大鼠髓核细胞体外衰老模型的建立

王厚磊; 卢伟; 李德芳; 丁磊; 吴靖平

doi:10.12025/j.issn.1008-6358.2016.20160103

大鼠髓核细胞体外衰老模型的建立

复旦大学附属金山医院骨科,上海 201508

基金项目: 上海市卫生和计划生育委员会科研项目(编号:2012-341)

计量
- 文章访问数: 2190
- HTML全文浏览量: 69
- PDF下载量: 1147
出版历程
- 刊出日期: 2016-02-24

Establishment of the Senescence Model of Rat Nucleus Pulposus Cell in Vitro

Department of Orthopedics, Jinshan Hospital,Fudan University, Shanghai 201508, China

摘要

摘要: 目的: 建立大鼠椎间盘髓核细胞体外衰老模型。方法: 提取大鼠椎间盘髓核细胞,在完全培养基中培养,作为对照组；在对照组基础上加入终浓度为100 μmol/L三丁基过氧化氢(t-BHP)培养2 h,构建髓核细胞体外衰老模型(衰老模型组)。采用Western印迹法检测两组髓核细胞的衰老相关指标微囊蛋白-1(caveolin-1)、β-半乳糖苷酶(SA-β-gal)的表达量,同时采用CCK-8细胞增殖实验检测两组的细胞活性。结果: 与对照组比较,t-BHP作用2 h后,衰老模型组caveolin-1、SA-β-gal表达量明显升高,且髓核细胞增殖速率减慢,细胞活性明显降低。结论: 采用t-BHP诱导的方法能成功构建髓核细胞体外衰老模型,caveolin-1在此过程中诱发了髓核细胞的衰老。
- 髓核细胞 /
- 衰老 /
- 三丁基过氧化氢
Abstract: Objective: To establish a senescence model of rat intervertebral disc nucleus pulposus cells. Methods: The nucleus pulposus cells, which were extracted from rat intervertebral discs and cultured in complete medium, were set as control group. The senescence model of nucleus pulposus cells in vitro(senescence model group) was established by additional culture for two hours on the basis of control group, to which tert-butyl hydroperoxide(t-BHP) was added with a final concentration of 100 μmol/L. The expression levels of senescence associated indexes, such as caveolin-1 and beta-galactosidase (SA-β-gal), in two groups were assessed by Western blotting, while the cell viability in two groups was detected by Cell Counting Kit-8(CCK-8) proliferation assay. Results: After two hours of t-BHP function, the expression levels of caveolin-1and SA-β-gal in senescence model group were significantly higher than those in control group, while the nucleus pulposus cells proliferation was slower and the cell viability was much lower. Conclusions: The senescence model of nucleus pulposus cells in vitro can be established successfully with the induction of t-BHP, and caveolin-1 induces the senescence of nucleus pulposus cells during the process.
- Nucleus pulposus cells /
- Senescence /
- Tert-butyl hydroperoxide

HTML全文

参考文献(21)

[1]	Tang X, Jing L, Chen J.Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging[J].PLoS One, 2012,7(12):e52020.
[2]	Sakai D.Future perspectives of cell-based therapy for intervertebral disc disease[J].Eur Spine J, 2008,7(Suppl 4):452-458.
[3]	Gruber HE, Ingram JA, Norton HJ, et al.Senescence in cells of the aging and degenerating intervertebral disc:immunolocalization of senescence-associated beta-galactosidase in human and sand rat discs[J].Spine (Phila Pa 1976), 2007,2(3):321-327.
[4]	Heathfield SK, Le Maitre CL, Hoyland JA.Caveolin-1 expression and stress-induced premature senescence in human intervertebral disc degeneration[J].Arthritis Res Ther, 2008,0(4):R87.
[5]	Gruber HE, Ingram JA, Davis DE, et al.Increased cell senescence is associated with decreased cell proliferation in vivo in the degenerating human annulus[J].Spine J, 2009,9(3):210-215.
[6]	Tang X, Jing L, Chen J.Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging[J].PLoS One, 2012,7(12):e52020.
[7]	Masaki H.Role of antioxidants in the skin:anti-aging effects[J].J Dermatol Sci, 2010,8(2):85-90.
[8]	Cheng H, Qiu L, Ma J, et al.Replicative senescence of human bone marrow and umbilical cord derived mesenchymal stem cells and their differentiation to adipocytes and osteoblasts[J].Mol Biol Rep, 2011,8(8):5161-5168.
[9]	Efimenko A, Dzhoyashvili N, Kalinina N, et al.Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential[J].Stem Cells Transl Med, 2014,3(1):32-41.
[10]	蔡大勇, 赵雁, 黄启福.D-半乳糖致原代培养神经元损伤模型的研究[J].中国病理生理杂志, 2002,8(7):794-797.
[11]	Szarka A, Bánhegyi G, Sümegi B.Mitochondria, oxidative stress and aging[J].Orv Hetil, 2014,5(12):447-452.
[12]	Zhou Y, Yang B, Yao X, et al.Establishment of an aging model of Sca-1+ hematopoietic stem cell and studies on its relative biological mechanisms[J].In Vitro Cell Dev Biol Anim, 2011,7(2):149-156.
[13]	Dimri GP.What has senescence got to do with cancer?[J].Cancer Cell, 2005,7(6):505-512.
[14]	Park WY, Park JS, Cho KA, et al.Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells[J].J Biol Chem, 2000,5(27):20847-20852.
[15]	Cho KA, Ryu SJ, Oh YS, et al.Morphological adjustment of senescent cells by modulating caveolin-1 status[J].J Biol Chem, 2004,9(40):42270-42278.
[16]	Cho KA, Park SC.Caveolin-1 as a prime modulator of aging:a new modality for phenotypic restoration?[J].Mech Ageing Dev, 2005,6(1):105-110.
[17]	Volonte D, Zhang K, Lisanti MP, et al.Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts[J].Mol Biol Cell, 2002,3(7):2502-2517.
[18]	Bartholomew JN, Volonte D, Galbiati F.Caveolin-1 regulates the antagonistic pleiotropic properties of cellular senescence through a novel Mdm2/p53-mediated pathway[J].Cancer Res, 2009,9(7):2878-2886.
[19]	Heathfield SK, Le Maitre CL, Hoyland JA.Caveolin-1 expression and stress-induced premature senescence in human intervertebral disc degeneration[J].Arthritis Res Ther, 2008,0(4):R87.
[20]	Cho KA, Park SC.Caveolin-1 as a prime modulator of aging:a new modality for phenotypic restoration?[J].Mech Ageing Dev, 2005,6(1):105-110.
[21]	Debacq-Chainiaux F, Erusalimsky JD, Campisi J, et al.Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo[J].Nat Protoc, 2009,4(12):1798-1806.