）以及两者联合对 BMSCs 的影响。我们从大鼠骨髓中分离出 BMSCs，并通过细胞术识别表面抗原进行确认。将 BMSCs 传代至第三代后，我们将其分别置于以下条件下处理：1%氧气和 OI、常氧和 OI、1%氧气但无 OI；常氧但无 OI 为对照条件。在第 3、7、14 和 21 天，我们通过 Western blot 检测缺氧诱导因子-1α和碱性磷酸酶的表达水平。通过 Hoechst 染色和末端脱氧核苷酸转移酶介导的 2'-脱氧尿苷，5'-三磷酸 nick 末端标记检测细胞凋亡；还检测了 caspase 活性。
当 BMSCs 在 1%氧气下生长时，缺氧诱导因子-1α的表达被诱导并上调。在缺氧加 OI 组中端粒末端脱氧核苷酸转移酶介导的 2'-脱氧尿苷，5'-三磷酸 nick 末端标记阳性细胞的发生率明显低于缺氧无 OI 组。Caspase 活性在第 3、7、14 和 21 天增加。BMSC 低氧组的 caspase 绝对值明显高于其他三组，且彼此之间的数值相似。
成骨诱导可以保护 BMSCs 免受缺氧诱导的凋亡。骨髓间充质干细胞可能是骨骼疾病细胞治疗的合适候选细胞。
