胰岛素样生长因子-1对大鼠纤维环细胞体外增殖活性的影响
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摘要
目的通过对椎间盘纤维环细胞的培养,细胞演变的观察,研究椎间盘纤维环细胞的生物学特性和退变机制,并为组织工程、基因治疗等提供方法和体外模型,胰岛素样生长因子-1(IGF-1)对大鼠纤维环细胞体外增殖活性的影响,并观察其量效和时效关系。
方法体外分离培养大鼠纤维环细胞,将传1代细胞种于96孔板,采用噻唑蓝(MTT)比色法,观察IGF-1在1%和10%血清浓度下对大鼠纤维环细胞体外增殖的调节作用及其剂量、时间与作用效果的关系,行HE染色,甲苯胺蓝,免疫细胞化学染色分析细胞的生物学特性。
结果原代纤维环细胞多为梭形,有伪足伸出,甲苯胺蓝染色,胞浆染为深蓝色,免疫细胞学方法检测表明纤维环细胞有Ⅰ型胶原表达,在1%血清条件下,IGF-1的作用不显著,在10%血清条件下,IGF-1能提高细胞的增殖活性,并且在有效浓度范围内呈剂量效应关系。
结论IGF-1能促进大鼠纤维环细胞的体外增殖,其效应在一定范围内与剂量和时间呈正相关,本实验为椎间盘纤维环细胞的培养提供了简单、有效的方法,为进一步研究椎间盘纤维环细胞生物学功能和调控研究打下了基础。
Objective To reseach the biologic specificity and degeneration of the anulus fibrosus(AF) cells by culturing AF cells and observing their continuous changes,meanwhile the methods and the cell models were provided for tissue engineering and gene therapy,to investigate the dose-dependent and time-dependent effect of insulinlike growth factor-1(IGF-1) on proliferation of anulus fibrosus (AF) cells in vitro
Methods Tissue samples of AF were obtained from rats,Cells were isolated and cultured in HAMF12/DMEM medium supplemented with 10%fetal bovine serum(FBS),Cells were seeded in 96-well plates at 1×10~5cells/well,After synchroni zation.,medium was replaced by HAMF12/DMEM containing 1%or 10%FBS with IGF-1,Dose-dependent and time- dependent effect were examined by MTT assay,After staining with the haematine eosin and the toluidine blue,the immunohistochemical method was used to study the synthesis of typeⅠcollagen.
Results The primary cells mostly appeared as flattened and spindle shape,with the pseudopodia being stretching out,The cytoplasm was profoundly blue after stained with the toluidine blue,The immunohistochemical test revealed that there existed the positive expression of typeⅠcollagen on the cells,In the presence of 1%FBS,the role of IGF-1 was less striking effect on cell proliferation,in 10%FBS,IGF-1 had statistically significant mitogenic effects,The dose-dependent effect of IGF-1 on cell proliferation was found within 100μg/L,The time-dependent effect showed a significant elevation
Conclusion IGF-1 was efficient to stimulate cell proliferation of rat NF cells in vitro in a dose-dependent and time-dependent manner,The experimental study provides a simple and effective method for the culture of anulus fibrosus cell in vitro,It also form a firm foundation for facilitating the furthur study of the manipulation and modulation of biologic function and biologic treatments of disc
方法体外分离培养大鼠纤维环细胞,将传1代细胞种于96孔板,采用噻唑蓝(MTT)比色法,观察IGF-1在1%和10%血清浓度下对大鼠纤维环细胞体外增殖的调节作用及其剂量、时间与作用效果的关系,行HE染色,甲苯胺蓝,免疫细胞化学染色分析细胞的生物学特性。
结果原代纤维环细胞多为梭形,有伪足伸出,甲苯胺蓝染色,胞浆染为深蓝色,免疫细胞学方法检测表明纤维环细胞有Ⅰ型胶原表达,在1%血清条件下,IGF-1的作用不显著,在10%血清条件下,IGF-1能提高细胞的增殖活性,并且在有效浓度范围内呈剂量效应关系。
结论IGF-1能促进大鼠纤维环细胞的体外增殖,其效应在一定范围内与剂量和时间呈正相关,本实验为椎间盘纤维环细胞的培养提供了简单、有效的方法,为进一步研究椎间盘纤维环细胞生物学功能和调控研究打下了基础。
Objective To reseach the biologic specificity and degeneration of the anulus fibrosus(AF) cells by culturing AF cells and observing their continuous changes,meanwhile the methods and the cell models were provided for tissue engineering and gene therapy,to investigate the dose-dependent and time-dependent effect of insulinlike growth factor-1(IGF-1) on proliferation of anulus fibrosus (AF) cells in vitro
Methods Tissue samples of AF were obtained from rats,Cells were isolated and cultured in HAMF12/DMEM medium supplemented with 10%fetal bovine serum(FBS),Cells were seeded in 96-well plates at 1×10~5cells/well,After synchroni zation.,medium was replaced by HAMF12/DMEM containing 1%or 10%FBS with IGF-1,Dose-dependent and time- dependent effect were examined by MTT assay,After staining with the haematine eosin and the toluidine blue,the immunohistochemical method was used to study the synthesis of typeⅠcollagen.
Results The primary cells mostly appeared as flattened and spindle shape,with the pseudopodia being stretching out,The cytoplasm was profoundly blue after stained with the toluidine blue,The immunohistochemical test revealed that there existed the positive expression of typeⅠcollagen on the cells,In the presence of 1%FBS,the role of IGF-1 was less striking effect on cell proliferation,in 10%FBS,IGF-1 had statistically significant mitogenic effects,The dose-dependent effect of IGF-1 on cell proliferation was found within 100μg/L,The time-dependent effect showed a significant elevation
Conclusion IGF-1 was efficient to stimulate cell proliferation of rat NF cells in vitro in a dose-dependent and time-dependent manner,The experimental study provides a simple and effective method for the culture of anulus fibrosus cell in vitro,It also form a firm foundation for facilitating the furthur study of the manipulation and modulation of biologic function and biologic treatments of disc
引文
[1] Poiraudeau S,Monteiro I,Anract P,et al.Phenotypic characteristics of rabbit intervertebral disc cells:Comparison with cartilage cells from the samea nimals [J].Spine,1999,24:837—844.
[2] Marouda A al.Factors involved in the nutrition of the human lumbar intervertebral disc : Cellula -rity and diffusion of glucose in vitro [J].J Anat,1975,120:113
[3] Le Maitre CL,Hoyland JA,Freemont AJ.Studies of human intervertebral disc cell function in a constrained in vitro tissue culture system[J].Spine,2004,29:1187-1195.
[4] Nerlich AG,Bachmeier BE,Boos N.Expression of fibronectin and TGF-βI mRNA and protein suggest altered regulation of extracellular matrix in degenerated disc tissue [J]. Eur Spine J.2005,14:17-26.
[5] Takegami K,An HS, Kumano F,et al.Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondromnase ABC-induced in vitro chemonucleolysis[J].Spine J,2005,5:231-238.
[6] Alini M,Li W,Markovic P,et al.The potential and limitations of a cell seeded collagen/ hyaluro nan scaffold to engineer:An intervertebral disc like matrix[J]. Spine , 2003,28:446-454
[7] Shingo Maeda,Shoichi Kokubun.Changes with age in proteoglycan synthesis in cells cultured in vitro from the inner and outer rabbit annulus fibrosus responses to inter- leukin-1 and interleukin-1 receptor antagonist protein[J].Spine,2000,25:166
[8] Gruber HE, Norton HJ, Hanley EN. Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro[J].Spine, 2000, 25 (17) : 2153-2157.
[9] GRUBER H , HANL EY E.Human disc cells in monolayer vs 3D culture :cell shape ,division and matrix formation [ J ]. BMC Musculoskelet Disord, 2000 ,1 (1) :1.
[10] Osada R,Ohshima H,Ishihara H,et al.Autocrine / paracrine mechanism of insulin- like growth factor-1 secretion.and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs[J].J Orthop Res, 1996.14(5): 690-699.
[11] Gruber H, Hanley E.Human disc ceils in monolayer VS 3D culture;cell shape, division and matrix formation[J].BMC Museuloskelet Disord,2000,1(1):1.
[12] Wang j.Bacr A,Kraus V,et al.lntervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture[J].Spine.2001.26(16): 1747-1751.
[13]Sato M,Kikuchi T,Asazuma T,et al.Glycosaminoylycan accumulation in primary culture of rabbit intervertebral disc cells[J].Spine,2002,26:2653-2660.
[14]Gan Jc,Ducheyne P Vresilovic EJ.lntervertebral disc tissue engineering I:cultures of nucleus pulposus cells[J].Clin Orthop,2003,6(411):315-324.
[15]Gruber HE,Leslie K,lngram J.Cell-based tissue engineering for the interverte- bral disc:in vitro studies of human disc cell gene expression and matrix production within selected cell carriers[J].Spine.2004.4(1):44-55.
[16]吕振华.椎间盘的生物化学.见:胡有谷,主编.腰椎间盘突出症.第二版.北京:人民卫生出版社,1995,84-86
[17]胡有谷,叶发刚.脊柱和椎间盘的胚胎发育.见:胡有谷,主编.腰椎间盘突出症.第二版.北京:人民卫生出版社,1995,7-8.
[18]Roberts S,Menage J,Duance V,et al.Collagen types around the cells of the inter- vertebral disc and cartilage end plate:An immunolocalization study[J].Spine,1991,16:1030-1038.
[19]Ichimura K,Tsuji H,Matsui H,et al.Cell culture of the intervertebral disc of rats:Factors influencing culture,proteoglycan,collagen and deoxybonucleic acid syn- thesis[J].J Spinal Disord,1991,4:428-436.
[20]Gruber HE,Hanley EN Jr.Human disc ceils in monolayer vs 3D culture:Cell shape,division and matrix formation[J].BMC Musculoskelet Disord,2000,1:1-10
[21]Ducheyne P,Vresilovic E,Shapiro IM.Bioactive glass serves as a substrate for maintenance of phenotype of nucleus pulposus cells of the intervertebral disc[J].J Biomed Mater Res,2000,51:596-604
[1] Sun YL,Hurtig M,Pilliar RM,et al. Characterization of nucleus pulposus tissue formed in vitro [J]. Orthop Res, 2001, 19(6): 1078-1084.
[2] Gruber HE,Hanley EN Jr,Human disc cells in monolayer VS 3D culture: cell-shaped division and matrix Formation[J].BMC Musculoskelet Disord,2O00,1(1):1-10.
[3] Sato M ,Kikuchi T, Asazuma T,et al. Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells [J]. Spine,2001, 26(24): 2653-2660.
[4] Ganey T, Libera J, Moos V, et al. Disc chondrocyte transplantation in a canine model: a treatment for degenerated or damaged intervertebral disc[J]. Spine, 2003, 28(23): 2609-2260.
[5] Mizuno H, Roy AK, Vacanti CA, et al.Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement [J]. Spine, 2004, 29(12): 1290-1297.
[6] Nomura T,Mochida J, Okuma M,et al.Nucleus pulposus allograft retarts intervertebral disc degeneration[J]. Clin Orthop,2001.389:94-101.
[7] Kusior LJ, Vacanti CA,BayleyJC,et al.Tissue engineering of nucleus pulposus[J].Trans Orthop Res Soc,1999,24: 807.
[8] Sato M,Asazuma T,Ishihara M,et al.An experimental study Of the regeneration of the intervertebral disc with an allograft of cultured anulus fibrosus cells using a tissue-engineering method[J].Spine 2003,28(6): 548-553.
[9] Korbling M,Estrov Z,Champlin R,et al.Adult stem cells and tissue repair[J].Bone Marrow Transplant,2003, 8(Suppl 1):23-24.
[10] Zuk PA,Zhu M,Ashjian P,et al.Human adipose tissue is a source of multipotent stem cells[J].Mol Biol Cell, 2002, 13(12):4279-4295.
[11] Sakai D, Mochida J, Yamamoto Y, et al. Transplantation of mesenchymal stem cells embedded in atelocollagen (R) gel to the intervertebral disc: a potential therapeutic model for disc degeneration[J].Biomaterials,2003,27(20):3531-3541.
[12]鞠小东,娄思权,马庆军,等.脂肪间充质干细胞的成骨诱导分化[J].骨与关节损伤杂志,2004,19(6):390-392.
[13]Ichimura K,Tsuji H,Matsui H,et al.Cell culture of the intervertebral disc of rats:factors influencing culture,proteoglyean,collagen,and deoxyrionucleic acid synthesis[J].J Spinal Disord,1991,4(4):428-436.
[14]Shinmei M,Kikuchi T,Yamagishi,et al.The role of interleukin on proteoglycanmetabolism of rabbit annulus fibrous cells cultured in vitro[J].Spine,1988,13(11):1284-1289.
[15]刘勇,胡有谷,吕振华.腰椎间盘细胞的培养及形态学观察[J].中华医学杂志,1999,79(2):109-111.
[16]Holy C,Shcichet M,Davies J.Engineering three dimension a bone tissue in vitro using biodegradable scaffolds in vestigation initadell seeding lens it and culture period[J].J Biomed Mater Res,2000,51(3):376-382.
[17]Botchwey E,Pollack S,Levine E,et al.Bone tissue engineering in arotating lioreactor using a microcarried matrix system[J].J Biomed MaterRes,2001,55(2):242-253.
[18]Qiu Q,Ducheyne P,Ayyaswamy P.3D bone tissue engineered with bioactive microsphersin simulated microgravity[J].In Vitro Cell Dev Bid Anim,2001,37(3):157-165.
[19]陈岩,胡有谷,齐宗华,等.转化生长因予β与椎间盘细胞Ⅰ型胶原基因调控的关系[J].中国矫形外科杂志,2000,7(2):151-153.
[20]Takegami K,Thonar EJ,An HS,et al.Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-l[J]:Spine,2002,27(12):1324-1325.
[21]Osada R,Ohshima H,Ishihara H,et al.Autocrine / paracrine mechanism of insulin-like growth factor-1 secretion,and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs[J].J Orthop Res,1996,14(5):690-699.
[22] Gruber HE, Norton HJ, Hanley EN Jr. Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro[J]. Spine, 2000, 25(17): 2153—2157.
[23] Nagano T,Yonenobu K,Miyamoto S,et al. Distribution of the basic fibroblast growth factor and its receptor gene expression in normal and degenerated rat intervertebral discs[J].Spine,1995,20(18): 1972-1978.
[24] Handa T,Ishihara H,Ohshima H,et al.Efects of hydrostatic pressure on matrix synthesis and matrix metal of proteinase production in the human lumber intervertebral discs[J]. Spine, 1997, 22(10): 1085-1091.
[25] Ishihara H.Mcnally D.Urban J.et al.Effects of hydrostatic pressure on matrix synthesis in different regions of the intervertebral disk [J].J Appl Physiol, 1996, 80(3): 839-846.
[26] Hutton WC, Elmer WA, Boden SD, et al. The effect of hydrostatic pressure on intervertebral disc metabolism[J].Spine, 1999. 24(15): 1507—1515.
[27] Hutton W,Elmer W,Bryce L,et al. Do the intervertebral disc cells respond to different levels of hydrostatic pressure[J]?Clinical Biomechanics,2001,16(9): 728—734.
[28] Liu GZ, Ishihara H, Osada R, et al. Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure[J].Spine, 2001, 26(2): 134—141.
[2] Marouda A al.Factors involved in the nutrition of the human lumbar intervertebral disc : Cellula -rity and diffusion of glucose in vitro [J].J Anat,1975,120:113
[3] Le Maitre CL,Hoyland JA,Freemont AJ.Studies of human intervertebral disc cell function in a constrained in vitro tissue culture system[J].Spine,2004,29:1187-1195.
[4] Nerlich AG,Bachmeier BE,Boos N.Expression of fibronectin and TGF-βI mRNA and protein suggest altered regulation of extracellular matrix in degenerated disc tissue [J]. Eur Spine J.2005,14:17-26.
[5] Takegami K,An HS, Kumano F,et al.Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondromnase ABC-induced in vitro chemonucleolysis[J].Spine J,2005,5:231-238.
[6] Alini M,Li W,Markovic P,et al.The potential and limitations of a cell seeded collagen/ hyaluro nan scaffold to engineer:An intervertebral disc like matrix[J]. Spine , 2003,28:446-454
[7] Shingo Maeda,Shoichi Kokubun.Changes with age in proteoglycan synthesis in cells cultured in vitro from the inner and outer rabbit annulus fibrosus responses to inter- leukin-1 and interleukin-1 receptor antagonist protein[J].Spine,2000,25:166
[8] Gruber HE, Norton HJ, Hanley EN. Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro[J].Spine, 2000, 25 (17) : 2153-2157.
[9] GRUBER H , HANL EY E.Human disc cells in monolayer vs 3D culture :cell shape ,division and matrix formation [ J ]. BMC Musculoskelet Disord, 2000 ,1 (1) :1.
[10] Osada R,Ohshima H,Ishihara H,et al.Autocrine / paracrine mechanism of insulin- like growth factor-1 secretion.and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs[J].J Orthop Res, 1996.14(5): 690-699.
[11] Gruber H, Hanley E.Human disc ceils in monolayer VS 3D culture;cell shape, division and matrix formation[J].BMC Museuloskelet Disord,2000,1(1):1.
[12] Wang j.Bacr A,Kraus V,et al.lntervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture[J].Spine.2001.26(16): 1747-1751.
[13]Sato M,Kikuchi T,Asazuma T,et al.Glycosaminoylycan accumulation in primary culture of rabbit intervertebral disc cells[J].Spine,2002,26:2653-2660.
[14]Gan Jc,Ducheyne P Vresilovic EJ.lntervertebral disc tissue engineering I:cultures of nucleus pulposus cells[J].Clin Orthop,2003,6(411):315-324.
[15]Gruber HE,Leslie K,lngram J.Cell-based tissue engineering for the interverte- bral disc:in vitro studies of human disc cell gene expression and matrix production within selected cell carriers[J].Spine.2004.4(1):44-55.
[16]吕振华.椎间盘的生物化学.见:胡有谷,主编.腰椎间盘突出症.第二版.北京:人民卫生出版社,1995,84-86
[17]胡有谷,叶发刚.脊柱和椎间盘的胚胎发育.见:胡有谷,主编.腰椎间盘突出症.第二版.北京:人民卫生出版社,1995,7-8.
[18]Roberts S,Menage J,Duance V,et al.Collagen types around the cells of the inter- vertebral disc and cartilage end plate:An immunolocalization study[J].Spine,1991,16:1030-1038.
[19]Ichimura K,Tsuji H,Matsui H,et al.Cell culture of the intervertebral disc of rats:Factors influencing culture,proteoglycan,collagen and deoxybonucleic acid syn- thesis[J].J Spinal Disord,1991,4:428-436.
[20]Gruber HE,Hanley EN Jr.Human disc ceils in monolayer vs 3D culture:Cell shape,division and matrix formation[J].BMC Musculoskelet Disord,2000,1:1-10
[21]Ducheyne P,Vresilovic E,Shapiro IM.Bioactive glass serves as a substrate for maintenance of phenotype of nucleus pulposus cells of the intervertebral disc[J].J Biomed Mater Res,2000,51:596-604
[1] Sun YL,Hurtig M,Pilliar RM,et al. Characterization of nucleus pulposus tissue formed in vitro [J]. Orthop Res, 2001, 19(6): 1078-1084.
[2] Gruber HE,Hanley EN Jr,Human disc cells in monolayer VS 3D culture: cell-shaped division and matrix Formation[J].BMC Musculoskelet Disord,2O00,1(1):1-10.
[3] Sato M ,Kikuchi T, Asazuma T,et al. Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells [J]. Spine,2001, 26(24): 2653-2660.
[4] Ganey T, Libera J, Moos V, et al. Disc chondrocyte transplantation in a canine model: a treatment for degenerated or damaged intervertebral disc[J]. Spine, 2003, 28(23): 2609-2260.
[5] Mizuno H, Roy AK, Vacanti CA, et al.Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement [J]. Spine, 2004, 29(12): 1290-1297.
[6] Nomura T,Mochida J, Okuma M,et al.Nucleus pulposus allograft retarts intervertebral disc degeneration[J]. Clin Orthop,2001.389:94-101.
[7] Kusior LJ, Vacanti CA,BayleyJC,et al.Tissue engineering of nucleus pulposus[J].Trans Orthop Res Soc,1999,24: 807.
[8] Sato M,Asazuma T,Ishihara M,et al.An experimental study Of the regeneration of the intervertebral disc with an allograft of cultured anulus fibrosus cells using a tissue-engineering method[J].Spine 2003,28(6): 548-553.
[9] Korbling M,Estrov Z,Champlin R,et al.Adult stem cells and tissue repair[J].Bone Marrow Transplant,2003, 8(Suppl 1):23-24.
[10] Zuk PA,Zhu M,Ashjian P,et al.Human adipose tissue is a source of multipotent stem cells[J].Mol Biol Cell, 2002, 13(12):4279-4295.
[11] Sakai D, Mochida J, Yamamoto Y, et al. Transplantation of mesenchymal stem cells embedded in atelocollagen (R) gel to the intervertebral disc: a potential therapeutic model for disc degeneration[J].Biomaterials,2003,27(20):3531-3541.
[12]鞠小东,娄思权,马庆军,等.脂肪间充质干细胞的成骨诱导分化[J].骨与关节损伤杂志,2004,19(6):390-392.
[13]Ichimura K,Tsuji H,Matsui H,et al.Cell culture of the intervertebral disc of rats:factors influencing culture,proteoglyean,collagen,and deoxyrionucleic acid synthesis[J].J Spinal Disord,1991,4(4):428-436.
[14]Shinmei M,Kikuchi T,Yamagishi,et al.The role of interleukin on proteoglycanmetabolism of rabbit annulus fibrous cells cultured in vitro[J].Spine,1988,13(11):1284-1289.
[15]刘勇,胡有谷,吕振华.腰椎间盘细胞的培养及形态学观察[J].中华医学杂志,1999,79(2):109-111.
[16]Holy C,Shcichet M,Davies J.Engineering three dimension a bone tissue in vitro using biodegradable scaffolds in vestigation initadell seeding lens it and culture period[J].J Biomed Mater Res,2000,51(3):376-382.
[17]Botchwey E,Pollack S,Levine E,et al.Bone tissue engineering in arotating lioreactor using a microcarried matrix system[J].J Biomed MaterRes,2001,55(2):242-253.
[18]Qiu Q,Ducheyne P,Ayyaswamy P.3D bone tissue engineered with bioactive microsphersin simulated microgravity[J].In Vitro Cell Dev Bid Anim,2001,37(3):157-165.
[19]陈岩,胡有谷,齐宗华,等.转化生长因予β与椎间盘细胞Ⅰ型胶原基因调控的关系[J].中国矫形外科杂志,2000,7(2):151-153.
[20]Takegami K,Thonar EJ,An HS,et al.Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-l[J]:Spine,2002,27(12):1324-1325.
[21]Osada R,Ohshima H,Ishihara H,et al.Autocrine / paracrine mechanism of insulin-like growth factor-1 secretion,and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs[J].J Orthop Res,1996,14(5):690-699.
[22] Gruber HE, Norton HJ, Hanley EN Jr. Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro[J]. Spine, 2000, 25(17): 2153—2157.
[23] Nagano T,Yonenobu K,Miyamoto S,et al. Distribution of the basic fibroblast growth factor and its receptor gene expression in normal and degenerated rat intervertebral discs[J].Spine,1995,20(18): 1972-1978.
[24] Handa T,Ishihara H,Ohshima H,et al.Efects of hydrostatic pressure on matrix synthesis and matrix metal of proteinase production in the human lumber intervertebral discs[J]. Spine, 1997, 22(10): 1085-1091.
[25] Ishihara H.Mcnally D.Urban J.et al.Effects of hydrostatic pressure on matrix synthesis in different regions of the intervertebral disk [J].J Appl Physiol, 1996, 80(3): 839-846.
[26] Hutton WC, Elmer WA, Boden SD, et al. The effect of hydrostatic pressure on intervertebral disc metabolism[J].Spine, 1999. 24(15): 1507—1515.
[27] Hutton W,Elmer W,Bryce L,et al. Do the intervertebral disc cells respond to different levels of hydrostatic pressure[J]?Clinical Biomechanics,2001,16(9): 728—734.
[28] Liu GZ, Ishihara H, Osada R, et al. Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure[J].Spine, 2001, 26(2): 134—141.