Human osteoblast- (hFOB 1.19 (ATCC CRL-1137) Transcriptome. Homo sapiens

objectives--Oxygen is a main participator of energy metabolism pathway energy in the human body. The degeneration of organ function may happen when Microcirculation doesn't get enough oxygen. And the consequence can be serious if there are no intervention for saving, sometimes even causing the patients’ death. Although hypoxia damage has been proved on many studies，we cannot ignore the physiopathology value of its existence. The cells will be activated by hypoxia when a mass of bioactive factors are released. Some of these may mediate the tissue healing and control the derailed inflammation response. However, the benefit will be restricted by cells death in excessive hypoxia. Based on this, we did a pre-primary experiments to explore how to exert the biggest influence on hypoxia.Methods-The third generation of human osteoblasts were distributed into three groups. Cells of control group were normally cultured (high-sugar DMEM, 10% FBS and 1% penicillin–streptomycin). Cells of hypoxia group were cultured in low-sugar DMEM (Invitrogen, USA) and 200μmol/L CoCl2 (sigma, USA) 4 hours and normally 4 hours. Cells of the Repeated brief hypoxia (RBH) group were under 3 times RBH (cultured in high-sugar DMEM, 10% FBS and 1% penicillin–streptomycin 10min and then were washed by phosphate buffer saline 3 times. Then cultured in low-sugar DMEM and 200μmol/L CoCl2 10min and were washed with phosphate buffer saline 3 times. The above management was RBH 1 time), then under hypoxia management 4 hours and normally 4 hours. In the end, the related miRNA were detected by high-throughput sequencing.Results- High-throughput sequencing of microRNA shown the expression of 2763 miRNAs. Further, we identified that hypoxia 4 hours made 60 and 56 target genes up-regulated and down-regulated respectively compared with normally cultured cells (pConclusion-Hypoxia 4 hours increased the cell death rate but promoted some bioactive factors secrete. High-throughput sequencing of microRNA shown that many miRNA participated in these effects to provide the basis for our future research.

研究目的：氧气是人体能量代谢通路的主要参与者。当微循环供氧不足时，可能引发器官功能退化；若未及时采取救治干预，病情可能进一步恶化，严重时甚至可导致患者死亡。尽管诸多研究已证实缺氧损伤的危害，但我们亦不能忽视缺氧状态本身的病理生理学意义。缺氧可激活细胞并释放大量生物活性因子，其中部分因子可介导组织修复、调控失控的炎症应答；但在过度缺氧状态下，细胞死亡会限制这类有益效应的发挥。基于此，我们开展了前期预实验，以探索如何最大化发挥缺氧的有益作用。
研究方法：将第三代人成骨细胞（human osteoblasts）分为三组。对照组细胞采用常规培养条件：高糖DMEM培养基、10%胎牛血清（FBS）及1%青霉素-链霉素混合液。缺氧组细胞采用低糖DMEM培养基（美国Invitrogen公司）+200μmol/L氯化钴（CoCl2，美国Sigma公司）培养4小时，随后更换为常规培养条件继续培养4小时。反复短时缺氧（Repeated brief hypoxia, RBH）组细胞先进行3次RBH处理：先用高糖DMEM、10%FBS及1%青霉素-链霉素培养10分钟，随后用磷酸盐缓冲液（phosphate buffer saline, PBS）洗涤3次；再更换为低糖DMEM+200μmol/L CoCl2培养10分钟，再次用PBS洗涤3次，上述完整流程即为1次RBH处理。完成3次RBH处理后，该组细胞再进行缺氧培养4小时，随后常规培养4小时。最后，采用高通量测序（high-throughput sequencing）检测相关微小RNA（microRNA, miRNA）的表达情况。
研究结果：高通量测序结果显示，共检测到2763种miRNA的表达。进一步分析发现，与常规培养的细胞相比，缺氧4小时处理组分别有60个靶基因上调、56个靶基因下调（P
研究结论：4小时缺氧处理可提升细胞死亡率，但同时可促进部分生物活性因子的分泌。高通量miRNA测序结果表明，多种miRNA参与了上述调控过程，为我们后续的研究提供了理论依据。