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

发布时间：2018-03-25 01:16

本文选题：细胞老化　切入点：生长因子　出处：《中国矫形外科杂志》2017年01期

【摘要】：[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(P0.05),特别是b FGF在SIPS模型中的表达显著高于RS模型(P0.05)。两种老化模型中的b FGF表达量显著高于正常组(P0.05)。[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。
[Abstract]:[objective] to construct the aging model of pulposus cells of nucleus pulposus of human intervertebral disc, and to explore the difference of growth factor expression in aging NPCs and its relationship with intervertebral disc degeneretion (IDD). The NPCs replicating senescence cells RSs model was induced, and different concentrations of hydrogen peroxide acted on the unaged cells to simulate the oxidative stress microenvironment of the intervertebral disc. The senescence-associated 尾 -galactosidase SA- 尾 -galactosidase (SA- 尾 -galactosidase) staining was used to detect the positive expression of NPCs. Flow cytometry was used to detect the percentage of cells in G1 phase and the rate of cell apoptosis. CCK-8 was used to detect the different generations of cells. Proliferative activity. Agilent expression microarray was used to screen the differentially expressed growth factors in normal and aging NPCs. Real-time fluorescent quantitative PCR was used to further verify the difference of expression of growth factors. [results] NPCs was subcultured continuously. The ratio of SA- 尾 -Gal positive cells in both aging models was higher than that in 70 G 1 phase cells, and the percentage of apoptosis was lower than that of basic fibroblast growth screened by 10%.Agilent expression microarray. VEGF-a and a(vascular endothelial growth factor FGFs were observed in different aging models. The expression of b FGF in SIPS model was significantly higher than that in RS model P0 05. The expression of b FGF in two aging models was significantly higher than that in normal group. [conclusion] oxidative stress induced by hydrogen peroxide can be induced by hydrogen peroxide. Aging NPCs upregulated bFGFs, and the upregulation rate in SIPS model was significantly higher than that in RSS-SIPS model. There may be differences in the expression of growth factor B FGF, which may be involved in disc revascularization and self-repair.
【作者单位】：东南大学医学院东南大学附属中大医院骨科;东南大学医学院东南大学附属中大医院普外科;
【基金】：国家自然科学基金资助项目(编号:81272035;81572170) 青年科学基金资助项目(编号:81201423)
【分类号】：R681.53

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