PDF(Pubmed)
Abstract：
BACKGROUND: Cartilage is a kind of avascular tissue, and it is difficult to repair itself when it is damaged. In this study, we investigated the regulation of chondrogenic differentiation and vascular formation in human jaw bone marrow mesenchymal stem cells (h-JBMMSCs) by the long-chain noncoding RNA small nucleolar RNA host gene 1 (SNHG1) during cartilage tissue regeneration.
METHODS: JBMMSCs were isolated from the jaws via the adherent method. The effects of lncRNA SNHG1 on the chondrogenic differentiation of JBMMSCs in vitro were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Pellet experiment, Alcian blue staining, Masson\'s trichrome staining, and modified Sirius red staining. RT-qPCR, matrix gel tube formation, and coculture experiments were used to determine the effect of lncRNA SNHG1 on the angiogenesis in JBMMSCs in vitro. A model of knee cartilage defects in New Zealand rabbits and a model of subcutaneous matrix rubber suppositories in nude mice were constructed for in vivo experiments. Changes in mitochondrial function were detected via RT-qPCR, dihydroethidium (DHE) staining, MitoSOX staining, tetramethyl rhodamine methyl ester (TMRM) staining, and adenosine triphosphate (ATP) detection. Western blotting was used to detect the phosphorylation level of signal transducer and activator of transcription 3 (STAT3).
RESULTS: Alcian blue staining, Masson\'s trichrome staining, and modified Sirius Red staining showed that lncRNA SNHG1 promoted chondrogenic differentiation. The lncRNA SNHG1 promoted angiogenesis in vitro and the formation of microvessels in vivo. The lncRNA SNHG1 promoted the repair and regeneration of rabbit knee cartilage tissue. Western blot and alcian blue staining showed that the JAK inhibitor reduced the increase of STAT3 phosphorylation level and staining deepening caused by SNHG1. Mitochondrial correlation analysis revealed that the lncRNA SNHG1 led to a decrease in reactive oxygen species (ROS) levels, an increase in mitochondrial membrane potential and an increase in ATP levels. Alcian blue staining showed that the ROS inhibitor significantly alleviated the decrease in blue fluorescence caused by SNHG1 knockdown.
CONCLUSIONS: The lncRNA SNHG1 promotes chondrogenic differentiation and angiogenesis of JBMMSCs. The lncRNA SNHG1 regulates the phosphorylation of STAT3, reduces the level of ROS, regulates mitochondrial energy metabolism, and ultimately promotes cartilage regeneration.
METHODS: JBMMSCs were isolated from the jaws via the adherent method. The effects of lncRNA SNHG1 on the chondrogenic differentiation of JBMMSCs in vitro were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Pellet experiment, Alcian blue staining, Masson\'s trichrome staining, and modified Sirius red staining. RT-qPCR, matrix gel tube formation, and coculture experiments were used to determine the effect of lncRNA SNHG1 on the angiogenesis in JBMMSCs in vitro. A model of knee cartilage defects in New Zealand rabbits and a model of subcutaneous matrix rubber suppositories in nude mice were constructed for in vivo experiments. Changes in mitochondrial function were detected via RT-qPCR, dihydroethidium (DHE) staining, MitoSOX staining, tetramethyl rhodamine methyl ester (TMRM) staining, and adenosine triphosphate (ATP) detection. Western blotting was used to detect the phosphorylation level of signal transducer and activator of transcription 3 (STAT3).
RESULTS: Alcian blue staining, Masson\'s trichrome staining, and modified Sirius Red staining showed that lncRNA SNHG1 promoted chondrogenic differentiation. The lncRNA SNHG1 promoted angiogenesis in vitro and the formation of microvessels in vivo. The lncRNA SNHG1 promoted the repair and regeneration of rabbit knee cartilage tissue. Western blot and alcian blue staining showed that the JAK inhibitor reduced the increase of STAT3 phosphorylation level and staining deepening caused by SNHG1. Mitochondrial correlation analysis revealed that the lncRNA SNHG1 led to a decrease in reactive oxygen species (ROS) levels, an increase in mitochondrial membrane potential and an increase in ATP levels. Alcian blue staining showed that the ROS inhibitor significantly alleviated the decrease in blue fluorescence caused by SNHG1 knockdown.
CONCLUSIONS: The lncRNA SNHG1 promotes chondrogenic differentiation and angiogenesis of JBMMSCs. The lncRNA SNHG1 regulates the phosphorylation of STAT3, reduces the level of ROS, regulates mitochondrial energy metabolism, and ultimately promotes cartilage regeneration.
摘要：
背景：软骨是一种无血管组织，当它被损坏时很难自我修复。在这项研究中,我们研究了软骨组织再生过程中长链非编码RNA小核仁RNA宿主基因1(SNHG1)对人颌骨骨髓间充质干细胞(h-JBMMSCs)软骨分化和血管形成的调控.
方法:通过贴壁方法从颌骨分离JBMMSCs。通过实时荧光定量聚合酶链反应(RT-qPCR)检测LncRNASNHG1对JBMMSCs软骨分化的影响,颗粒实验，阿尔辛蓝染色，马森三色染色，和改良天狼星红染色。RT-qPCR,基质凝胶管形成，和共培养实验用于确定lncRNASNHG1对JBMMSCs体外血管生成的影响。建立了新西兰兔膝关节软骨缺损模型和裸鼠皮下基质橡胶栓剂模型，用于体内实验。通过RT-qPCR检测线粒体功能的变化，二氢乙锭(DHE)染色,MitoSOX染色,四甲基罗丹明甲酯（TMRM）染色，和三磷酸腺苷（ATP）检测。Western印迹法检测信号转导和转录激活因子3(STAT3)的磷酸化水平。
结果：阿尔辛蓝染色，马森三色染色，和修饰的天狼星红染色显示lncRNASNHG1促进软骨分化。lncRNASNHG1促进体外血管生成和体内微血管形成。lncRNASNHG1促进兔膝关节软骨组织的修复和再生。Westernblot和Alcian蓝染色显示JAK抑制剂降低了SNHG1引起的STAT3磷酸化水平增加和染色加深。线粒体相关性分析显示，lncRNASNHG1导致活性氧（ROS）水平降低，线粒体膜电位的增加和ATP水平的增加。Alcian蓝染色显示，ROS抑制剂显著缓解了SNHG1敲低引起的蓝色荧光下降。
结论：lncRNASNHG1促进JBMMSCs的软骨分化和血管生成。lncRNASNHG1调节STAT3的磷酸化，降低ROS的水平，调节线粒体能量代谢，并最终促进软骨再生。
方法:通过贴壁方法从颌骨分离JBMMSCs。通过实时荧光定量聚合酶链反应(RT-qPCR)检测LncRNASNHG1对JBMMSCs软骨分化的影响,颗粒实验，阿尔辛蓝染色，马森三色染色，和改良天狼星红染色。RT-qPCR,基质凝胶管形成，和共培养实验用于确定lncRNASNHG1对JBMMSCs体外血管生成的影响。建立了新西兰兔膝关节软骨缺损模型和裸鼠皮下基质橡胶栓剂模型，用于体内实验。通过RT-qPCR检测线粒体功能的变化，二氢乙锭(DHE)染色,MitoSOX染色,四甲基罗丹明甲酯（TMRM）染色，和三磷酸腺苷（ATP）检测。Western印迹法检测信号转导和转录激活因子3(STAT3)的磷酸化水平。
结果：阿尔辛蓝染色，马森三色染色，和修饰的天狼星红染色显示lncRNASNHG1促进软骨分化。lncRNASNHG1促进体外血管生成和体内微血管形成。lncRNASNHG1促进兔膝关节软骨组织的修复和再生。Westernblot和Alcian蓝染色显示JAK抑制剂降低了SNHG1引起的STAT3磷酸化水平增加和染色加深。线粒体相关性分析显示，lncRNASNHG1导致活性氧（ROS）水平降低，线粒体膜电位的增加和ATP水平的增加。Alcian蓝染色显示，ROS抑制剂显著缓解了SNHG1敲低引起的蓝色荧光下降。
结论：lncRNASNHG1促进JBMMSCs的软骨分化和血管生成。lncRNASNHG1调节STAT3的磷酸化，降低ROS的水平，调节线粒体能量代谢，并最终促进软骨再生。
