Notch-1基因敲除兔骨髓间充质干细胞阻止兔椎间盘退行性变的实验研究
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摘要
目的研究小分子干扰RNA(siRNA)靶向沉默兔骨髓间充质干细胞(MSC)中Notch-1基因,并通过TGF-β1诱导Notch-1基因沉默MSC向髓核样细胞定向分化,观测Notch-1基因在MSC向类软骨细胞定向分化时的调节作用。
方法(1)4-6周龄新西兰兔麻醉后,取股骨骨髓,以密度梯度离心法分离培养MSC；(2)根据siRNA原理针对Notch-1设计短发卡状RNA (shRNA),瞬时转染入MSC,转化生长因子.-β1(TGF-β1)诱导转染MSC分化；(3)实验分组为TGF-β1诱导无转染组MSC、TGF-β1诱导转染无意siRNA空质粒组MSC、TGF-β1诱导转染siRNA-Notch-1质粒组MSC三组,甲苯胺蓝染色及Ⅱ型胶原染色检测MSC的细胞表型的改变情况；(4) RT-PCR及Western blot法检测三组细胞中Ⅱ型胶原(COL2)及蛋白聚糖(ACAN)基因表达水平。
结果(1)光镜下观察MSC细胞形态主要为梭形或椭圆形,呈巢状或漩涡状生长；(2)转染后MSC中Notch-1基因沉默效率为47%(P<0.001),即所选位点具有明显的沉默效果。(3)甲苯胺蓝染色TGF-β1诱导转染siRNA-Notch-1质粒组MSC染色阳性表达明显高于其余两组。(4) RT-PCR检测发现TGF-β1诱导转染siRNA-Notch-1质粒组MSC的COL2表达为TGF-β1诱导无转染组MSC的2.55倍(P<0.001),为TGF-β1诱导转染无意siRNA空质粒组MSC的2.53倍(P<0.001)。ACAN表达分别为1.51倍(P<0.01)和1.53倍(P<0.01)。Western blot检测发现TGF-β1诱导转染siRNA-Notch-1质粒组MSC的COL2表达为TGF-β1诱导无转染组MSC的1.25倍(P<0.05),为TGF-β1诱导转染无意siRNA空质粒组MSC的1.37倍(P<0.05)。ACAN表达分别为1.99倍(P<0.05)和1.46倍(P<0.05)。
结论(1)使用密度梯度离心法可以分离出纯度较高的MSC。(2)三组细胞经TGF-β1诱导均可产生COL2及ACAN,表现出类软骨细胞表型；TGF-β1诱导转染siRNA-Notch-1质粒MSC表型基因表达较转染空质粒MSC及未转染MSC显著提高。
目的使用纤维环穿刺抽吸法造模兔椎间盘退行性变,移植转化生长因子p1(TGF-β1)诱导Notch-1基因沉默兔骨髓间充质干细胞(MSCs)到退变的椎间盘内,观测退变椎间盘的变化。
方法(1)4只体重0.4-0.5kg新西兰兔麻醉后,取股骨骨髓,以密度梯度离心法分离培养MSCs;(2)将针对Notch-1的短发卡状RNA (shRNA)及无意空质粒shRNA,瞬时转染入MSCs, TGF-β1诱导转染MSCs分化：(3)体重1.0-1.5kg新西兰兔10只,手术对兔脊柱L3-4、L4-5、L5-6三个椎间盘行穿刺抽吸髓核组织造模,2周后行核磁共振成像(MRI)检查观测造模情况；将细胞分为TGF-β1诱导转染空质粒组、TGF-β1诱导转染shRNA-Notch-1质粒组、TGF-β1诱导无转染组三组细胞,造模2周后分别移植入L3-4、L4-5、L5-6三个椎间盘,4周后行MRI检查观测细胞治疗情况；(4)动物MRI检查后处死行髓核组织的甲苯胺蓝染色观测蛋白多糖表达情况,RT-PCR及Western-blot检测退变椎间盘组织Ⅱ型胶原(COL2)及蛋白多糖(ACAN)改变情况。
结果(1)细胞移植4周后MRI检测发现,L4-5椎间盘%T2加权像扫描(%ST2WI)值增加最为明显,与其他2组有明显差异；L3-4、L5-6椎间盘%ST2WI值增加,两组间无统计学差异。(2)甲苯胺蓝染色：与空白对照组及阴性对照组相比较,实验组椎间盘组织内蛋白多糖的表达显著提高。(3) RT-PCR检测发现实验组椎间盘内Ⅱ型胶原及蛋白多糖的表达明显高于其余两组,存在显著性差异,阴性对照组与空白对照组间无显著性差异(4) Western blot检测发现现实验组椎间盘内Ⅱ型胶原及蛋名糖的表达明显高于其余两组,存在显著性差异,阴性对照组与空白对照组间无显著性差异。结论兔退变椎间盘内移植Notch-1基因敲除兔骨髓间充质干细胞,可有效的修复退变椎间盘组织。
Objective To observe the effects of Notchl gene on the directional differentiation of rabbit marrow mesenchymal stem cells (MSCs) into a nucleus pulposus-like phenotype through silenced Northl gene expression by small interfering RNA (siRNA) and induced by TGF-β1.
Methods MSCs were obtained from New Zealand rabbits (4-6week-old) femur bone marrow and purified by discontinuous gradient density centrifugation. The short hairpin RNA (shRNA) aimed at Notchl gene was designed according to the siRNA principle, and transient transfect into MSCs. Cells were divided into three groups:induced by TGF-β1only (group1); induced by TGF-β1and transfect by nonsense siRNA (group2); induced by TGF-β1and transfect by shRNA-Notchl (group3). The expressions of proteoglycan were detected by toluidine blue staining. The expressions of collagen Ⅱ and proteoglycan were examined in gene and protein level with RT-PCR and Western blot.
Results The MSCs were ellipse or fusiform shape and gathered in nest or swirl. Notchl expression was down-regulated by shRNA-Notchl, and the silencing rate was47%(P<0.001). The positive expressions in shRNA-Notchl group were higher than the others by toluidine blue staining. RT-PCR showed that the expression of collagen Ⅱ in group1was2.55times than group1(P<0.001),2.53times than group2(P<0.001), and the expression of proteoglycan in group3was1.51times than group1(P<0.01),1.53times than group2(P<0.01). Western-blot showed that the expression of collagen Ⅱ in group1was1.25times than group1(P<0.05),1.37times than group2(P<0.05), and the expression of proteoglycan in group3was1.99times than group1(P<0.05),1.46times than group2(P<0.05).
Conclusions This study demonstrates that MSCs could be purified by discontinuous gradient density centrifugation. TGF-β1can induce the MSCs to excrete collagen Ⅱand proteoglycan, and the down-regulation of Notchl gene could increase these excretion.
Objective To investigated the effects of Notchl knockdown and treatment with TGF-β1on the regulation of the directional differentiation of mesenchymal stem cells (MSCs) in suppressing the degeneration of punctured intervertebral discs in rabbits.
Methods MSCs were isolated from the femur bone of New Zealand rabbits and purified with discontinuous gradient density centrifugation. Notchl siRNAs were designed, synthesized, and transiently transfected into these MSCs, and then treated with TGF-β1.Ten New Zealand rabbits were invited in this study.The rabbits'discs in L3/4, L4/5and L5/6were stabbed by a needle. Two weeks after the operation, the discs were examined with Magnetic Resonance Images(MRI). And the L3/4discs were treated by pYr1.1transfected MSCs treated with TGF-β1, L4/5discs treated by shRNA-Notch-1transfected MSCs treated with TGF-β1, L5/6discs were treated by the MSCs treated with TGF-β1only. Four weeks after the treatment, the discs were examined with MRI. The expression of proteoglycan in nucleus pulposus was evaluated by using toluidine blue, and the expressions of collagen II and proteoglycan were examined with RT-PCR and Western-blot.
Results Four weeks after the treatment, the signal intensity of T2weightedimages of the L4/5discs was higher than the others, with a significant difference found between them. The toluidine blue staining showed that the expression of proteoglycan in the L4/5discs was higher than that in the others. RT-PCR and Western-blot showed that the expression of collagen Ⅱ and proteoglycan mRNA and protein in the L4/5discs were significant higher than that in the L3/4and L5/6disc.
Conclusion Transplantation of shRNA-Notch-1transfected MSCs treated with TGF-β1restrain the degeneration of punctured discs.
方法(1)4-6周龄新西兰兔麻醉后,取股骨骨髓,以密度梯度离心法分离培养MSC；(2)根据siRNA原理针对Notch-1设计短发卡状RNA (shRNA),瞬时转染入MSC,转化生长因子.-β1(TGF-β1)诱导转染MSC分化；(3)实验分组为TGF-β1诱导无转染组MSC、TGF-β1诱导转染无意siRNA空质粒组MSC、TGF-β1诱导转染siRNA-Notch-1质粒组MSC三组,甲苯胺蓝染色及Ⅱ型胶原染色检测MSC的细胞表型的改变情况；(4) RT-PCR及Western blot法检测三组细胞中Ⅱ型胶原(COL2)及蛋白聚糖(ACAN)基因表达水平。
结果(1)光镜下观察MSC细胞形态主要为梭形或椭圆形,呈巢状或漩涡状生长；(2)转染后MSC中Notch-1基因沉默效率为47%(P<0.001),即所选位点具有明显的沉默效果。(3)甲苯胺蓝染色TGF-β1诱导转染siRNA-Notch-1质粒组MSC染色阳性表达明显高于其余两组。(4) RT-PCR检测发现TGF-β1诱导转染siRNA-Notch-1质粒组MSC的COL2表达为TGF-β1诱导无转染组MSC的2.55倍(P<0.001),为TGF-β1诱导转染无意siRNA空质粒组MSC的2.53倍(P<0.001)。ACAN表达分别为1.51倍(P<0.01)和1.53倍(P<0.01)。Western blot检测发现TGF-β1诱导转染siRNA-Notch-1质粒组MSC的COL2表达为TGF-β1诱导无转染组MSC的1.25倍(P<0.05),为TGF-β1诱导转染无意siRNA空质粒组MSC的1.37倍(P<0.05)。ACAN表达分别为1.99倍(P<0.05)和1.46倍(P<0.05)。
结论(1)使用密度梯度离心法可以分离出纯度较高的MSC。(2)三组细胞经TGF-β1诱导均可产生COL2及ACAN,表现出类软骨细胞表型；TGF-β1诱导转染siRNA-Notch-1质粒MSC表型基因表达较转染空质粒MSC及未转染MSC显著提高。
目的使用纤维环穿刺抽吸法造模兔椎间盘退行性变,移植转化生长因子p1(TGF-β1)诱导Notch-1基因沉默兔骨髓间充质干细胞(MSCs)到退变的椎间盘内,观测退变椎间盘的变化。
方法(1)4只体重0.4-0.5kg新西兰兔麻醉后,取股骨骨髓,以密度梯度离心法分离培养MSCs;(2)将针对Notch-1的短发卡状RNA (shRNA)及无意空质粒shRNA,瞬时转染入MSCs, TGF-β1诱导转染MSCs分化：(3)体重1.0-1.5kg新西兰兔10只,手术对兔脊柱L3-4、L4-5、L5-6三个椎间盘行穿刺抽吸髓核组织造模,2周后行核磁共振成像(MRI)检查观测造模情况；将细胞分为TGF-β1诱导转染空质粒组、TGF-β1诱导转染shRNA-Notch-1质粒组、TGF-β1诱导无转染组三组细胞,造模2周后分别移植入L3-4、L4-5、L5-6三个椎间盘,4周后行MRI检查观测细胞治疗情况；(4)动物MRI检查后处死行髓核组织的甲苯胺蓝染色观测蛋白多糖表达情况,RT-PCR及Western-blot检测退变椎间盘组织Ⅱ型胶原(COL2)及蛋白多糖(ACAN)改变情况。
结果(1)细胞移植4周后MRI检测发现,L4-5椎间盘%T2加权像扫描(%ST2WI)值增加最为明显,与其他2组有明显差异；L3-4、L5-6椎间盘%ST2WI值增加,两组间无统计学差异。(2)甲苯胺蓝染色：与空白对照组及阴性对照组相比较,实验组椎间盘组织内蛋白多糖的表达显著提高。(3) RT-PCR检测发现实验组椎间盘内Ⅱ型胶原及蛋白多糖的表达明显高于其余两组,存在显著性差异,阴性对照组与空白对照组间无显著性差异(4) Western blot检测发现现实验组椎间盘内Ⅱ型胶原及蛋名糖的表达明显高于其余两组,存在显著性差异,阴性对照组与空白对照组间无显著性差异。结论兔退变椎间盘内移植Notch-1基因敲除兔骨髓间充质干细胞,可有效的修复退变椎间盘组织。
Objective To observe the effects of Notchl gene on the directional differentiation of rabbit marrow mesenchymal stem cells (MSCs) into a nucleus pulposus-like phenotype through silenced Northl gene expression by small interfering RNA (siRNA) and induced by TGF-β1.
Methods MSCs were obtained from New Zealand rabbits (4-6week-old) femur bone marrow and purified by discontinuous gradient density centrifugation. The short hairpin RNA (shRNA) aimed at Notchl gene was designed according to the siRNA principle, and transient transfect into MSCs. Cells were divided into three groups:induced by TGF-β1only (group1); induced by TGF-β1and transfect by nonsense siRNA (group2); induced by TGF-β1and transfect by shRNA-Notchl (group3). The expressions of proteoglycan were detected by toluidine blue staining. The expressions of collagen Ⅱ and proteoglycan were examined in gene and protein level with RT-PCR and Western blot.
Results The MSCs were ellipse or fusiform shape and gathered in nest or swirl. Notchl expression was down-regulated by shRNA-Notchl, and the silencing rate was47%(P<0.001). The positive expressions in shRNA-Notchl group were higher than the others by toluidine blue staining. RT-PCR showed that the expression of collagen Ⅱ in group1was2.55times than group1(P<0.001),2.53times than group2(P<0.001), and the expression of proteoglycan in group3was1.51times than group1(P<0.01),1.53times than group2(P<0.01). Western-blot showed that the expression of collagen Ⅱ in group1was1.25times than group1(P<0.05),1.37times than group2(P<0.05), and the expression of proteoglycan in group3was1.99times than group1(P<0.05),1.46times than group2(P<0.05).
Conclusions This study demonstrates that MSCs could be purified by discontinuous gradient density centrifugation. TGF-β1can induce the MSCs to excrete collagen Ⅱand proteoglycan, and the down-regulation of Notchl gene could increase these excretion.
Objective To investigated the effects of Notchl knockdown and treatment with TGF-β1on the regulation of the directional differentiation of mesenchymal stem cells (MSCs) in suppressing the degeneration of punctured intervertebral discs in rabbits.
Methods MSCs were isolated from the femur bone of New Zealand rabbits and purified with discontinuous gradient density centrifugation. Notchl siRNAs were designed, synthesized, and transiently transfected into these MSCs, and then treated with TGF-β1.Ten New Zealand rabbits were invited in this study.The rabbits'discs in L3/4, L4/5and L5/6were stabbed by a needle. Two weeks after the operation, the discs were examined with Magnetic Resonance Images(MRI). And the L3/4discs were treated by pYr1.1transfected MSCs treated with TGF-β1, L4/5discs treated by shRNA-Notch-1transfected MSCs treated with TGF-β1, L5/6discs were treated by the MSCs treated with TGF-β1only. Four weeks after the treatment, the discs were examined with MRI. The expression of proteoglycan in nucleus pulposus was evaluated by using toluidine blue, and the expressions of collagen II and proteoglycan were examined with RT-PCR and Western-blot.
Results Four weeks after the treatment, the signal intensity of T2weightedimages of the L4/5discs was higher than the others, with a significant difference found between them. The toluidine blue staining showed that the expression of proteoglycan in the L4/5discs was higher than that in the others. RT-PCR and Western-blot showed that the expression of collagen Ⅱ and proteoglycan mRNA and protein in the L4/5discs were significant higher than that in the L3/4and L5/6disc.
Conclusion Transplantation of shRNA-Notch-1transfected MSCs treated with TGF-β1restrain the degeneration of punctured discs.
引文
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12. Wuertz K,Godburn K and Neidl inger.Behavior of mesenchymal stem cells in the chemical microenvironment of the intervertebral disc. Spine,2008,33(17):1843-1849.
13. Moore KA, Lemischka IR. Stem cells and their niches.Science,2006,311:1880-1885.
14. Wuertz,K Godburn,K and Neidlinger-Wilke C. Behavior of Mesenchymal Stem Cells in the Chemical Microenvironment of the Intervertebral Disc. Spine (Phila Pa 1976),2008,33(17):1843-1849.
15. Sulkowski S, Wincewicz A, Sulkowska M and Koda M. Transforming growth factor-betaland regulators of apoptosis. Ann N Y Acad Sci 2009,1171:116-123.
16. Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M and Fujinaga T. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels:influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol Bioeng 2006,93(6):1152-1163.
17. Steck E, Bertram H, Abel R, Chen B, Winter A and Richter W. Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells 2005,23(3):403-411.
1. MW Lively.Sports medicine approach to low back pain.South Med,2002,95,642-646
2. Adams MA, Roughley PJ. What is intervertebral disc degeneration, and what causes it? Spine,2006,31:2151-2161
3. A Maetzel,L Li.The economic burden of low back pain:A review of studies published between 1996 and 2001. Best Pract Res Clin Rheumatol,2002,16,23-30
4. Haefeli M,Kalberer F,Saegesser D,et al.The course of macroscopic degeneration in the human lumbar intervertebral disc.Spine,2006,31,1522-1531
5. Adams MA,Freeman BJ,Morrison HP,et al.Mechanical initiation of intervertebral disc degeneration.Spine,2000,25,1625-1636
6. Bibby SR,Urban JP.Effect of nutrient deprivation on the viability of intervertebral disc cells.Eur Spine J,2004,13,695-701
7. Freemont AJ.The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain.Rheumatology (Oxford),2009,48,5-10
8. Paesold QNerlich AQBoos N.Biological treatment strategies for disc degeneration: potentials and shortcomings.Eur Spine J,2007,16,447-468
9. Leung VY,Tam V,Chan D,et al.Tissue engineering for intervertebral disk degeneration.Orthop Clin North Am,2011,Oct,42(4),575-583
10. Masuda K,Lotz JC.New challenges for intervertebral disc treatment using regenerative medicine.Tissue Eng Part B Rev,2010,Feb,16(1),147-158
11. Ehlicke F,Freimark D,Heil B,et al.Intervertebral disc regeneration:influence of growth factors on differentiation of human mesenchymal stem cells (hMSC).Artif Organs,2010, 33(4),244-252
12. Nerurkar NL,Elliott DM,Mauck RL.Mechanical design criteria for intervertebral disc tissue engineering.J Biomech,2010,Apr,19,43(6),1017-1030
13. Richardson SM,Hoyland JA,Mobasheri R,et al.Mesenchymal stem cells in regenerative medicine:opportunities and challenges for articular cartilage and intervertebral disc tissue engineering.J Cell Physiol,2010,222(1),23-32
14. Yang H,Wu J,Ebraheim M,et al.Transplanted mesenchymal stem cells with pure fibrinous gelatin-transforming growth factor-betal decrease rabbit intervertebral disc degeneration. Spine,2010,10(9),802-810
15. Park SH,Gil ES,Cho H,et al.Intervertebral disk tissue engineering using biphasic silk composite scaffolds.Tissue Eng Part A,2012 Mar,18(5-6),447-458
16. O'Halloran DM,Pandit AS.Tissue-engineering approach to regenerating the intervertebral disc.Tissue Eng,2007,13,1927-1954
17. Xu HH,Simon CG Jr.Self-hardening calcium phosphate composite scaffold for bone tissue engineering. Orthop Res,2004,22(3),535-543
18. Hutmacher DW.Scaffolds in Tissue Engineering Bone and Cartilage.Biomaterials,20-00,21,2529-2543
19. Halloran DO,Grad S,Stoddart M,et al.An injectable cross-linked scaffold for nucleus pulposus regeneration.Biomaterials.2008,29(4),438-447
20. Lu ZF,Zandieh Doulabi B,Wuisman PI.Differentiation of adipose stem cells by nucleus pulposus cells:configuration effect. Biochem Biophys Res Commun,2007,359(4),9-91-996
21. Yang S,Leong KF,Du Z,et al.The design of scaffolds for use in tissue engineering (Part II):rapid prototyping techniques.Tissue Eng,2002,8(l),1-11
22. BP Chan,KW Leong. Scaffolding in tissue engineering:general approaches and tissue-specific considerations.Eur Spine J,2008,December,17(Suppl 4),467-479
23. Melrose J,Ghosh P,Taylor TK.A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican) and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral disc.J Anat,2001,198,3-15.
24. Adam M,Deyl Z.Degeneratedannulus fibrosus of the intervertebral disc contains collagen type II. Ann Rheum Dis,1984,43,258-263
25. Watt SL,Lunstrum GP,McDonough AM,et al.Characterization of collagen types XII and XIV from fetal bovine cartilage.J Biol Chem,1992,Oct 5,267(28),20093-20099
26. Alini M,Li W,Markovic P,et al.The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine,20-03,Mar, 1,28(5),446-454
27. Huang B,Li CQ,Zhou Y,et al.Collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering.Biomed Mater Res B Appl Biomater,2010, Feb,92(2),322-331
28. Sakai D,Mochida J,Iwashina T,et al.Atelocollagen for culture of human nucleus pulposus cells forming nucleus pulposus-like tissue in vitro:influence on the proliferation and proteoglycan production of HNPSV-1 cells.Biomaterials,2006,27-(3),346-353
29. 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 chondroitinase ABC-induced in vitro chemonucleolysis.Spine,2005,5(3),231-238
30. Iwashina T,Mochida J,Miyazaki T,et al.Low-intensity pulsed ultrasound stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells cultured in alginate.Biomaterials,2006,27(3),354-361
31. Chou AI,Nicoll SB.Characterization of photocrosslinked alginate hydrogels for nucleus pulposus cell encapsulation.Biomed Mater Res A,2009,91,187-194
32. Reza AT,Nicoll SB.Characterization of novel photocrosslinked carboxymethylcellulose hydrogels for encapsulation of nucleus pulposus cells. Acta Biomater.2010,Jan,6(1),179-186
33. KS Kim,JH Lee,HH Ahn,et al.The osteogenic differentiation of rat muscle-derived stem cells in vivo within in situ-forming chitosan scaffolds.Biomaterials.2008,29,4420-4428
34. Anderson DQRisbud MV,Shapiro IM,et al.Cell-based therapy for disc repair.Spine,2005,5,297-303
35. Roughley P,Hoemann C,DesRosiers E,et al.The potential of chitosan-based gels containing intervertebral disc cells for nucleus pulposus supplementation. Biomaterials,2006,27,388-396
36. Richardson SM,Hughes N,Hunt JA,et al.Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels.Biomaterials,2008,29,85-93
37. Mizuno H,Roy AK,Vacanti CA,et al.Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.Spine,2004,29(12),1290-1297
38. A Abbushi.Regeneration of intervertebral disc tissue by resorbable cell-free polyglycolic acid-based implants in a rabbit model of disc degeneration. Spine,2008,Jun 15,33(14),1527-1532
39. Razaq S,Wilkins RJ,Urban JP.The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus.Eur Spine J,2003,12(4),341-349
40. Le Maitre CL,Freemont AJ,Hoyland JA.Accelerated cellular senescence in degenerate intervertebral discs:a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther,2007,9(3),R45.
41. Gaetani P,Torre ML,Klinger M.Adipose-derived stem cell therapy for intervertebral disc regeneration:an in vitro reconstructed tissue in alginate capsules.Tissue Eng Part A,2008,14(8),1415-1423.
42. O'Halloran DM,Pandit AS.Tissue-engineering approach to regenerating the intervertebral disc.Tissue Eng,2007,13,1927-1954
43. Battie MC,Videman T,Parent E.Lumbar disc degeneration:epidemiology and genetic influences. Spine,2004,29,2679-2690
44. Sambrook PN,MacGregor AJ,Spector TD.Genetic influences on cervical and lumbar disc degeneration:a magnetic resonance imaging study in twins. Arthritis Rheum,1999,42,366-372
2. Ruan D,He Q and Ding Y. Intervertebral disc transplantation in the treatment of degenerative spine disease:a preliminary study. Lancet 2007,369:993-999.
3. 肖剑,贾连顺,程岚.兔腰椎间盘髓核细胞活力测定及外源性基因在其中的表达.中国临床康复,2002,6(20)：3023-3024.
4. Erwin WM, Ashman K and O'Donnel P. Nucleu pulposus notochord cells secrete connective tissue growth factor and up-regulate proteoglycan expression by intervertebral disc chondrocytes. Arthritis Rheum 2006,54(12):3859-3867.
5.胡资兵,曾荣,郭伟韬,林颢.骨髓间充质干细胞诱导分化特征.中国组织工程研究与临床康复,2008,43：8561-8566.
6. Baksh D, Song L and Tuan RS. Adult mesenchymal stem cells:characterization, differentiation, and application in cell and gene therapy. Cell Mol Med 2004, 8:301-316.
7. Indrawattana N, Chen G and Tadokoro M.Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun 2004,320(3):914-919.
8. Schwanbeck R, Schroeder T, Henning K, Kohlhof H, Rieber N, Erfurth ML and Just U. Notch signaling in embryonic and adult myelopoiesis. Cells Tissues Organs 2008,188(1-2):91-102.
9. Hayes AJ, Dowthwaite GP, Webster SV and Archer CW. The distribution of Notch receptors and their ligands during articular cartilage development. J Anat 2003,202(6):495-502.
10. Andersson ER, Sandberg R and Lendahl U. Notch signaling:simplicity in design, versatility in function. Development 2011,138(17):3593-3612.
11. Wu L, Sun T, Kobayashi K, Gao P and Griffin JD. Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors. Mol Cell Biol 2002,22(21):7688-7700.
12. Iso T, Kedes L and Hamamori Y. HES and HERP families:multiple effectors of the Notch signaling pathway. J Cell Physiol 2003,194(3):237-255.
13. Hiyama A, Skubutyte R, Markova D, Anderson DG, Yadla S, Sakai D, Mochida J, Albert TJ, Shapiro IM and Risbud MV. Hypoxia activates the notch signaling pathway in cells of the intervertebral disc:implications in degenerative disc disease. Arthritis Rheum 2011,63(5):1355-1364.
14. Colombini A, Lombardi G and Corsi MM. Pathophysiology of the human intervertebral disc. Biochem Cell Biol 2008,40 (5):837-842.
15. Mochida J. New strategies for disc repair:novel preclinical trials. J Orthop Sci 2005,10(1):112-118.
16. Strassburg S, Richardson SM, Freemont AJ and Hoyland JA. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med 2010,5(5):701-711.
17. Gogate SS, Fujita N, Skubutyte R, Shapiro IM and Risbud MV. TonEBP and HIF coordinate Hsp70 expression in hypoxic nucleus pulposus cells:Role of Hsp70 in HIF-1α degradation. J Bone Miner Res 2012,27(5):1106-1117.
18. Risbud MV, Albert TJ, Guttapalli A, Vresilovic EJ, Hillibrand AS, Vaccaro AR and Shapiro IM. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro:implications for cell-based transplantation therapy. Spine 2004,29(23):2627-2632.
19. Bertolo A, Mehr M, Aebli N, Baur M, Ferguson SJ and Stoyanov JV. Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells. Eur Spine J 2011,8. [Epub ahead of print]
20. Acosta FL Jr, Metz L, Adkisson HD, Liu J, Carruthers-Liebenberg E, Milliman C, Maloney M and Lotz JC. Porcine intervertebral disc repair using allogeneic juvenile articular chondrocytes or mesenchymal stem cells. Tissue Eng Part A 2011,17(23-24):3045-3055.
21. Sulkowski S, Wincewicz A, Sulkowska M and Koda M. Transforming growth factor-betal and regulators of apoptosis. Ann N Y Acad Sci 2009,1171:116-123.
22. Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M and Fujinaga T. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels:influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol Bioeng 2006,93(6):1152-1163.
23. Steck E, Bertram H, Abel R, Chen B, Winter A and Richter W. Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells 2005,23(3):403-411.
1. Colombini A, Lombardi G and Corsi MM. Pathophysiology of the human intervertebral disc. Biochem Cell Biol 2008,40 (5):837-842.
2. Mochida J. New strategies for disc repair:novel preclinical trials. J Orthop Sci 2005,10(1):112-118.
3. Strassburg S, Richardson SM, Freemont AJ and Hoyland JA. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med 2010,5(5):701-711.
4. Gogate SS, Fujita N, Skubutyte R, Shapiro IM and Risbud MV. TonEBP and HIF coordinate Hsp70 expression in hypoxic nucleus pulposus cells:Role of Hsp70 in HIF-1α degradation. J Bone Miner Res 2012,27(5):1106-1117.
5. Erwin WM, Inman RD. Notochord cells regulate intervertebral disc chondrocyte proteoglycan production and cell proliferation. Spine (Phila Pa 1976). 2006,31(10):1094-1099.
6. Risbud MV, Albert TJ, Guttapalli A, Vresilovic EJ, Hillibrand AS, Vaccaro AR and Shapiro IM. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro:implications for cell-based transplantation therapy. Spine 2004,29(23):2627-2632.
7. Bertolo A, Mehr M, Aebli N, Baur M, Ferguson SJ and Stoyanov JV. Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells. Eur Spine J 2011,8. [Epub ahead of print]
8. Richardson SM,Curran JM and Chen R.The differentiation of bone marrow mesenchymal stem cells into chondrocyte like cells on poly-L-lactic acid (PLLA) scaffolds. Biomaterials.2006,27(22):4069-4078.
9. Sakai D,Mochidal J and Yamamoto Y.Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc:a potential therapeutic model for disc degeneration. Biomaterials,2003,24(20):3531-3541.
10. Acosta FL Jr, Metz L, Adkisson HD, Liu J, Carruthers-Liebenberg E, Milliman C, Maloney M and Lotz JC. Porcine intervertebral disc repair using allogeneic juvenile articular chondrocytes or mesenchymal stem cells. Tissue Eng Part A 2011,17(23-24):3045-3055.
11.王佳,陈晓禾,黄永灿等.缺氧对hBMSCs和胎盘来源MSCs增殖的影响.中国修复重建外科杂志,2009,23(2)：136-139.
12. Wuertz K,Godburn K and Neidl inger.Behavior of mesenchymal stem cells in the chemical microenvironment of the intervertebral disc. Spine,2008,33(17):1843-1849.
13. Moore KA, Lemischka IR. Stem cells and their niches.Science,2006,311:1880-1885.
14. Wuertz,K Godburn,K and Neidlinger-Wilke C. Behavior of Mesenchymal Stem Cells in the Chemical Microenvironment of the Intervertebral Disc. Spine (Phila Pa 1976),2008,33(17):1843-1849.
15. Sulkowski S, Wincewicz A, Sulkowska M and Koda M. Transforming growth factor-betaland regulators of apoptosis. Ann N Y Acad Sci 2009,1171:116-123.
16. Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M and Fujinaga T. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels:influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol Bioeng 2006,93(6):1152-1163.
17. Steck E, Bertram H, Abel R, Chen B, Winter A and Richter W. Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells 2005,23(3):403-411.
1. MW Lively.Sports medicine approach to low back pain.South Med,2002,95,642-646
2. Adams MA, Roughley PJ. What is intervertebral disc degeneration, and what causes it? Spine,2006,31:2151-2161
3. A Maetzel,L Li.The economic burden of low back pain:A review of studies published between 1996 and 2001. Best Pract Res Clin Rheumatol,2002,16,23-30
4. Haefeli M,Kalberer F,Saegesser D,et al.The course of macroscopic degeneration in the human lumbar intervertebral disc.Spine,2006,31,1522-1531
5. Adams MA,Freeman BJ,Morrison HP,et al.Mechanical initiation of intervertebral disc degeneration.Spine,2000,25,1625-1636
6. Bibby SR,Urban JP.Effect of nutrient deprivation on the viability of intervertebral disc cells.Eur Spine J,2004,13,695-701
7. Freemont AJ.The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain.Rheumatology (Oxford),2009,48,5-10
8. Paesold QNerlich AQBoos N.Biological treatment strategies for disc degeneration: potentials and shortcomings.Eur Spine J,2007,16,447-468
9. Leung VY,Tam V,Chan D,et al.Tissue engineering for intervertebral disk degeneration.Orthop Clin North Am,2011,Oct,42(4),575-583
10. Masuda K,Lotz JC.New challenges for intervertebral disc treatment using regenerative medicine.Tissue Eng Part B Rev,2010,Feb,16(1),147-158
11. Ehlicke F,Freimark D,Heil B,et al.Intervertebral disc regeneration:influence of growth factors on differentiation of human mesenchymal stem cells (hMSC).Artif Organs,2010, 33(4),244-252
12. Nerurkar NL,Elliott DM,Mauck RL.Mechanical design criteria for intervertebral disc tissue engineering.J Biomech,2010,Apr,19,43(6),1017-1030
13. Richardson SM,Hoyland JA,Mobasheri R,et al.Mesenchymal stem cells in regenerative medicine:opportunities and challenges for articular cartilage and intervertebral disc tissue engineering.J Cell Physiol,2010,222(1),23-32
14. Yang H,Wu J,Ebraheim M,et al.Transplanted mesenchymal stem cells with pure fibrinous gelatin-transforming growth factor-betal decrease rabbit intervertebral disc degeneration. Spine,2010,10(9),802-810
15. Park SH,Gil ES,Cho H,et al.Intervertebral disk tissue engineering using biphasic silk composite scaffolds.Tissue Eng Part A,2012 Mar,18(5-6),447-458
16. O'Halloran DM,Pandit AS.Tissue-engineering approach to regenerating the intervertebral disc.Tissue Eng,2007,13,1927-1954
17. Xu HH,Simon CG Jr.Self-hardening calcium phosphate composite scaffold for bone tissue engineering. Orthop Res,2004,22(3),535-543
18. Hutmacher DW.Scaffolds in Tissue Engineering Bone and Cartilage.Biomaterials,20-00,21,2529-2543
19. Halloran DO,Grad S,Stoddart M,et al.An injectable cross-linked scaffold for nucleus pulposus regeneration.Biomaterials.2008,29(4),438-447
20. Lu ZF,Zandieh Doulabi B,Wuisman PI.Differentiation of adipose stem cells by nucleus pulposus cells:configuration effect. Biochem Biophys Res Commun,2007,359(4),9-91-996
21. Yang S,Leong KF,Du Z,et al.The design of scaffolds for use in tissue engineering (Part II):rapid prototyping techniques.Tissue Eng,2002,8(l),1-11
22. BP Chan,KW Leong. Scaffolding in tissue engineering:general approaches and tissue-specific considerations.Eur Spine J,2008,December,17(Suppl 4),467-479
23. Melrose J,Ghosh P,Taylor TK.A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican) and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral disc.J Anat,2001,198,3-15.
24. Adam M,Deyl Z.Degeneratedannulus fibrosus of the intervertebral disc contains collagen type II. Ann Rheum Dis,1984,43,258-263
25. Watt SL,Lunstrum GP,McDonough AM,et al.Characterization of collagen types XII and XIV from fetal bovine cartilage.J Biol Chem,1992,Oct 5,267(28),20093-20099
26. Alini M,Li W,Markovic P,et al.The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine,20-03,Mar, 1,28(5),446-454
27. Huang B,Li CQ,Zhou Y,et al.Collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering.Biomed Mater Res B Appl Biomater,2010, Feb,92(2),322-331
28. Sakai D,Mochida J,Iwashina T,et al.Atelocollagen for culture of human nucleus pulposus cells forming nucleus pulposus-like tissue in vitro:influence on the proliferation and proteoglycan production of HNPSV-1 cells.Biomaterials,2006,27-(3),346-353
29. 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 chondroitinase ABC-induced in vitro chemonucleolysis.Spine,2005,5(3),231-238
30. Iwashina T,Mochida J,Miyazaki T,et al.Low-intensity pulsed ultrasound stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells cultured in alginate.Biomaterials,2006,27(3),354-361
31. Chou AI,Nicoll SB.Characterization of photocrosslinked alginate hydrogels for nucleus pulposus cell encapsulation.Biomed Mater Res A,2009,91,187-194
32. Reza AT,Nicoll SB.Characterization of novel photocrosslinked carboxymethylcellulose hydrogels for encapsulation of nucleus pulposus cells. Acta Biomater.2010,Jan,6(1),179-186
33. KS Kim,JH Lee,HH Ahn,et al.The osteogenic differentiation of rat muscle-derived stem cells in vivo within in situ-forming chitosan scaffolds.Biomaterials.2008,29,4420-4428
34. Anderson DQRisbud MV,Shapiro IM,et al.Cell-based therapy for disc repair.Spine,2005,5,297-303
35. Roughley P,Hoemann C,DesRosiers E,et al.The potential of chitosan-based gels containing intervertebral disc cells for nucleus pulposus supplementation. Biomaterials,2006,27,388-396
36. Richardson SM,Hughes N,Hunt JA,et al.Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels.Biomaterials,2008,29,85-93
37. Mizuno H,Roy AK,Vacanti CA,et al.Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.Spine,2004,29(12),1290-1297
38. A Abbushi.Regeneration of intervertebral disc tissue by resorbable cell-free polyglycolic acid-based implants in a rabbit model of disc degeneration. Spine,2008,Jun 15,33(14),1527-1532
39. Razaq S,Wilkins RJ,Urban JP.The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus.Eur Spine J,2003,12(4),341-349
40. Le Maitre CL,Freemont AJ,Hoyland JA.Accelerated cellular senescence in degenerate intervertebral discs:a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther,2007,9(3),R45.
41. Gaetani P,Torre ML,Klinger M.Adipose-derived stem cell therapy for intervertebral disc regeneration:an in vitro reconstructed tissue in alginate capsules.Tissue Eng Part A,2008,14(8),1415-1423.
42. O'Halloran DM,Pandit AS.Tissue-engineering approach to regenerating the intervertebral disc.Tissue Eng,2007,13,1927-1954
43. Battie MC,Videman T,Parent E.Lumbar disc degeneration:epidemiology and genetic influences. Spine,2004,29,2679-2690
44. Sambrook PN,MacGregor AJ,Spector TD.Genetic influences on cervical and lumbar disc degeneration:a magnetic resonance imaging study in twins. Arthritis Rheum,1999,42,366-372