Objective: To investigate the effect of LOC103693069 on hypoxic apoptosis of bone marrow mesenchymal stem cells (BMSCs).
Methods: BMSCs from 1-week-old Sprague Dawley rat bone marrow were isolated, cultured, and passaged by the whole bone marrow adherent culture method. After identification of adipogenic, chondrogenic, and osteogenic differentiation, the 3rd generation cells were treated with hypoxia under 5%O 2, 1%O 2, and anaerobic conditions. After 48 hours, the cell viability, apoptosis, and apoptosis-related proteins [hypoxia inducible factor 1α (HIF-1α), Caspase-3, B cell lymphoma/leukemia 2 (Bcl-2)] expressions were detected, and normal BMSCs were used as controls. Based on the research results, the concentration group with the most obvious apoptosis was selected and used for subsequent experiments. After 48 hours of hypoxia treatment, BMSCs were taken and analyzed by gene chip and real-time fluorescence quantitative PCR (qRT-PCR) to screen the most significantly down-regulated gene and construct their high-expression, low-expression, and negative control lentiviruses; BMSCs were transfected with the different lentiviruses, respectively. After qRT-PCR detection confirmed that the transfection was successful, the BMSCs were treated with hypoxia for 48 hours to observe the cell viability and the expressions of apoptosis-related proteins.
Results: After cell viability, apoptosis, and apoptosis-related proteins were detected, cell apoptosis was the most significant under anaerobic conditions after 48 hours. The above indicators were significantly different from other groups ( P<0.05), and this group was used for treatment conditions for subsequent experiments. Gene chip analysis showed that after 48 hours of hypoxia treatment, AC125847.1, LOC102547753, AABR07017208.2, and LOC103693069 were significantly down-regulated in BMSCs, and the expressions of LOC103693069 was the most significant down-regulation detected by qRT-PCR ( P<0.05). It was selected to construct lentivirus and transfect BMSCs. Afterwards, qRT-PCR detection showed the successful transfection into the cells. After hypoxia treatment, the apoptosis rate and the expressions of apoptosis-related proteins of BMSCs overexpressed by the gene were significantly reduced ( P<0.05).
Conclusion: LOC103693069 can relieve the hypoxic apoptosis of BMSCs.
目的: 探讨LOC103693069对BMSCs缺氧性凋亡的作用。.
方法: 取1周龄SD大鼠骨髓，采用全骨髓贴壁培养法分离培养BMSCs并传代，经成脂、成软骨和成骨诱导分化鉴定后，取第3代细胞分别采用5%O 2、1%O 2及无氧条件处理48 h，检测细胞活性、凋亡以及凋亡相关蛋白 ［低氧诱导因子 1α（hypoxia inducible factor 1α，HIF-1α）、半胱氨酸蛋白酶 3（Caspase-3）、B细胞淋巴瘤/白血病基因2（B cell lymphoma/leukemia 2，Bcl-2）］ 表达，以正常BMSCs作为对照，确定细胞凋亡最明显的浓度组用于后续实验。取缺氧处理48 h后BMSCs，通过基因芯片以及实时荧光定量PCR（real-time fluorescence quantitative PCR，qRT-PCR）分析，筛选表达下调最显著基因并构建其高表达、低表达以及阴性对照慢病毒并转染BMSCs，经qRT-PCR检测明确转染成功后，缺氧处理48 h观察细胞活性及凋亡相关蛋白表达。.
结果: 经细胞活性、凋亡以及凋亡相关蛋白检测，以无氧条件培养48 h后细胞凋亡最显著，上述指标与其他组比较差异均有统计学意义（ P<0.05），以该组处理条件进行后续实验。基因芯片分析示缺氧处理48 h后，BMSCs中AC125847.1、LOC102547753、AABR07017208.2、LOC103693069明显下调，qRT-PCR检测LOC103693069表达下调最显著（ P<0.05）；选择其构建慢病毒并转染BMSCs，qRT-PCR检测示成功转染至细胞；经缺氧处理后检测显示该基因高表达的BMSCs凋亡率和凋亡相关蛋白表达明显降低（ P<0.05）。.
结论: LOC103693069可改善BMSCs缺氧性凋亡。.
Keywords: Bone marrow mesenchymal stem cells; hypoxic apoptosis; long non-coding RNA; rat.