目的:血管生成是成功愈合牙髓损伤的关键事件,缺氧是牙髓血管生成的主要刺激因子。在这项研究中,我们研究了缺氧对人牙髓干细胞(hDPSCs)促血管生成潜能的影响以及miR-143-5p在此过程中的作用.
方法:分离人牙髓干细胞,体外培养和表征。氯化钴(CoCl2)用于在hDPSC中诱导缺氧。CCK-8和Transwell测定用于确定缺氧对hDPSC增殖和迁移的影响。定量实时聚合酶链反应(qRT-PCR),进行蛋白质印迹(WB)和ELISA以评估hDPSC中HIF-1α和血管生成细胞因子的mRNA和蛋白质水平。使用Matrigel管形成和鸡绒毛尿囊膜(CAM)测定在体外测量缺氧对hDPSC促血管生成潜能的影响。构建重组慢病毒载体以在hDPSC中稳定过表达或抑制miR-143-5p,并使用qRT-PCR评估促血管生成作用,WB,和试管形成测定。使用生物信息学预测工具鉴定和验证miR-143-5p靶基因,双荧光素酶报告基因测定和RNA下拉实验。最后,我们使用裸鼠皮下移植模型来确定缺氧处理和miR-143-5p过表达/抑制hDPSC在牙髓再生中的作用.
结果:低氧促进hDPSC增殖,迁移和促血管生成潜力。体内实验表明,缺氧处理(50和100μMCoCl2)可促进牙髓血管生成和牙本质形成。与促血管生成因子的水平相反,miR-143-5p水平随着CoCl2浓度的增加而降低。miR-143-5p抑制显著促进hDPSC的促血管生成潜能,而miR-143-5p过表达在体外抑制血管生成。双荧光素酶报告基因测定将视黄酸受体相关的孤儿受体α(RORA)鉴定为hDPSC中的miR-143-5p靶基因。RNA下拉实验表明,HIF-1α和RORA被生物素标记的miR-143-5p拉低,缺氧组HIF-1α和RORA与miR-143-5p结合的水平低于常氧组。hDPSC中miR-143-5p表达的抑制促进异位牙髓组织再生。
结论:CoCl2诱导的缺氧促进hDPSC驱动的旁分泌血管生成和牙髓再生。miR-143-5p的抑制通过直接靶向HIF-1α和RORA上调缺氧条件下hDPSC的促血管生成潜能。
OBJECTIVE: Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR-143-5p in the process.
METHODS: Human dental pulp stem cells were isolated, cultured and characterized in vitro. Cobalt chloride (CoCl2) was used to induce hypoxia in hDPSCs. CCK-8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF-1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured in vitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR-143-5p in hDPSCs, and the proangiogenic effects were assessed using qRT-PCR, WB, and tube formation assays. miR-143-5p target genes were identified and verified using bioinformatics prediction tools, dual-luciferase reporter assays and RNA pull-down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR-143-5p overexpression/inhibition in hDPSCs in dental pulp regeneration.
RESULTS: Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The in vivo experiments showed that hypoxia treatment (50 and 100 μM CoCl2) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR-143-5p levels decreased with increasing CoCl2 concentration. miR-143-5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR-143-5p overexpression inhibited angiogenesis in vitro. Dual-luciferase reporter assay identified retinoic acid receptor-related orphan receptor alpha (
RORA) as an miR-143-5p target gene in hDPSCs. RNA pull-down experiments demonstrated that HIF-1α and
RORA were pulled down by biotin-labelled miR-143-5p, and the levels of HIF-1α and
RORA bound to miR-143-5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR-143-5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration.
CONCLUSIONS: CoCl2-induced hypoxia promotes hDPSCs-driven paracrine angiogenesis and pulp regeneration. The inhibition of miR-143-5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF-1α and
RORA.