SDF1/CXCR4信号轴在退变髓核细胞诱导血管新生中的作用
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摘要
目的探讨髓核细胞(NPCs)分泌的基质细胞衍生因子1(SDF1)对退变椎间盘血管化的影响及相关分子机制。方法培养退变椎间盘NPCs,病毒转染分别上调和下调SDF1表达,根据SDF1表达情况分为DOWN、D和UP 3组;培养人脐静脉内皮细胞(HUVECs),将不同组NPCs或其条件培养基与HUVECs进行共培养,期间还在共培养体系中加入CXCR4受体抑制剂AMD3100,CCK8检测、细胞迁移实验、管腔形成实验观察HUVECs成血管能力。结果各组NPCs构建成功;与HUVECs共培养后,利用CCK8法、细胞迁移实验、管腔形成实验检测结果显示随SDF1表达升高,内皮细胞活力、迁移能力、管腔形成能力也随之升高(P<0.05);当加入AMD3100后,内皮细胞活力、迁移能力、管腔形成能力在D组和UP组均受到显著抑制(P<0.05)。结论人退变椎间盘NPCs表达SDF1。SDF1/CXCR4信号轴影响内皮细胞活力、迁移能力和成管能力,并在退变椎间盘血管化中发挥重要作用。
Objective To investigate the influence and relative mechannism of stromal cell derived factor 1(SDF1)secreted by nucleus pulposus cells(NPCs) on the vascularization of degenerated intervertebral disc. MethodsNPCs of degenerated intervertebral disc were obtained and cultured. The expression of SDF1 in NPCs was up regulated and down regulated by transfected virus, then NPCs were classified as DOWN(down regulation),D(degeneration) and UP(up regulation) groups according to the expression of SDF1. Human umbilical vein endothelial cells(VECs) were cultured. Different groups of NPCs or conditional medium from NPCs were respectively co-cultured with VECs, during the period, AMD3100, the CXCR4 receptor inhibitor was also added into the co-culture system. The CCK8, cell migration assay and tube formation assay were used to detect the angiogenesis ability of VECs. Results Each group of NPCs was successfully constructed. After co-cultured with VECs, the CCK8, cell migration assay and tube formation assay showed that VECs cell vitality, migration ability and tube formation ability wereenhanced as the expression of SDF1 was increased( P<0. 05). After adding AMD3100,the cell vitality,migration ability and tube formation ability of VECs were significantly suppressed in D and UP groups( P<0. 05). Conclusions SDF1 is secreted by NPCs of degenerated intervertebral disc,the SDF1/CXCR4 axis may influence the ability of vitality,migration and tube formation of VECs,and may thus play an important role in vascularization of human degenerated intervertebral disc.
Objective To investigate the influence and relative mechannism of stromal cell derived factor 1(SDF1)secreted by nucleus pulposus cells(NPCs) on the vascularization of degenerated intervertebral disc. MethodsNPCs of degenerated intervertebral disc were obtained and cultured. The expression of SDF1 in NPCs was up regulated and down regulated by transfected virus, then NPCs were classified as DOWN(down regulation),D(degeneration) and UP(up regulation) groups according to the expression of SDF1. Human umbilical vein endothelial cells(VECs) were cultured. Different groups of NPCs or conditional medium from NPCs were respectively co-cultured with VECs, during the period, AMD3100, the CXCR4 receptor inhibitor was also added into the co-culture system. The CCK8, cell migration assay and tube formation assay were used to detect the angiogenesis ability of VECs. Results Each group of NPCs was successfully constructed. After co-cultured with VECs, the CCK8, cell migration assay and tube formation assay showed that VECs cell vitality, migration ability and tube formation ability wereenhanced as the expression of SDF1 was increased( P<0. 05). After adding AMD3100,the cell vitality,migration ability and tube formation ability of VECs were significantly suppressed in D and UP groups( P<0. 05). Conclusions SDF1 is secreted by NPCs of degenerated intervertebral disc,the SDF1/CXCR4 axis may influence the ability of vitality,migration and tube formation of VECs,and may thus play an important role in vascularization of human degenerated intervertebral disc.
引文
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[6] Pfirrmann CW,Metzdorf A,Zanetti M,et al.Magnetic resonance classification of lumbar intervertebral disc degeneration[J].Spine (Phila Pa 1976),2001,26:1873-1878.
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[11]Johnson WE,Caterson B,Eisenstein SM,et al.Human intervertebral disc aggrecan inhibits endothelial cell adhe-sion and cell migration in vitro[J].Spine(Phila Pa 1976),2005,30:1139-1147.
[12] Peng B,Chen J,Kuang Z,et al.Expression and role of connective tissue growth factor in painful disc fibrosis and degeneration[J].Spine(Phila Pa 1976),2009,34:E178-E182.
[13] R?sep T,Minajeva A,Asser T.Relationship between neovascularization and degenerative changes in herniated lumbar intervertebral discs[J].Eur Spine J,2013,22:2474-2480.
[14] Yang W,Yu XH,Wang C,et al.Interleukin-1β in intervertebral disk degeneration[J].Clin Chim Acta,2015,450:262-272.
[15] Gupta SK,Lysko PG,Pillarisetti K,et al.Chemokine receptors in human endothelial cells.Functional expression of CXCR4 and its transcriptional regulation by inflam-matory cytokines[J].J Biol Chem,1998,273:4282-4287.