摘要
目的研究低氧培养对大鼠髓核间质干细胞(NPMSCs)增殖的影响。方法分离大鼠尾椎髓核获得NPMSCs并进行体外扩增培养,观察细胞形态,流式细胞仪鉴定细胞免疫表型。取第3代NPMSCs分为常氧组和2%低氧组,分别培养7 d及14 d,观察细胞形态;MTT法检测细胞增殖;流式细胞仪检测细胞活力和RT-q PCR检测低氧诱导因子1α(HIF-1α)、葡萄糖转运蛋白1(GLUT-1)、血管内皮生长因子(VEGF)、沉默信息调节蛋白1(SIRT1)及SIRT6的mRNA表达情况。结果原代NPMSCs细胞早期可形成葵花样细胞集落,传代后细胞增殖明显加快,细胞形态以纺锤形为主。第3代细胞高表达干细胞相关阳性表面抗原分子,低表达干细胞相关阴性表面抗原分子。低氧组细胞形态以多角形为主,细胞更容易聚集成团块,且细胞增殖速度明显加快(P<0.05),细胞凋亡率明显下降(P<0.05)。低氧组HIF-1α、VEGF、GLUT-1、SIRT1及SIRT6表达量明显高于常氧组(P<0.05)。结论 2%O2的低氧环境促进髓核间质干细胞增殖,其机制可能与SIRT1及SIRT6介导的HIF-1α信号通路有关。
Objective To investigate the effect of hypoxia on the proliferation of nucleus pulposus-derived mesenchymal stem cells（NPMSCs） in vitro and explore its possible mechanism.Methods NPMSCs were isolated from nucleus pulposus of Sprague-Dawley rats.Cellular morphology was observed and expression of CD11 b,CD45,CD73,CD90 and CD105 was detected using flow cytometry.The third generation NPMSCs were cultured under normoxia（20% O2） and hypoxia（2% O2） for 14 days.Cell viability was determined by the annexin-V-FITC/propidium iodide doublestaining assay and cell proliferation was measured by MTT assay.The expressions of hypoxia-inducible factor-1α（HIF-1α）,glucose transporter 1（GLUT-1）,vascular endothelial growth factor（VEGF）,silentinformation regulator protein 1（SIRT1） and silent information regulator protein 6（SIRT6） at the mRNA level were examined by semi-quantitative reverse transcription polymerase chain reaction（RT-q PCR）.Results NPMSCs formed sunflower-like colony and the third passage NPMSCs became homogeneous and exhibited spindle-like morphology.Meanwhile,high expression level of stem cell-related positive antigen molecules and low expression levels of negative antigen molecules.Hypoxia promoted proliferation of NPMSCs and promoted gene expression of HIF-1α,GLUT-1,VEGF,SIRT1 and SIRT6 significantly（P〈0.05）.Conclusions Hypoxia has a significant impact on the proliferation of NPMSCs and SIRT1,SIRT6 mediated HIF-1α pathway is potentially involved in the mechanism.
出处
《基础医学与临床》
CSCD
2017年第11期1546-1551,共6页
Basic and Clinical Medicine
基金
国家自然科学基金青年基金(81401830)
中国博士后科学基金二等资助(2015M571714)
江苏省自然科学基金青年基金(BK20140496)
关键词
髓核间质干细胞
低氧
增殖
缺氧诱导因子-1Α
沉默信息调节蛋白
nucleus pulposus mesenchymal stem cells
hypoxia
proliferation
HIF- 1α
silent information regulator protein
