摘要
椎间盘退变是引起成人腰腿痛、颈肩痛的主要原因。随着人均寿命的延长和社会工作压力增大,椎间盘退变性疾病的发病率明显升高,而目前的保守治疗和外科手术治疗均不能从根本上,解决患者的病情。通过动物实验及临床初步应用证明,椎间盘移植后可以异体存活,并能完全缓解患者的临床症状,就其生物力学方面亦可满足生理活动需要,但植入的异体椎间盘在生化代谢及组织学上有明显的退行性改变,这将导致临床症状的再次出现,并可能出现椎间盘活动度消失等并发症。最近的研究结果显示椎间盘内细胞活性的提高能延缓甚至阻止椎间盘退变性改变。因此本实验拟通过以异体椎间盘为支架材料,以表达外源性人BMP7的髓核细胞为种子细胞,体外构建组织工程椎间盘,并观察组织工程椎间盘的生物活性及功能情况。
1.深低温储存温度及时间对异体椎间盘生物活性的影响
目的:探讨不同的深低温储存温度及储存时间对异体椎间盘生物活性影响,获得最适合临床应用的保存条件。
方法:取犬椎间盘52只,分为对照组、液氮保存组和-80℃保存组。在冷冻保存液中分别储存于液氮及-80℃低温冰箱中,在储存的2w、1m、2m、4m、6m及12m时,通过EB/FAD法检测椎间盘组织中不同部位的细胞存活率,以DMMB法检测髓核组织蛋白多糖(PG)含量,以羟脯氨酸法检测髓核组织中的总胶原含量。
结果:各检测时间点两组间外层纤维环细胞活性明显高于内层纤维环及髓核组织(p<0.05),内层纤维环及髓核组织间细胞活性未见明显不同;在内层纤维环及髓核组织中,细胞存活率在储存2w时,可见液氮组明显高于-80℃组(p<0.05),自储存1月起至12个月,两组间均无明显差别(p>0.05)。在储存期间两储存组蛋白多糖含量均逐渐降低,在1m至12m储存期间,液氮保存组蛋白多糖含量均高于-80℃保存组。而两组间总胶原蛋白含量在储存期间随时间逐渐降低,但两组间未见明显差异。
结论:利用液氮保存椎间盘组织,无论在细胞总的存活率及PG含量上均优于-80℃,1年的液氮保存期间,细胞存活率及功能蛋白PG含量均在一个恒定的水平,能满足异体椎间盘移植的基本要求,可作为组织工程椎间盘的支架材料。
2.重组腺相关病毒人BMP7(rAAV2-hBMP7)病毒载体的构建及鉴定
目的:探讨能否利用pSNAV2.0质粒构建系统构建pSNAV2.0-hBMP7穿梭质粒,并包装纯化成可供实验用rAAV-hBMP7病毒载体。
方法:利用AgeI和NheI酶切pDC316-hBMP7-IRES-EGFP质粒,回收纯化hBMP7基因片段,以AgeI和NheI酶切载体质粒pSNAV2.0-LacZa,回收纯化pSNAV2.0载体质粒片段,利用T4 DNA连接酶将纯化回收的hBMP7基因与pSNAV2.0载体质粒片段连接构建,并转化入感受态大肠杆菌DH-5α进行扩增纯化,测序鉴定;用Lipofectamine 2000将序列正确的pSNAV2.0- hBMP7质粒转染至BHK-21细胞,G418选择培养,并大量扩增至所需并用HSV1-rc/ΔUL2辅助病毒感染,以PEG8000/NaCl沉淀,以氯仿纯化浓缩、检测滴度及纯度。
结果:成功构建了pSNAV2.0- hBMP7质粒,经PCR、酶切和测序鉴定构建正确。利用Lipofectamine 2000将序列正确的pSNAV2.0- hBMP7质粒转染至BHK-21细胞,并通过含有G418的培养基,选择性扩增,并浓缩纯化出了滴度达5.1×1011 v.g、纯度>97%的rAAV-hBMP7病毒载体。
结论:利用pSNAV2.0质粒包转系统能成功将外源性基因hBMP7包装成滴度及纯度满足实验要求的rAAV-hBMP7病毒载体。
3.介导人骨形成蛋白-7的2型腺相关病毒转染髓核细胞及其对髓核细胞表型的影响
目的:以介导外源性人骨形成蛋白-7(hBMP7)基因的2型腺相关病毒(recombinant adeno-associated virus type-2,rAAV2)载体转染犬髓核细胞(canine nucleus pulposus cell, cNP cell),观察转染后髓核细胞hBMP7蛋白表达,并分析髓核细胞生物功能变化。
方法:实验组以最佳感染复数(MOI)1×105 vg/cell转染犬髓核细胞,对照组以相同MOI的不含hBMP7基因的rAAV2-EGFP病毒感染。在转染后4d、7d和14d分别以RT-PCR、Wenstenbloting方法分别对两组髓核细胞中hBMP7的mRNA转录及蛋白表达进行检测。在转染后的第7天,检测两组细胞增殖能力。在转染后的4、7和14d,利用real-time PCR、DMMB及Elisa法分别检测蛋白多糖、Ⅰ和Ⅱ胶原的mRNA含量、蛋白多糖含量及Ⅰ和Ⅱ型胶原蛋白含量。
结果:在以1×105 vg/cell转染犬髓核细胞后,在4d、7d和14d时均可检测到实验组髓核细胞中高表达hBMP7 mRNA,而对照组,无论在何时间点均不能检测到hBMP7 mRNA的转录,半定量分析显示,7d时mRNA含量较高。Westenbloting蛋白检测结果显示:7d、14d时,实验组均可检测到分子量为55kd特异性hBMP7蛋白的表达,而对照组均未观察到阳性蛋白条带。半定量分析显示:转染后7d时hBMP7蛋白含量相对较高。在转染7天后,转染组与未转染组细胞增殖能力未见明显区别。对实验组髓核细胞的Real-time PCR定量分析发现:蛋白多糖、Ⅱ型胶原mRNA在转染后第4天无明显增加,但在第7天和14天均明显增高。而Ⅰ型胶原mRNA在4、7和14天均未见明显变化。实验组蛋白多糖含量第4天未见明显变化,而第7、14天较对照组分别增高42%及77%。而Ⅰ胶原含量两组间未见明显增加,实验组Ⅱ型胶原含量在第4天未见明显增加,而第7、14天分别较对照组增高63%及94%。
结论: rAAV2-hBMP7病毒载体能有效地转染犬髓核细胞并长期稳定表达人骨形成蛋白-7,并能提高犬髓核细胞蛋白多糖及Ⅱ型胶原含量,这将能为组织工程椎间盘提供可选择的基因修饰的种子细胞。
4.组织工程椎间盘的体外构建
目的:探讨利用液氮储存异体椎间盘及表达外源性人BMP7的髓核细胞体外构建组织工程椎间盘的方法
方法:取储存于液氮中2个月的犬椎间盘24只,分为EGFP对照组、1×104、1×105和1×106四组,每组6只。对照组用16G注射器自椎间盘后正中注入1×105的表达EGFP的髓核细胞20ul, 1×104组注入PKH-26标记的表达hBMP7的髓核细胞20ul,其总细胞数为1×104,1×105组为总细胞数1×105的PKH-26标记的表达hBMP7的髓核细胞20ul,1×106组为细胞总数1×106的PKH-26标记的表达hBMP7的髓核细胞20ul。注射后立即置入50ml离心管中,加入30ml完全培养基,分别于培养4、7、14天,从椎间盘形态、细胞存活、及髓核细胞荧光强度、蛋白多糖及胶原含量等方面进行评价。
结果:在储存的第4天及7天,椎间盘外形基本未见明显改变,而在储存至14天时,可见椎间盘上下终板有钙质脱落。在各时间点均可见表达绿色荧光蛋白髓核细胞。对不同组、不同时间点的组织工程椎间盘中荧光强度定量后发现:培养第7和14天时,1×105组荧光强度明显高于1×106组和1×104组。而且1×105组培养第7和14天时蛋白多糖及总胶原含量均较另外三组明显增高。
结论:利用液氮储存异体椎间盘同1×105转染髓核细胞复合,体外培养7天能构建出较理想的组织工程椎间盘。
小结:
1.利用液氮保存椎间盘组织,无论在细胞总的存活率及PG含量上均优于-80℃,1年的液氮保存期间,细胞存活率及功能蛋白PG含量均在一个恒定的水平,能满足异体椎间盘移植的基本要求,可作为组织工程椎间盘的支架材料。
2.利用pSNAV2.0质粒包转系统能成功将外源性基因hBMP7包装成滴度及纯度满足实验要求的rAAV-hBMP7病毒载体。
3. rAAV2-hBMP7病毒载体能有效地转染犬髓核细胞并长期稳定表达人骨形成蛋白-7,并能提高犬髓核细胞蛋白多糖及Ⅱ型胶原含量,这将能为组织工程椎间盘提供可选择的基因修饰的种子细胞。
4.利用液氮储存异体椎间盘同1×105转染髓核细胞复合,体外培养7天能构建出生物活性较高组织工程椎间盘。这将能被用于进一步动物实验,以研究组织工程椎间盘的生物活性。
Human intervertebral disc degeneration is mainly reason for low back pain and neck-should pain. The ratio of degenerative disc disease step up obviously with lengthening average longevity of human and increasing social work stress. Moreover, the expectant treatment and surgical treatment couldn’t thoroughly solve clinical problem at present. It has been proved that the frozen allogenic intervertebral discs could survive in animal study and clinical trail. Furthermore, the allogenic intervertebral disc could completely relieve clinical symptoms in clinical trail. The motion and stability of the spinal unit is preserved after transplantation of frozen allogenic intervertebral discs. But signs of mild disc degeneration were observed in allogenic intervertebral disc transplantation. This mild disc degeneration will cause clinical symptoms emergence once more. The present study results showed that the change of intervertebral disc degeneration could be prevent, even repair, by increasing the active of nucleus pulposus cell. So in this study, tissue engineering intervertebral disc were constructed by using allograft intervertebral disc with the cannie nucleus pulposus cell expressing bone morphogenetic protein-7. Furthermore, the bioactive and function were also investigate in this study.
1. Effects of different storage temperatures and times on cell viability of cryopreserved intervertebral disc
Objectives. To investigate effects of different storage temperatures and times on cell viability of cryopreserved intervertebral disc and obtain the optimal storage condition.
Methods. The 52 canine intervertebral discs were divided into the control group, liquid nitrogen group and -80℃group. The intervertebral discs in liquid nitrogen group and -80℃group were immerged into the freezing preservation medium and storage in liquid nitrogen and -80℃low temperature refrigerator. At the time-points of 2w、1m、2m、4m、6m and 12m, the ratio of different position cell survival were detected by EB/FDA dying method, the PG content of NP tissue were detected by DMMB method, and the collagen content were detected by hydroxyproline method.
Results. The ratio of cell survival in outer layer annulus fibrosus is higher than that in inner layer annulus fibrosus and nucleus pulposus in both groups (p<0.05). The ratio of cell survival is not significantly different in inner layer annulus fibrosus and nucleus pulposus. The ratio of cell survival in inner layer annulus fibrosus in liquid nitrogen group is higher that in -80℃group in the 2nd storage week (p<0.05). The ratio of cell survival in the both groups was not different from 1st to 12th storage month (p>0.05). The PG content reduced gradually with storage duration. From 1st to 12th storage month, the PG content in liquid nitrogen is higher than that in -80℃group. The collagen content in both groups reduced gradually in storage duration, and was not different(p>0.05). Conclusion:Both the ratio of cell survival and PG content of intervertebral disc storaged in liquid nitrogen is higher than that of in -80℃. In 1 year storage duration, intervertebral disc storaged in liquid nitrogen maintains the stable ratio of cell survival and PG content, is able to suitable for the basic requirement to allogenic transplantation, is able to be the scaffold of tissue engineering.
2. Construction and identification of recombination adeno-associated virus type-2 vector carrying human bone morphogenetic protein-7
Objectives: To construct and identification of the recombinant adeno-associated virus type-2 vector carrying human bone morphogenetic protein ( rAAV2-hBMP7).
Methods: The recombinant AAV2 packaging plasmid pSNAV2.0 and full-length hBMP7 cDNA was respectively obtained by the simultaneous digestion of plasmid pDC316-BMP7-IRES-EGFP with AgeIase and NheIase. The full-length hBMP7 cDNA and recombinant AAV2 packaging plasmid pSNAV2.0 were then integrated using T4 DNA ligase. The integrated product was used to transfect the competence Bacillus coli DH-5α. The transfected bacillus coli DH-5αwas cultured in agarose culture medium containing ampicil. Monoclonal colonies were selected and inoculated into Luria Broth (LB) culture medium containing ampicil. After 12 hours in culture, plasmid pSNAV2.0-hBMP7 was extracted and the base sequence was analyzed. After constructed and identified plasmid pSNAV2.0-hBMP7, rAAV2-hBMP7 vector were constructed, amplifying and purifying by AAVMaxTM package and purification system. The optimal multiplicity of infection (MOI) for hNP cell were detected by rAAV vector carrying enhanced green fluorescent protein r(AAV2-EGFP).
Results: Plasmid pSNAV2.0-hBMP7 was successfully constructed by integrating the full-length hBMP-7 cDNA and recombinant AAV2 packaging plasmid pSNAV2.0 using T4 DNA ligase. Plasmid pSNAV2.0-hBMP7 base sequence was correct by PCR detection and assay of base seguence. The rAAV2-hBMP7 vector was packaged by pSNAV2.0-hBMP7 plasmid using the standard calcium phosphate precipitation method. The titer was determined using quantitative DNA dot blots and purity was examined using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Titers averaged approximately 5.1×1011 vg/ml and purity was >97%.
Conclusion:The rAAV2-hBMP7 vector can be successfully constructed by plasmid pSNAV2.0 package system. The titers and purity of the rAAV2-hBMP7 vector is suitable for next experiments.
3.Effects of adeno-associated virus-2 mediated human BMP-7 gene transfection on the chondrocytic phenotype of nucleus pulposus cellsObjectives: To investigate the effects of adeno-associated virus-2 expressing human bone morphogenetic protein-7 (rAAV2-hBMP7) on canine nucleus pulposus cells.
Methods: Following infection with rAAV-BMP7 vector at multiplicities of infection of 1×105 genomes per cell and subsequent culture, nucleus pulposus cells transferred with hBMP7 gene were assessed semi-qualitatively for BMP7 expression with real-time PCR. At 7 days post-transfection, proliferative ability of nucleus pulposus cells were comparative in the transfection and no-transfection cells. Aggrecan, typeⅠand typeⅡcollagen secreted by nucleus pulposus cells were qualitatively accessed at 4, 7 and 14 days post-transfection in the transfection and control groups.
Results: The adeno-associated virus can successfully transfer human BMP7 into the canine NP cell. The NP cell transfected by rAAV-hBMP7 vector express human BMP7 at least 14 days. The expressed human BMP7 promote remarkably accumulation of proteoglycans 42% and 77% (p<0.05) higher than no-transfection cells at 7 and 14 days post-transfection, and typeⅡcollagen 63% and 94% (p<0.05).
Conclusion: The rAAV-based gene delivery approach is capable of promoting the expression of proteoglycans and typeⅡcollagen of nucleus pulposus. This approach may be applied for the treatment of degenerative disc disease in the future.
4. Construction of Tissue Engineering Intervertebral Disc Using allograft Intervertebral Disc with the Cannie Nucleus Pulposus Cell Expressing Bone morphogenetic protein-7
Objective: To construct the tissue engineering intervertebral disc using allograft intervertebral disc with the cannie nucleus pulposus cell expressing hBMP7
Methods: The 24 intervertebral discs storage in liquid nitrogen for 2 months were divided into EGFP, 1×104、1×105 and 1×106 groups. EGFP group: 20ul cell suspension including 1×105 nucleus pulposus cells expressing EGFP were injected into intervertebral disc. 1×104 group: 20ul cell suspension including 1×104 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. 1×105 group: 20ul cell suspension including 1×105 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. 1×106 group: 20ul cell suspension including 1×106 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. Intervertebral discs injected were immerged into 30 ml culture medium. The morphous, cell survival, cell fluorescence intensity, PG and collagen content of intervertebral discs in every group were assessed at 4, 7, 14 culture days.
Results: The morphous of intervertebral discs in every group didn’t change at 4 and 7 culture days. However, the calcium content in the lamina terminalis of intervertebral disc lost at 14 culture days. In EGFP group, nucleus pulposus cells expressing hBMP7 could be observed at any time points. Results showed that cell fluorescence intensity in 1×105 group was higher than that of 1×106 and 1×104 groups. Moreover, the PG and collagen contents in 1×105group were higher than that of 1×106 and 1×104 groups.
Conclusion: The ideal tissue engineering intervertebral disc could be constructed using allograft intervertebral disc storaged in liquid combined with the cannie nucleus pulposus cell expressing hBMP7, and then be cultured for 7days in vitro.
Summary:
1. Both the ratio of cell survival and PG content of intervertebral disc storaged in liquid nitrogen is higher than that of in -80℃. In 1 year storage duration, intervertebral disc storaged in liquid nitrogen maintains the stable ratio of cell survival and PG content, is able to suitable for the basic requirement to allogenic transplantation, is able to be the scaffold of tissue engineering.
2. The rAAV2-hBMP7 vector can be successfully constructed by Plasmid pSNAV2.0 package system. The titers and purity of the rAAV2-hBMP7 vector is suitable for next experiments.
3. The rAAV-based gene delivery approach is capable of promoting the expression of proteoglycans and typeⅡcollagen of nucleus pulposus. This approach may be applied for the treatment of degenerative disc disease in the future.
4. The ideal tissue engineering intervertebral disc could be constructed using allograft intervertebral disc storaged in liquid combined with the cannie nucleus pulposus cell expressing hBMP7, and then be cultured for 7days in vitro.
引文
1.胡有谷.腰椎间盘突出症.北京,人民卫生出版社, 2001, 4
2.Mixter WJ, Barr JS. Rupture of the intervertebral disc eith involvement of the spinal canal, New Eng J Med, 1934, 211:210
3.Frei H, Oxland TR, Rathonyi GC, et al. The effect of nucleotomy on lumbar spine mechanics in compression and shear loading. Spine 2001;26:2080–9
4.Natarajan RN, Andersson GB, Patwardhan AG, et al. Effect of annular incision type on the change in biomechanical properties in a herniated lumbar intervertebral disc. J Biomech Eng 2002;124:229–36
5.Goffin J, Geusens E, Vantomme N, et al. Long-Term Follow-Up After Interbody Fusion of the Cervical Spine, J Spinal Disord Tech. 2004,17(2):79-85
6.魏见伟,王德春,胡有谷.腺相关病毒载体介导的TIMP-1对人退变腰椎间盘髓核细胞蛋白多糖的影响.中国脊柱脊髓杂志,2008;18(9):680-683
7. 1e Maitre CL, Freemont AJ, Hoyland JA, Localization of degradative enzymesand their inhihitops inthe degenerate humanintervertebral disc, J Pathol, 2004, 204(1):47-54
8. Haro H, Crawford HC, Fingleton B, et a1. Matrix metalloproteinase-3-dependent generation of a macrophage chemoattractant in a model of herniated disc resorption. J Clin Invest, 2000, 105(2):133-141
9. Sobajima S, Shimer AI, Chadderdon RC, et a1. Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction.Spine J, 2005, 5(1):14-23)
10.魏见伟,王德春,胡有谷.基质金属蛋白酶及其抑制因子与腰椎间盘退变关系的研究进展.中国脊柱脊髓杂志,2006,16(4):304-3
11. le Maitre CL, Freemont AJ, Hoyland JA, The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration. Arthritis Res Ther, 2005, 7(4):R732-R745
12. Seguin CA, Pilliar RM, Roughley PJ, et a1. Tumor necrosis factor-alpha modulates matrix production and catabolism in nudeus pulposus tissue. Spine,2005,30(17):1940-1948
13. Specchia N, Pagnotta A, Toesca A, et a1. Cytokines and growth factors in the protruded intervertebral disc of the lumbar spine. Eur Spine J, 2002,11(2):145- 151
14. Pattison ST,Ma lrose J,Ghosh P,et a1.Regulation of gdatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead cultureby transforring growth factor-beta(1)and insulin like growth factor-I.(ⅫBiol Int,2001;25(7):679-689
15. Tsuji T, Chiba K, Imabayashi H, et al. Age-related changes in expression of tissue inhihitor of metalloproteinase-3 assodated with transition from the notochordal nucleus pulposus to the fibrocartilaginous nucleus pulposus in rabbit intervertebral disc. Spine, 2007,32(8):849_856
16. Li J,Yoon ST, Hutton WC. Effect of bone morphogenefic protein-2(BMP-2) on matrix production, other BMPs,and BMP receptors in rat intervertebral disc cel1. Spinal Disord Tech, 2004, 17(5):423_428
17. 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, Spine. 2001 Jan 15;26(2):134-41.
18. Kohyama K, Saura R, Doita M,et al, Intervertebral disc cell apoptosis by nitric oxide: biological understanding of intervertebral disc degeneration, Kobe J Med Sci. 2000,46(6):283-95
19. Furusawa N, Baba H, Miyoshi N,et al, Herniation of cervical intervertebral disc: immunohistochemical examination and measurement of nitric oxide production, Spine, 2001 May 15;26(10):1110-6.
20. Rannou F, Richette P, Benallaoua M, et a1. Cyclic tensile stretch modulates proteoglycan production by intervertebral disc annulus fibrosus cells through production of nitrite oxide. J cel1 Biochem, 2003, 90(1):l48- l57
21. Akool el-S, Doller A, Muller R, et a1. Nitric oxide induces TIMP-l expression by activating the transforming growth factor beta-Smad signaling pathway. J Biol Chem, 2005,280(47):39403-39416.
22. BattiéMC, Haynor DR, Fisher LD,et al, Similarities in degenerative findings on magnetic resonance images of the lumbar spines of identical twins, J Bone Joint Surg Am. 1995, 77(11):1662-70
23. Mann V, Hobson EE, Li B, et al, A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality, J Clin Invest. 2001, 107(7):899-907
24. Sive JI, Baird P, Jeziorsk M, Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs, Mol Pathol. 2002, 55(2):91-7
25. Annunen S, Paassilta P, Lohiniva J,et al, An allele of COL9A2 associated with intervertebral disc disease, Science. 1999, 16;285(5426):409-12
26. Kanemoto M, Hukuda S, Komiya Y, Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs, Spine, 1996, 1;21(1):1-8.
27. Takahashi M, Haro H, Wakabayashi Y, The association of degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human matrix metalloproteinase-3 gene, J Bone Joint Surg Br. 2001, 83(4):491-5
28. Sun M, Li Z, Zhang Y , Homology modeling and docking study of cyclin-dependent kinase (CDK) 10, Bioorg Med Chem Lett, 2005, 2;15(11):2851-6.
29. Roughley PJ, Alini M, Antoniou J,The role of proteoglycans in aging, degeneration and repair of the intervertebral disc, Biochem Soc Trans. 2002,30(Pt 6):869-74
30. Kawaguchi Y, Osada R, Kanamori M, Association between an aggrecan gene polymorphism and lumbar disc degeneration, Spine, 1999, 24(23):2456-60.
31. Jones G, White C, Sambrook P, Allelic variation in the vitamin D receptor, lifestyle factors and lumbar spinal degenerative disease, Ann Rheum Dis, 1998, 57(2):94-9
32. Kawaguchi Y, Kanamori M, Ishihara H, The association of lumbar disc disease with vitamin-D receptor gene polymorphism, J Bone Joint Surg Am, 2002, 84-A(11):2022-8
33.张宁,陈维善,LMP-1基因与椎间盘退变,国际骨科学杂志,2008,29(4):270-271
34. Handa T, Ishihara H, Ohshima H, Effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc, Spine, 1997, 15;22(10):1085-91
35. Ishihara H, McNally DS, Urban JP,et al, Effects of hydrostatic pressure on matrix synthesis in different regions of the intervertebral disk, J Appl Physiol, 1996, 80(3):839-46.
36. Cinotti G, Della Rocca C, Romeo S,et al, Degenerative changes of porcine intervertebral disc induced by vertebral endplate injuries Spine (Phila Pa 1976). 2005 Jan 15;30(2):174-80.
37.陈立,詹红生,应航等,异常应力环境下兔颈椎软骨终板蛋白聚糖变化的实验研究,中国矫形外科杂志,2003,11(2):110-112
38. Ishihara H, Urban JP, Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc, J Orthop Res, 1999,17(6):829-35.
39. Kurunlahti M, Karppinen J, Haapea M, et al, Three-year follow-up of lumbar artery occlusion with magnetic resonance angiography in patients with sciatica: associations between occlusion and patient-reported symptoms, Spine, 2004, 15;29(16):1804-8
40.金谷,赵凤东,范顺武,腹主动脉粥样硬化与腰椎间盘退变,国际骨科学杂志,2006,27(3)779-786
41. Magnier C, Boiron O, Wendling-Mansuy S,et al, Nutrient distribution and metabolism in the intervertebral disc in the unloaded state: a parametric study, J Biomech. 2009,42(2):100-8
42.草国永,周跃,滕海军等,水通道蛋白在正常大鼠椎间盘组织中的表达与分布,中国矫形外科杂志,2005,13(13):996-999
43.郭昊,李学军,细胞膜上的水通道-2003年诺贝尔化学奖工作介绍,生理学研究进展,207,38(3):283-288
44. Oda J.Intervertebral disc change with ageing of human cervical Vetebra.Spine, 1988, 13:201-205
45.刘洪.16岁以下少年儿童腰椎间盘突出症的远期随访.中国矫形外科杂志。2005,13(5):330
46. Masuda K, Oegema TR Jr, An HS (2004) Growth factors and treatment of intervertebral disc degeneration. Spine 29:2757–2769
47. Masuda K, An HS (2004) Growth factors and the intervertebral disc. Spine J 4:330S–340S
48. Ahn SH, Cho YW, Ahn MW, Jang SH, Sohn YK, Kim HS (2002) mRNA expression of cytokines and chemokines in herniated lumbar intervertebral discs. Spine 27:911–917
49. Burke JG, Watson RW, Conhyea D, McCormack D, Dowling FE, Walsh MG, Fitzpatrick JM (2003) Human nucleus pulposus can respond to a pro-inflammatory stimulus. Spine 28:2685–2693
50. Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF 3rd, Evans CH (1996) Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine 21:271–277
51. Weiler C, Nerlich AG, Bachmeier BE, Boos N (2005) Expression and distribution of tumor necrosis factor alpha in human lumbar intervertebral discs: a study in surgical specimen and autopsy controls. Spine 30:44–53
52. Igarashi T, Kikuchi S, Shubayev V, Myers RR (2000) 2000 Volvo Award winner in basic science studies: exogenous tumor necrosis factor-alpha mimics nucleus pulposus-induced neuropathology. Molecular, histologic, and behavioral comparisons in rats. Spine 25:2975–2980
53. Olmarker K, Larsson K (1998) Tumor necrosis factor alpha and nucleus-pulposus-induced nerve root injury. Spine 23:2538–2544
54. Le Maitre CL, Freemont AJ, Hoyland JA (2005) The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration. Arthritis Res Ther 7:R732–R745
55. Seguin CA, Pilliar RM, Roughley PJ, Kandel RA (2005) Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine 30:1940–1948
56. Masuda K, An HS (2006) Prevention of disc degeneration with growth factors. Eur Spine J 15(Suppl 15):422–432 .
57. Setton LA, Bonassar LJ, Masuda K (2007) Regeneration and replacement of the intervertebral disc. In: Robert L, Robert L, Joseph V (eds) Principles of tissue engineering, 3rd edn. Academic Press, Burlington, pp 877–896 .
58. Masuda K, An H (2008) Growth factors for intervertebral disc regeneration. In: Yue J, Bertagnoli R, McAfee P, An H (eds) Motion preservation surgery of the spine—advanced techniques and controversies. Elsevier, Amsterdam.
59. Masuda K, Imai Y, Okuma M, Muehleman C, Nakagawa K, Akeda K, Thonar E, Andersson G, An HS (2006) Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model. Spine 31:742–754
60. Iwashina, Mochida, et al. Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration[J]. Spine, 2006, 31:1177—1186.
61. Chung, Sylvia A, Wei, et a1. Nucleus pulposus cellular longevity by telomerase gene therapy[J]. Spine, 2007, 32:1188-1196.
62. Liu H, Kemeny DM, Heng BC, et a1. The immnogenieity and immunomodulatory function of osteogenie cells differentiated from mesenchymal stem cells[J], Immunol, 2006, 176:2864-2871
63. Risbud MV, Izzo MW, Adams CS, et a1. Mesenchymal stem cells respond to their microenvironment in vitro to assume nucleus pulposus-like phenotype[R]. International Society for the Study of the Lumbar Spine,2003.
64. Richardson SM, Walker RV, Parker S, et a1. Intervertebral disc cell mediated mesenchymal stem cell differentiation[J]. Stem Cells, 2006, 24:707—716
65. Thompson JP, Oegema TJ, Bradford DS (1991) Stimulation of mature canine intervertebral disc by growth factors. Spine 16:253–260
66. Gruber HE, Fisher EC Jr, Desai B, Stasky AA, Hoelscher G, Hanley EN Jr (1997) Human intervertebral disc cells from the annulus: three-dimensional culture in agarose oralginate and responsiveness to TGF-beta1. Exp Cell Res 235:13–21
67. Osada R, Ohshima H, Ishihara H, Yudoh K, Sakai K, Matsui H, Tsuji H (1996) 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 Orthop Res 14:690–699
68. Gruber HE, Norton HJ, Hanley EN Jr (2000) Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro. Spine 25:2153–2157
69. Masuda K, Takegami K, An H, Kumano F, Chiba K, Andersson GB, Schmid T, Thonar E (2003) Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads. J Orthop Res 21:922–930
70. Yoon TS, Su Kim K, Li J, Soo Park J, Akamaru T, Elmer WA, Hutton WC (2003) The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro. Spine 28:1773–1780
71. Kim DJ, Moon SH, Kim H, Kwon UH, Park MS, Han KJ, Hahn SB, Lee HM (2003) Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells. Spine 28:2679–2684
72. Gilbertson L, Ahn SH, Teng PN, Studer RK, Niyibizi C, Kang JD (2008) The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells. Spine J 8:449–456.
73. Wehling P, Antiapoptotic and antidegenerative effect of an autologous IL-1ra/IGF-1/PDGF combination on human intervertebral disc cells in vivo. In: Proceeding of the international society for the study of the lumbar spine, 29th Annual Meeting,Cleveland, OH, 2002, p24.
74. Akeda K,An Hs,Pichika R,et a1.Masuda Plateletrieh plasma(PRP) stimulates the extracellular matrix metabolism of porcinenucleus pulposus and annulus fibrosus cells cultured in alginate beads[J].Spine,2006,31:959—966.
75. Nagae M,Ikeda T,Mikami Y,et a1.Intervertebral disc regeneration using platelet rich plasma and biodegradable gelatin hydrogel microspheres[J].Tissue Eng,2007,13:147—158.
76. Chujo,Takehide A,Howard S,et a1.Effects of growth differentiation factor-5 on the intervertebral disc in vitro bovine study and in vivo rabbit disc degeneration model study[J].Spine,2006,3 1:2909—2917
77. Li X, Leo BM, Beck G, Balian G, Anderson GD (2004) Collagen and proteoglycan abnormalities in the GDF-5-deficient mice and molecular changes when treating disk cells with recombinant growth factor. Spine 29:2229–2234
78. Chujo T, An HS, Akeda K, Miyamoto K, Muehleman C, Attawia M, Andersson G, Masuda K (2006) Effects of growth differentiation factor-5 on the intervertebral disc—in vitro bovine study and in vivo rabbit disc degeneration model study. Spine 31:2909–2917
79. Walsh AJ, Bradford DS, Lotz JC (2004) In vivo growth factor treatment of degenerated intervertebral discs. Spine 29:156–163
80. An HS, Takegami K, Kamada H, Nguyen CM, Thonar EJ, Singh K, Andersson GB, Masuda K (2005) Intradiscal administration of osteogenic protein-1 increases intervertebral disc height and proteoglycan content in the nucleus pulposus in normal adolescent rabbits. Spine 30:25–31
81. Kim KW, Lim TH, Kim JG, Jeong ST, Masuda K, An HS (2003) The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine 28:982–990
82. Scott NA, Harris PF, Bagnall KM (1980) A morphological and histological study of the postnatal development of intervertebral discs in the lumbar spine of the rabbit. J Anat 130:75–81
83. Chujo T, An H, Asanuma K, Takatori R, Inoue N, Attawia M, Lee C, Muehleman C, Masuda K (2007) A single injection of recombinant human GDF-5 effectively restores mature rabbit of intervertebral discs degenerated by anular puncture. Trans Orthop Res Soc 32:267 .
84. Huang KY, Yan JJ, Hsieh CC, Chang MS, Lin RM (2007) The in vivo biological effects of intradiscal recombinant human bone morphogenetic protein-2 on the injured intervertebral disc: an animal experiment. Spine 32:1174–1180
85. Larson JW 3rd, Levicoff EA, Gilbertson LG, Kang JD Biologic modification of animal models of intervertebral disc degeneration. J Bone Joint Surg Am, 2006, 88(Suppl 2):83–87
86. Klein RG, Eek BC, Oneill CW, et a1. Biochemical injection treatment for discogenic low back pain:a pilot study[J]. Spine. 2003, 3:220 -226
87. Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, Aebi M, Alini M (1996) The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing,and degeneration. J Clin Invest 98:996–1003
88. Alini M, Roughley PJ, Antoniou J, Stoll T, Aebi M (2002) A biological approach to treating disc degeneration: not for today, but may be for tomorrow. Eur Spine J 11:S215–S220
89. Buckwalter JA (1995) Aging and degeneration of the human intervertebral disc. Spine 20:1307–1314
9O. 0kurrm M,Mochida J,Nishimura K,et aL J Orthop Res,2000;18(6):988~997
91. Yamamoto Y, Mochida J, Sakai D, Nakai T, Nishimura K, Kawada H, Hotta T (2004) Upregulation of the viability of nucleus pulposus cells by bone-marrow-derived stromal cells: significance of direct cell-to-cell contact in co-culture system. Spine 29:1508–1514 [PubMed]
92. Nomura T,Mochida J,Okuma M,et a1.Nucleus pulposus allograft retards intervertebral disc degeneration, Clin Orthop Relat Res. 2001 Aug;(389):94-101 CIin Onhop,2001;389:94~101
93. Gruber HE,Johnson TL,Leslie K,et a1.Autologous intervertebral disc cell implantation: a model using Psammomys obesus, the sand rat, Spine,2002;27(15):1626~1633
94. Gorensek M, Jaksimovi? C, Kregar-Velikonja N, Gorensek M, Knezevic M, Jeras M, Pavlovcic V, C?r A Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes. Cell Mol Biol Lett, 2004, 9:363–373
95. Mwale F, Roughley P, Antoniou J, Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc. Eur Cell Mater, 2004, 8:58–63
96. Sakai D, Mochida J, Yamamoto Y et al. Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration[J]. Biomaterials; 2003(24):3531–3541
97. Sakai D, Mochida J, Iwashina T et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc[J]. Biomaterials; 2006 (27):335–345
98. Sakai D, Mochida J, Iwashina T et al. Differentiation of mesenchymal stem cells transplanted to a rabbit degenerative disc model: potential and limitations for stem cell therapy in disc regeneration[J]. Spine; 2005 (30):2379–2387
99. Hiyama A, Mochida J, Iwashina T et al. Transplantation of mesenchymal stem cellsin a canine disc degeneration model[J]. J Orthop Res. 2008 (26):589-600.
100. Zhang YG, Guo X, Xu P et al. Bone mesenchymal stem cells transplanted into rabbit intervertebral discs can increase proteoglycans[J]. Clin Orthop Relat Res; 2005 (430):219–226
101. Crevensten G, Walsh AJ, Ananthakrishnan D et al. Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs[J]. Ann Biomed Eng; 2004 (32):430–434
102. Washina, Tom, Mochida, et a1. Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration[J]. Spine, 2006, 31:1177—1186
103. Chung, Sylvia A, Wei, et a1. Nucleus pulposus cellular longevity by telomerase gene therapy[J]. Spine, 2007, 32:1188—1196
104. Jesmin S, Zaedj S, Maeda S, et a1. ReversaI of Elevated Cardiac Expression of TGF-beta-l and Endothelin-1 in Diabetic Rats by Long-Acting Calcium Antagonist. Exp Biol Med 2006, 231(6):907—992
105. Sato M,Kikuchi T,Asazuma T,ei a1.Glycosaminoglycan accu—mu1arion in primary culture of rabbit inter'vertebral disc cells.Spine,2001,26:2653—2660
106. Sun Y1,Hurtig M,Pilliar RM,et a1.Characterization of nucleus pulposus tissue formed in vitro[J].Orthop Res,2001,6:1078—1084.
107. Gruber HE, Hanley EN, Human disc cells in monolayer VS 3D culture:cell-shaped division and matrix formation[J]. BMC Musculoskelet Disord, 2000, 1:1—10
108. Ganey T, Libera J, Moos V, et a1. Disc chondrocyte transplantation in a can ine model: a treatment for degenerated or damaged intervertebral disc[J]. Spine, 2003, 23:2609-2260
109. Mizuno H, Roy AK, Vacanti CA, et a1. Tissue engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement[J]. Spine, 2004, 12:1290—1297
110. Kusior LJ, Vacanti CA, Bayley JC, et a1. Tissue engineering of nucleus pulposus[J]. Trans Orthop Res Soc, 1999,24:807
111. Sato M, Asazuma T, Ishihara M, et a1. 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, 6:548-553
112. Tamai N. Myouj A. Hirao M, et al、A new biotechnoleg Y for articular ca rtilage repair:subchondraI implantation of a composite of interconnected poroushydroxyapatite synthetic polymer (PLA-PEG)and bone morphogenetic protein-2 (rhBMP-2). Osteoarthdtis Cartilage,2005,13(5):405-417
113. Gomes ME, Bossano CM, Johnston CM, et a1. In vitro Iocalization of bone growth factors in constructs of biodegradable scafolds seeded with marrow stromal cells and cultured in a flow perfusion bioreactor, Tissue Eng, 2006:12(11:177-188
114. Luk KD, Ruan DK, Lu DS, el a1. Fresh frozen intervertebraI disc allografting in a bipedal animaI model, Spine, 2003,28(9):864-869
115. Nishida K, James D, Kang J, et a1, Modulation of the biologic activity of the rabbit intervertebral disc transfer of the human transforming growth factor-βl encoding gene[J]. Spine, 1999, 24(23):2419-2425
116. Moon SH, Gilbertson LG, Nishida K, et a1, Human intervertebral disc cells are genetically modifiable by adenovirus- mediated gene transfer implications for the clinical management of intervertebral disc disorders, Spine, 2000, 25:2573—2579
117. Risbud MV, AIbe rt TJ, Guttapalli A, et a1, Diferentiation of mesenchymal stem cells towards a nucleus pulposus-like In vitro:implications for cel1-based transplantation therapy, Spine 2004, 29 (12):2627-2632
118. Nakagawa H, Akita S, Fukuj M, et a1, Human mesenchymal stem cells uccessfully improve skin-substitute wound healing, Br J Dermatol, 2005;153(1):29—36
119. Juncosa—Melvin N, Boivin GP, Gooch C, et a1, The effect of autoIlgous mesenchymaI stem cells on the biomechanics and histology of gel-co llagen spo nge constructs used for rabbit patellar tendon repair, Tissue Eng 2OO6:12(2):369—379
120. Guilak F, Loft KE, Awad HA, et a1, Clonal analysis of the differentiation Potential of human adipose-derived adult stem cells, J CelI Physiol, 2006, 206(1):229-237
121. Wang JY, Bacr AE, Kraus VB, et a1. Intervertebral disc cells exhibit diferences in gene expression in alginate and monolayer culture[J], Spine, 2001, 26(16):1747~1751
122. Homer HA, Roberts S, Bielby RC, et a1. Cells from diferent regions of the intervertebral disc:effect of culture system on matrix expression and cell phenotype[J], Spine, 2002, 27(10):1018—1028
123. Perka C, Amold U, Spitzer RS, et a1, The use of fibrin beads for tissue engineering and subsequential transplantation[J], Tissue Eng, 2001, 7(3):359~361
124. Gruber HE, Leslie K, Ingram J, et al, Cell-based tissue engineering for the intervertebral disc:in vitro studies of human disc cell gene expression and matrix production with in selected cell carriers, Spine J, 2004,4:44—55
125. Sakai D, Mochida J, 1washina T, ei a1, Atelocollagen for culture of human -101-nucleus pulposus cells forming nucleus pulposus—like tissue in vitro:influence on the proliferation and proteoglycan production of HNPSV一1 cells.Biomaterials.2006,27:346—353
126. Alini M, Li W, Markovic P, et a1, The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix [J], Spine, 2003, 28 (5):446—454
127. Mwale F, Iordanova M, Demers CN, et a1, Biological evaluation of chitosan salts Cross-linked to genlpin as a cell scaffold for disk tissue engineering, Tissue Eng, 2005, 1 1:130—140
128. Di, Martino A, Sittinger M, Risbud MV, Chitosan:a versatile lfiopolymer for orthopaedic tissue-engineering, Biomaterials, 2005, 26:5983—5990
129. Brown RQ, Mount A, Burg KJ, Evaluation of polymer scaffolds to be used in a composite injectable system for intervertebral disc tissue engineering, J Biomed Mater Res A, 2005, 74:32—39
130. Yang SH, Chen PQ, Chen YF, ei a1, Gelatin/chondroitin-6-sulfate copolymer scaffold for culturing human nucleus pulposus cells in vitro with production of extracellular matrix, J Biomed Mater Res B Appl Biomater, 2005, 74:488—494
131.潘勇,周跃,李长青,新型组织工程椎间盘纤维环支架材料的制备和形态学观察,重庆医学,2007,36(2)152-155
132. Le Visage C, Yang SH, Kadakia L, et al, Small intestinal submucosa as a potential bioscaffold for intervertebral disc regeneration, Spine, 2006, 31(21):2423-30
133. Sims TJ, Mancl LA, Braham PH, Antigenic variation and cross-reactivity in Bacteroides forsythus clinical isolates detected by western blot, Spine, 2006, 31(21):2423-30; discussion 2431
134. Stern S, Lindenhayn K, Schultz O, et al, Cultivation of porcine cells from the nucleus pulposus in a fibrin/hyaluronic acid matrix, Acta Orthop Scand. 2000 Oct;71(5):496-502
136. Crevensten G, Walsh AJ, Ananthakrishtmn D, et a1, Intervertebral disc cell therapy for regeneration mesenchmal stem cell implantation into intervertebral discs, Ann Bion Eng, 2O04, 32:430—434
137. Sato M,Kikuchi M,Ishihara M,et a1.Tissue engineering of the intervertebral disc with cultured annulus fibrosus cells using atelocollagen honeycomb—shaped scafold with a membrane seal(ACHMS scafold).Med Biol Eng Comput,2003,41:365—371
138. Richardson SM, Hughes N, Hunt JA, et a1, Human mesenchymal stem ce11dif_ferentiation to NP-like cells in chitosan glycerophosphate hydrogels[J], Biomaterials, 2008, 29:85—93
139. Richards0n SM, Curran JM, Chen R, et a1, The differentiation of bone marrow mesenchymal stem cells into ehondrocyte-like cells on poly-L—lactic acid(PLLA)scafolds[J], Biomaterials, 2006, 27:4069-4078
140.Nishida Y, Knudson CB, Eger W, et al. Osteogenic protein 1 stimulates cells-associated matrix assembly by normal human articular chondrocytes: Up-regulation of hyaluronan synthase, CD44, and aggrecan. Arthritis Rheum , 2000; 43:206–14
141. Takegami K,Thonar EJ,An HS,et al.Osteogenic protein 1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin1.Spine,2002,27(12):1318-1325。
142. Luk KD;Ruan DK;Chow DH;Leong JC. Intervertebral disc autografting in a bipedal animal model. Clin Orthop Relat Res 1997:33:13-26
143. Convery FR, Akeson WH, Amiel D, et al. Long-term survival of chondrocytes in an osteochondral articular cartilage allograft. J Bone Joint Surg Am 1996;78:1082–1088
144. Matsuzaki H, Wakabayashi K, Ishihara K, et al. Allografting intervertebral discs in dogs: a possible clinical application. Spine 1996;21:178–83
145. Dike Ruan, Qing He, Yu Ding, et al. Intervertebral disc transplantation in the treatment of degenerative spine disease: a preliminary study. Lancet 2007; 369: 993–99
146.阮狄克,沈根际,周勇等.同种异体椎间盘移植的实验研究.中华骨科杂志1996;16:511-516
147. Flynn J, Rudert MJ, Olson E, et al. The effects of freezing or freeze-drying on the biomechanical properties of the canine intervertebral disc. Spine 1990; 15:567–70
148. Frick SL, Hanley EN Jr, Meyer RA Jr, et al. Lumbar intervertebral disc transfer: a canine study. Spine 1994;19:1826–34
149. Katsuura A, Hukuda S. Experimental study of intervertebral disc allografting in the dog. Spine 1994;19:2426–32
150. Olson EJ, Hanley EN Jr, Rudert MJ, et al. Vertebral column allografts for the treatment of segmental spine defects: an experimental investigation in dogs. Spine 1991;16:1081–8
151. Matsuzaki, Hiromi MD; Wakabayashi,et at, Allografting Intervertebral Discs in Dogs: A Possible Clinical Application, Spine, 1996, 21(2):178-183
152.吴小兵,董小岩,伍志坚,屈建国,侯云德.一种快速高效分离和纯化重组腺病毒伴随病毒载体的方法.科学通报. 2000,45(19):2071-2075
153. J.萨姆布鲁克, E.F.弗里奇, T.曼尼阿蒂斯著.金冬雁,黎孟枫,译.分子克隆实验指南.北京:科学出版社,1992
154. Synder RO, Xiao X, Samulski RJ. Production of recombinant adeno-associated viral vectors. In: Dracopoli N (ed). Current Protocols in Human Genetics. John Wiley: New York, 1996. 12.1.1-12.1.20.
155. McCown TJ, Xiao X, Li J, Breese GR, Samulski RJ (1996) Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res 713(1-2): 99-107
156. Koeberl DD, Alexander IE, Halbert CL, Russell DW, Miller AD (1997) Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors. Proc Natl Acad Sci 94(4): 1426-1431
157. Wu ZJ, Wu XB, Hou YD (1999) Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated virus. Chin Sci Bull 44: 715-719
158. Wu XB, Dong XY, Wu ZJ, Cao H, Niu DB, Qu JG, et al (2000) A novel method for purification of recombinant adeno-associated virus vectors on a large scale. Chin Sci Bull 45: 2071-2075
159.李连华翁习生邱贵兴等,2型重组腺相关病毒转染新西兰兔关节软骨细胞的体外研究,中华医学杂志,2006,6,86(21)1489-92
160. Asahina I, Sampath TK, Hauschka PV (1996) Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic differentiation of clonal murine target cells. Exp Cell Res 222:38–47
161. Zhang Y, An HS, Song S, Toofanfard M, Masuda K, Andersson GB et al (2004) Growth factor osteogenic protein-1: differing effects on cells from three distinct zones in the bovine intervertebral disc. Am J Phys Med Rehabil 83:515–21
162. Paul R, Haydon RC, Cheng H, Ishikawa A, Nenadovich N, Jiang W, et al (2003) Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine 28:755–763
163. Yoon ST, Park JS, Kim KS, Li J, Attallah-Wasif ES, Hutton WC, et al (2004) ISSLS prize winner: LMP-1 upregulates intervertebral disc cell production of proteoglycans and BMPs in vitro and in vivo. Spine 29:2603–2611
164. Zhang Y, Phillips FM, Thonar EJ, Oegema T, An HS, Roman-Blas JA, et al (2008) Cell therapy using articular chondrocytes overexpressing BMP7 or BMP-10 in a rabbit disc organ culture model. Spine 15;33(8):831-8
165. Zhang Y, Markova D, Im HJ, Hu W, Thonar EJ, He TC, et al (2009) Primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 BMPs and Sox9 maintain appropriate phenotype. Am J Phys Med Rehabil 88(6):455-63
166. Zhang Y, An HS, Thonar EJ, Chubinskaya S, He TC, Phillips FM (2006) Comparative effects of bone morphogenetic proteins and sox9 overexpression on extracellular matrix metabolism of bovine nucleus pulposus cells. Spine 31(19):2173-9
167. Schwarz EM (2000) The adeno-associated virus vector for orthopaedic gene therapy. Clin Orthop 379:S31-S39
168. Qu FJ, Liu Y, Zhu DL (2008) Construction of Tissue-engineered Cartilage with BMP7 Gene-transfected Chondrocytes. The Journal of International Medical Research 36: 837– 847