低氧可以促进人骨髓间充质干细胞(human bone marrow-derived mesenchymal stem cells,hMSCs)增殖。为探讨其可能机制,本实验采用cDNA芯片技术动态检测低氧促进hMSCs增殖过程中基因表达的变化,用RT-PCR验证芯片结果。结果显示,在含21 329条基因探针的芯片上,检测到282个基因差异表达,其中代谢类基因最多;差异表达基因的数目随低氧时间不同而变化,其中24 h时差异表达基因的数目最多。差异表达基因中4个为已知的低氧诱导因子-1(hypoxia- inducible factor 1,HIF-1)靶基因,在低氧处理36 h时都基本上调。此外,差异表达基因中有10个连续变化的基因,这些基因中既有上调基因也有下调基因。4个HIF-1靶基因和连续变化的基因的RT1-PCR结果大部分与cDNA芯片结果一致。结果提示,低氧促进hMSCs增殖是多基因参与的过程,可能与HIF-1及其下游信号通路有关。
Our previous study demonstrates that hypoxia promotes human bone marrow-derived mesenchymal stem cell (hMSC) proliferation. The aim of the present study was to investigate the gene profile involved in this process by using cDNA microarray. Cultured hMSCs were treated with hypoxia (3% 02) for4 h, 12 h, 24 h, 36 h, 48 h and 72 h, respectively. Then these cells were collected to prepare total RNA. Hypoxia-induced gene expression profile was examined and analyzed by GenePix Pro 4.0 software. Some of cDNA microarray results were confirmed by RT-PCR. Microarray analysis identified that 282 genes expressed differentially, of which most were involved in metabolism. The number of differentially expressed genes at different hypoxia time points was different, and most genes were regulated after 24-hour hypoxia. Among the 282 differentially expressed genes, 4 hypoxia-inducible factor 1 (HIF-1) targeted genes and 10 genes that changed at 3 continuous time points were found. The results obtained indicated that 4 HIF-1 targeted genes, i.e., transforming growth factor β3 (TGFβ3), phospho-glycerate kinase 1 (PGK 1 ), insulin-like growth factor binding protein 3 (IGFBP3) and BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), displayed up-regulated pattern at 36 h under hypoxia. BNIP3 displayed a dynamically up-regulated pattern at 12, 36 and 72 h under hypoxia. However, TGFβ3 and PGK1 were downregulated at 72 h. In addition, the gene expressions of adenylate kinase 3-like 1 (HAC), neurofilament light polypeptide 68 kDa (NEFL), N-myc downstream regultated gene 1 (NDRG1), discoidin domain receptor family member 1 (DDR1), tribbles homolog 3 (TRIB3), nucleoprotein (AHNAK) and eukaryotic elongation factor selenocyteine-tRNA-specific (EESTS) were up-regulated. Moreover, the gene expressions of EESTS, NEFL were up-regulated at 5 different time points under hypoxia. Furthermore, it was found that the gene expressions of histone cluster 1 (HIS 1) and transferring recepto