Wu Dongjin, Liu Liyan, Fu Shenglong, Zhang Jun
Department of Spine Surgery, The Second Hospital of Shandong University, Shandong, China.
Department of Nephrology, The Fifth People's Hospital of Jinan, Shandong, China.
Biochem Biophys Res Commun. 2022 May 28;606:100-107. doi: 10.1016/j.bbrc.2022.02.085. Epub 2022 Feb 23.
Hypoxia conditions induced by bone defects would prolong the duration of bone regeneration. The effect of osteostatin (OST) on the osteogenic differentiation of mesenchymal stem cells (MSCs) and angiogenesis under hypoxia conditions remain unexplored.
SPF mice were obtained, and MSCs were isolated from bone marrow. MSCs were treated with 1% oxygen for hypoxia induction, and 200 nM of OST was used to treat cells under nomorxia or hypoxia conditions. Cell proliferation was evaluated using CCK8 assay, and trypan blue staining was implemented for determining cell death ratio. Alkaline phosphatase activity and alizarin redS staining was conducted to histologically evaluated osteogenic differentiation. Flow cytometry was used for the detection of CD31Emcn cells (Type H ECs), whose migration was detected by Transwell assay and angiogenesis was measured by tube formation assay. Protein level was measured by western blotting and mRNA level was monitored via RT-qPCR.
The MSC proliferation was enhanced by OST under hypoxia conditions. The osteogenic differentiation of MSCs was decreased under hypoxia conditions, and treatment of OST significantly reversed its inhibitory effect. The hypoxia treated culture medium of MSCs promoted the proliferation, migration, and angiogenesis of type H ECs, while the effects were further strengthened by OST addition. HIF-1α was found to be upregulated in hypoxia treated MSCs, whereas silencing of HIF-1α had reversed effects on the angiogenic capacity of Type H ECs.
OST improved the proliferation and osteogenic differentiation of MSCs and further promoted angiogenesis of type H ECs through upregulating HIF-1α expression.
骨缺损诱导的缺氧状态会延长骨再生的持续时间。骨抑素（OST）在缺氧条件下对间充质干细胞（MSCs）成骨分化和血管生成的影响尚未得到探索。
获取无特定病原体（SPF）小鼠，并从骨髓中分离出MSCs。将MSCs置于1%氧气环境中进行缺氧诱导，使用200 nM的OST在常氧或缺氧条件下处理细胞。采用CCK8法评估细胞增殖，并进行台盼蓝染色以确定细胞死亡率。进行碱性磷酸酶活性和茜素红S染色以组织学评估成骨分化。使用流式细胞术检测CD31Emcn细胞（H型内皮细胞），通过Transwell试验检测其迁移能力，并通过管腔形成试验测量血管生成。通过蛋白质印迹法测量蛋白质水平，并通过逆转录定量聚合酶链反应（RT-qPCR）监测mRNA水平。
在缺氧条件下，OST增强了MSCs的增殖。在缺氧条件下，MSCs的成骨分化降低，而OST处理显著逆转了其抑制作用。缺氧处理的MSCs培养基促进了H型内皮细胞的增殖、迁移和血管生成，而添加OST进一步增强了这些作用。发现缺氧处理的MSCs中缺氧诱导因子-1α（HIF-1α）上调，而沉默HIF-1α对H型内皮细胞的血管生成能力有相反的影响。
OST通过上调HIF-1α表达改善了MSCs的增殖和成骨分化，并进一步促进了H型内皮细胞的血管生成。
