人老化椎间盘髓核细胞体外老化模型的建立与细胞老化表型的初步研究

Senescence of nucleus pulposus cells in human aging intervertebral discs: in vitro model establishment and preliminary phenotype study

[目的]构建人椎间盘髓核细胞(nucleus pulposus cells,NPCs)老化模型,探讨老化NPCs中生长因子的表达差异及其与椎间盘退变(intervertebral disc degeneretion,IDD)的关系。[方法]体外单层培养人正常NPCs系(Scien Cell 4800),连续1:2传代培养,诱导建立NPCs复制性老化(replication senescence cells,RS)模型;不同浓度双氧水作用于未老化细胞,模拟椎间盘氧化应激微环境,诱导建立应激诱导过早老化(stress induced premature senescence cells,SIPS)模型。细胞衰老β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-gal)染色检测NPCs阳性表达率。流式细胞仪检测G1期细胞比例及细胞凋亡率。CCK-8检测不同代次细胞增殖活性。应用Agilent表达谱芯片初步筛选出正常与老化NPCs中差异性表达的生长因子,实时荧光定量PCR进一步验证所筛选生长因子的表达差异。[结果]NPCs经连续传代、双氧水诱导可分别构建RS模型及SIPS模型。两种老化模型SA-β-Gal阳性表达的细胞比例均高于70%,G1期细胞比例均高于80%,凋亡率均低于10%。Agilent表达谱芯片筛选出的碱性成纤维生长因子(basic fibroblast growth factor,b FGF)与血管内皮生长因子a(vascular endothelial growth factor a,VEGF-a)在不同老化模型中,表达差异有统计学意义(P<0.05),特别是b FGF在SIPS模型中的表达显著高于RS模型(P<0.05)。两种老化模型中的b FGF表达量显著高于正常组(P<0.05)。[结论]双氧水诱导所引起的氧化应激可促进细胞过早老化。老化NPCs上调b FGF,且SIPS模型中的上调幅度显著高于RS,SIPS可能比RS更加上调b FGF。不同老化诱导条件下,生长因子的表达可能存在差异,b FGF可能参与椎间盘再血管化及自我修复。

[Objective] To establish a model of senescence of nucleus pulposus cells （NPCs） in human intervertebral discs,and to investigate the correlation of expression differentiation of growth factors in aging NPCs and intervertebral disc de- generation. [Methods] Normal human NPCs were serially passaged in monolayer culture with a split ratio of 1：2, to establish a NPC model of replication senescence （RS） . Then, the intervertebral disc oxidative stress micro-environment was simulated by stimulating non-aging cells with different concentration of hydrogen peroxide,to establish a model of stress-induced prema- ture senescence （SIPS） . The senescence-associated β-galactosidase （SA-β-gal） staining was used to determine the per- centage of SA-β-Gal-positive NPCs. Flow cytometry was used to measure the percent age of G1 cells and the apoptosis rate. The CCK-8 assay was then applied to evaluate cell proliferation in different generations. Agilent microarray was used to prelimi- narily screen growth factors with different expressions between normal and aging NPCs. Quantitative real-time PCR was used to further confirm the differences in the expression of selected growth factors. [Results] The models of RS and SIPS were estab- lished by serial passage and hydrogen peroxide treatment of NPCs, respectively. In both models, the percentages of SA-β-Gal-positive cells and G1 cells were higher than 70% and 80%, respectively, while the apoptosis rates were lower than 10%. There were significant differences in the expression of two growth factors selected by agilent microarray, basic fibroblast growth factor （bFGF） and vascular endothelial growth fac-tot a （VEGF-a） ,between the two models （P 〈0.05） . Particularly, the expression of bFGF was significantly higher in the model of SIPS than in the model of RS （P 〈0.05） . The expression of bFGF was significantly higher in both models than in the normal group （P 〈0.05） . [Conclusions] Oxidative stress induced by hydrogen peroxide can promote premature senescence in cells. Aging NPCs have upregulated expression of bFGF,and the increase in expression of bFGFis substantially greater in the model of SIPS than in the model of RS, suggesting a greater upregulation of bFGF expression in the model of SIPS than in the model of RS. The expression of growth factors may differ in senescence induced by different factors, bFGF may play a role in re- vascularization and self-healing of intervertebral discs.