当归注射液对体外培养兔软骨细胞增殖和代谢的影响
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摘要
目的：
探讨兔关节软骨细胞分离培养的方法和生物学特性，观察中药当归注射液对体外培养兔关节软骨细胞的增殖以及对肿瘤坏死因子-α诱导的软骨细胞损伤的保护作用，探讨其治疗骨关节炎的机制。
方法：
体外培养从3周龄新西兰兔关节分离培养的软骨细胞，倒置显微镜下观察原代及传代培养的细胞形态学变化，用组织化学甲苯胺蓝染色法鉴定软骨细胞。将传一代的软骨细胞接种于96孔培养板，细胞贴壁后，将其分成当归组A—F组与空白对照组G组，分别用含10％，5％，2.5％，1.25％，0.625％，0.312％和0％的当归注射液的含10％血清DMEM培养基进行培养，H组未接种细胞，只加无血清DMEM培养基做为调零组。培养48h后用MTT比色法测定各组软骨细胞的增殖情况。取第二代软骨细胞接种到24孔培养板中，培养5d后将细胞随机分为8组，分别换用不同的培养液。当归A—F组分别以含10％，5％，2.5％，1.25％，0.625％，0.312％当归注射液的含10％血清DMEM培养基培养，并在每孔加入100U肿瘤坏死因子-α诱导细胞损伤。实验对照组不加当归注射液，仅以含10％血清DMEM培养基培养细胞，并在每孔加入100U肿瘤坏死因子-α。空白对照组只以不含当归注射液的含10％血清DMEM培养基培养。培养72h后收集细胞上清液，用化学比色法测定SOD及NOS含量，观察当归注射液对软骨细胞的保护作用。
结果：
成功建立体外培养兔关节软骨细胞的实验方法。原代培养的软骨细胞呈多角形，传代3次后出现去分化。形态学、组织化学染色显示细胞培养3代以内可以保持表型的稳定。5％，2.5％，1.25％，0.625％，0.312％的当归注射液对软骨细胞的增殖有明显的促进作用，与对照组相比有非常显著性的差异。其中0.625％当归组作用最强，10％当归组对软骨细胞的增殖无促进作用，与对照组相比无显著性差异。肿瘤坏死因子-α可显著升高软骨细胞中NOS活性，降低SOD活性，当归注射液组能对抗上述结果并呈浓度依赖性。
结论：
本实验建立的体外培养关节软骨细胞的方法简单可行。细胞具有良好的生物学活性，细胞表型可保持3代稳定。药物实验显示中药当归注射液可有效的促进软骨细胞的增殖，并可有效的防止和保护肿瘤坏死因子-α诱导的细胞损伤作用，调节软骨细胞代谢，减缓骨关节炎关节软骨的退变。从而证实当归注射液可有效的防治骨关节炎。
Objective:
To investigate the method of isolation and culture of rabbit articular chondrocytes for biological characteristic studies, To observe the effect of DangGui injection on proliferation of rabbit articular chondrocyte and the protective effect of DangGui injection on chondrocyte lesion induced by TNF-α, and discuss the mechanism of DangGui injection for treating Osteoarthritis.
Methods:
Articular chondrocytes were isolated from the cartilages of 3 week-old New Zealand rabbits were cultured in vitro. The morphological changes and growth feature of primary culture and subculture of chondrocytes were observed under the inverted microscope each day. Toluidine blue staining was used to indentify the chondrocytes .The first subculture chondrocytes were inoculated into 96- hole culture plate and divided into the experimental groups DangGui A - F group and the G group, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25 %, 0.625%, 0.312%and 0% of DangGui injection, respectively. G Group is the blank control group were cultured in 10% serum DMEM medium, H Group were cultured in serum-free DMEM medium and no chondrocyte. After cultured for 48 hours,chondrocytes proliferation was assessed by MTT chromometry. The second subculture chondrocytes were inoculated into two 24-hole culture plates. After incubation for five days, chondrocytes were divided into 8 groups randomly with different medium. DangGui A-F groups, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25%, 0.625%, 0.312% of Dang Gui injection, with100U/ml TNF-α, respectively. Experimental group were cultured in 10% serum medium with 100U/ml TNF-α, Blank control group were cultured in 10% serum DMEM only, after incubation for 72 hours, culture supernatants were collected for SOD and NOS measurement.
Results:
The primarily cultured chondrocytes was in polygonal shape, and became dedifferentiation after 3 passage. The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages . Compared with the control, 5%, 2.5%, 1.25%, 0.625%, 0.312% of DangGui injection could promote the proliferation of chondrocytes with significant difference. 0.625% DangGui injection had the greatest function in the six doses with a significant difference from other groups. 10% DanShen injection had no significant promotion effect difference. TNF-αcould promote the NOS activity and depress SOD activity in chondrocytes. DangGui injection could altered the above mentioned results in a concentration dependent manner.
Conclusions:
The method used in this work for isolation and culture of chondrocytes is simple and feasible. The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the first 3 passages. The following experiment suggested that DangGui injection can effectively promote the proliferation of chondrocytes and prevent and protect against chondrocytes lesion induced by TNF-α, and regulate anabolism of chondrocytes, thus it can defer the degeneration of osteoarthritis cartilage. It demonstrated that DangGui injection was effective to prevent and cure osteoarthritis.
探讨兔关节软骨细胞分离培养的方法和生物学特性，观察中药当归注射液对体外培养兔关节软骨细胞的增殖以及对肿瘤坏死因子-α诱导的软骨细胞损伤的保护作用，探讨其治疗骨关节炎的机制。
方法：
体外培养从3周龄新西兰兔关节分离培养的软骨细胞，倒置显微镜下观察原代及传代培养的细胞形态学变化，用组织化学甲苯胺蓝染色法鉴定软骨细胞。将传一代的软骨细胞接种于96孔培养板，细胞贴壁后，将其分成当归组A—F组与空白对照组G组，分别用含10％，5％，2.5％，1.25％，0.625％，0.312％和0％的当归注射液的含10％血清DMEM培养基进行培养，H组未接种细胞，只加无血清DMEM培养基做为调零组。培养48h后用MTT比色法测定各组软骨细胞的增殖情况。取第二代软骨细胞接种到24孔培养板中，培养5d后将细胞随机分为8组，分别换用不同的培养液。当归A—F组分别以含10％，5％，2.5％，1.25％，0.625％，0.312％当归注射液的含10％血清DMEM培养基培养，并在每孔加入100U肿瘤坏死因子-α诱导细胞损伤。实验对照组不加当归注射液，仅以含10％血清DMEM培养基培养细胞，并在每孔加入100U肿瘤坏死因子-α。空白对照组只以不含当归注射液的含10％血清DMEM培养基培养。培养72h后收集细胞上清液，用化学比色法测定SOD及NOS含量，观察当归注射液对软骨细胞的保护作用。
结果：
成功建立体外培养兔关节软骨细胞的实验方法。原代培养的软骨细胞呈多角形，传代3次后出现去分化。形态学、组织化学染色显示细胞培养3代以内可以保持表型的稳定。5％，2.5％，1.25％，0.625％，0.312％的当归注射液对软骨细胞的增殖有明显的促进作用，与对照组相比有非常显著性的差异。其中0.625％当归组作用最强，10％当归组对软骨细胞的增殖无促进作用，与对照组相比无显著性差异。肿瘤坏死因子-α可显著升高软骨细胞中NOS活性，降低SOD活性，当归注射液组能对抗上述结果并呈浓度依赖性。
结论：
本实验建立的体外培养关节软骨细胞的方法简单可行。细胞具有良好的生物学活性，细胞表型可保持3代稳定。药物实验显示中药当归注射液可有效的促进软骨细胞的增殖，并可有效的防止和保护肿瘤坏死因子-α诱导的细胞损伤作用，调节软骨细胞代谢，减缓骨关节炎关节软骨的退变。从而证实当归注射液可有效的防治骨关节炎。
Objective:
To investigate the method of isolation and culture of rabbit articular chondrocytes for biological characteristic studies, To observe the effect of DangGui injection on proliferation of rabbit articular chondrocyte and the protective effect of DangGui injection on chondrocyte lesion induced by TNF-α, and discuss the mechanism of DangGui injection for treating Osteoarthritis.
Methods:
Articular chondrocytes were isolated from the cartilages of 3 week-old New Zealand rabbits were cultured in vitro. The morphological changes and growth feature of primary culture and subculture of chondrocytes were observed under the inverted microscope each day. Toluidine blue staining was used to indentify the chondrocytes .The first subculture chondrocytes were inoculated into 96- hole culture plate and divided into the experimental groups DangGui A - F group and the G group, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25 %, 0.625%, 0.312%and 0% of DangGui injection, respectively. G Group is the blank control group were cultured in 10% serum DMEM medium, H Group were cultured in serum-free DMEM medium and no chondrocyte. After cultured for 48 hours,chondrocytes proliferation was assessed by MTT chromometry. The second subculture chondrocytes were inoculated into two 24-hole culture plates. After incubation for five days, chondrocytes were divided into 8 groups randomly with different medium. DangGui A-F groups, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25%, 0.625%, 0.312% of Dang Gui injection, with100U/ml TNF-α, respectively. Experimental group were cultured in 10% serum medium with 100U/ml TNF-α, Blank control group were cultured in 10% serum DMEM only, after incubation for 72 hours, culture supernatants were collected for SOD and NOS measurement.
Results:
The primarily cultured chondrocytes was in polygonal shape, and became dedifferentiation after 3 passage. The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages . Compared with the control, 5%, 2.5%, 1.25%, 0.625%, 0.312% of DangGui injection could promote the proliferation of chondrocytes with significant difference. 0.625% DangGui injection had the greatest function in the six doses with a significant difference from other groups. 10% DanShen injection had no significant promotion effect difference. TNF-αcould promote the NOS activity and depress SOD activity in chondrocytes. DangGui injection could altered the above mentioned results in a concentration dependent manner.
Conclusions:
The method used in this work for isolation and culture of chondrocytes is simple and feasible. The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the first 3 passages. The following experiment suggested that DangGui injection can effectively promote the proliferation of chondrocytes and prevent and protect against chondrocytes lesion induced by TNF-α, and regulate anabolism of chondrocytes, thus it can defer the degeneration of osteoarthritis cartilage. It demonstrated that DangGui injection was effective to prevent and cure osteoarthritis.
引文
1. Jackson JP. Surgery of the knee joint[C].London, Chapmam and Hall: HD,1984, 285-290.
2. Creamer P, HochbergMC. Osteoarthritis[R]. Lancet. 1997. 350: 503-508.
3. Anderson J, Felson D. Factors associated with Knee osteoarthritis (OA) in a national survey [J]. The Framingham study Arthritis Rheum, 1986,29:256
4. Blot L, Marcelis A, Derogelaer JP, et al. Effects diclofenac, aceclofenac and meloxicam on the metabolism ofproteoglycans and hyaluronan in osteoarthritic human cartilage [J]. Br J Pharmacol, Dec 2000,131(7):14-21.
5．黄枫，谢国平．中医药治疗膝骨关节炎实验研究进展[J]．中国中医药信息志，2005，12(8)：109-111．
6. Pelletier JP.[J]. osteoarthritis Cartilage, 1999,7(3):308-309.
7．黄涛，徐传毅，邹季．活血止痛汤治疗早期骨性关节炎的实验研究[J]．中医正骨，1999，11(3)：3-5．
8．徐国钧，何宏贤，徐珞珊．中国药材学[M]．北京：中国医药科技出版社，1996．332-338．
9．郑红。明清医家论治骨痹(骨关节炎)用药规律研究[J]．山东中医药大学学报，2003，27(2)：95-97．
10．方洪钜．当归成分的研究[J]．药学学报，1997，(14)：617-621．
11．夏泉，张平，李绍平．当归的药理作用进展[J]．时珍国医国药，2004，15：164-166．
12．靖会，姚凌云，李教社．国内阿魏酸钠的药理研究进展[J]．西北药学杂志，2002．17：236-238．
13．陶学勤．当归对超氧化物歧化酶的作用[J]．南京中医学院学报，1992，8：103-106．
14．胡永欣，陈红，瞿美英．几种天然药物清除超氧阴离子自由基的作用[J]．第二军医大学学报，1993，14(1)：48-50．
15．吴建龙，王大元．阿魏酸钠的药理研究进展[J]．中国药学杂志，1993， 28：267．
16．欧阳静萍，王雄，Muller S，Stoltz J F．TNF—α导致细胞骨架重排和当归的逆转作用[J]．中国病理生理杂志，2000，16(10)：981-982．
17．刘保新，陈廖斌，汪晖．当归、阿魏酸钠对骨关节炎软骨退变与自由基的影响[J]．武汉大学学报(医学版)，2006，(05)：626-628．
18．宋手中，蒋清涛，赵大庆．关节内注射当归注射液治疗膝关节骨性关节炎40例观察[J]．中国中西医结合杂志，1993，13(7)：425．
19．司徒镇强，吴军正．细胞培养[M]．西安：世界图书出版公司，1996年4月，第一版．
20. Plumb MS, Aspden RM. The response of elderly human articular cartilage to mechanical stimuli in vitro [J]. Osteoarthritis Cartilage, 2005,13(12):1084-1091
21. 李保林，王海彬，周正新．兔软骨细胞的分离与培养[J]．广州医药，2005，(1)：2-4．
22．张志光，郑卫平，苏凯．兔关节软骨细胞的分离培养和形态学特征[J]．中山大学报：医学科学版，2004，25(1)：63．
23. Ishizaki Y, Julia FB, Martin CR. Autocrine signals enable chondrocytes to survive in culture[J].J Cell Biolol, 1994,26 (4): 1069.
24. Kart sogiannis V, Ng KW. Cell lines and primary cell cultures in the study of bone cell biology [J].Mol Ceil Endocrinol, 2004, 228 (122) : 79.
25. Garnero P, Rousseau JC, Delmas PD. Molecular basis and clinical use of biochemical markers of bone, cartilage, and synovium in joint diseases [J]. Arthritis Rheum, 2000,43(5):953-968.
26. Archer CW, Francis-West P. The chondrocyte [J]. Int J Biochem Cell Biol,2003,35(4) :401-404.
27. Terry DE, Chopra RK, Ovenden J, et al. Differential use of alcian blue and toluidine blue dyes for quantification and isolation of anionic glycoconjugates from cell culture :application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes [J]. Analytical Biochemistry, 2000, 285:211-219.
28. Gueme P A, Sublet A, Lotz M. Growth factor responsiveness of human articular chondrocytes: distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts[J]. J Cell Phsiol, 1994,158 (3):476-484.
29. Marlovits S, Zeller P, Singer P, et al. Cartilage repair:generations of autologous chondrocyte transplantation [J]. Eur J Radiol, 2006,57(1):24-31.
30. Tallheden T, Bengtsson C, Brantsing C, et al.Proliferation and differentiation potential of chondrocytes from osteoarthritic patients [J]. Arth Res Ther, 2005, 7(3):560-568.
31. Tamamura Y, Iwamoto M. Rapid phenotypic changes in passaged articular chondrocyte subpopulations[J]. Clin Calcium, 2004,14(7): 15.
32. Davis MA, Ettinger WH, Neuhans JM, Hauck WW. The association of knee injury and obesity with unilateral and bilateral osteoarthritis of the knee. Am J Epidemiol, 1999, 130:278-288.
33．冯伟，石印玉，沈培芝．MTT法分析中药含药血清对体外软骨细胞增殖影响的研究[J]．上海中医药大学学报．2000，14(1)：43-45．
34．吕存贤，王维佳．膝骨关节炎病因病理研究及中医治疗概况[J]．浙江中医学院学报，2005，29(1)：82-84．
35．林木南，刘献祥．骨性关节炎中细胞因子的协同效应[J]．福建中医学院学报，2006，16(2)：69-71．
36．陈百强，张洪斌，张汉杰．生长因子对软骨细胞的作用[J]．中华骨科杂志．1999，19(2)：746-747．
37. Arner EC, Tortorella MD. Signal transduction through chondrocyte integrin receptors induces matrix metalloproteinase synthesis and synergizes with interleukin-1 [J]. Arthritis Rheum, 1995,38:1304.
38. Kanyama M, Kuboki T,Kojima S, et al. Matrix metalloproteinases andtessue inhibitors of metalloproteinases in synovial fluids of patients with temporomandibular joint osteoarthritis[J]. Orofac Pain,2000,14(1):20.
39．解志杰，许建中．关节软骨的结构和代谢与年龄相关的变化[J]．中华老年医学杂志．2000：19(5)：394-396．
40. Salvatierra J, Escames G, Hernandez P, et al. Cartilageand serum levels of nitric oxide inpatients with hip osteoarthritis[J]. Rheumatol,1999, 26(9):2015.
41．薛延．骨质疏松症诊断与治疗指南[M]．北京：科学出版社，1999年9月第一版，314-315．
42．邓廉夫，柴本甫，杨庆铭．骨关节炎滑膜细胞分泌肿瘤坏死因子生物学特征研究[J]．中华骨科杂志1999，19(12)：726-728．
43. Westacott TI, Atkins RM, Dieppe PA, et al. Tumor necrosis factor-α receptor expression on chondrocytes isolated from human articular cartilage. J Rheumatol, 1994, 21:1710-1715.
44. Homandberg GA, Umadi V, Kang H, et al. High molecular weight hyaluronan promotes of IL-1 beta damaged explants from both young and old bovines[J].Osteoarthritis Cartilage,2003,11(3): 177-186.
45．田华，娄思权，党耕町．肿瘤坏死因子-α导致软骨损伤的机制探讨[J]．中华外科杂志，2000，38(1)：76-77．
46. Arend WP, Dayer JM.Inhibition of the production and effects of IL-1 and TNF-α in rheumatoid arthritis [J]. Arthritis Rheum, 1995, 38:151-160.
47．吴蠡荪．自由基与临床医学[J]．微循环学杂志，1998，8：24-26．
48. Frederiks WM, Bosch KS. Localization of superoxide dismutase activity in rat tissue[J]. Free Radic Biol Med, 1997, 22:241-248.
49．孙材江，王慧，杨锡兰．退行性膝关节炎患者氧自由基代谢的观察[J]．中华骨科杂志．1992，12(6)：433-435．
50．许鹏，蔡乾坤．氧自由基对软骨代谢的影响[J]．中国矫形外科杂志，2000，7(10)：1008-1010．
51. Regan E, Flannelly J,Bowler R, et al.Ext racellular superoxide dismutase and oxidant damage in osteoarthritis [J].Arthritis Rheum, 2005,11:3479-3491.
52. Ostalowska A, Birkner E, Wiecha M, et al. Lipid peroxidation and antioxidant enzymes in synovial fluid of patients with primary and secondary osteoarthritis of the knee joint [J]. Osteoarthritis Cartilage, 2006, 2:139-145.
53．李松，曹峻岭，郝家骥．在培养的人胚软骨细胞中硒抗氧自由基损伤作 用观察[J]．中国国矫形外科杂志2000，7(4)：362-365．
54. Martin JA, Brown TD, Heiner AD, et al, Chondrocyte senescence,joint loading and osteoarthritis[J]. Olin Orthop Relat Res, 2004, 427(Suppl): 96-103.
55. Yudoh K, Nguyen T, Nakamura H, et al. Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function[J]. Arthritis Res Ther, 2005, 7: 380-391.
56．李松，黄石登，曹峻岭．自由基对体外培养人胚关节软骨细胞的影响[J]．中华骨科杂志．1998，18(7)：426-428．
57．马建兵，刘德玉，李堪印．中药对家兔实验性膝关节骨性关节炎氧自由基代谢的影响[J]．中医正骨．2000，12(1)：8-9．
58．王耶，刘建宇，宋艳．丹参和透明质酸钠注射液对骨性关节炎治疗作用的实验研究[J]．中国生化药物杂志．1998，19(5)：248-225．
59．谢林，周尊谦，孙达武．丹紫康冲剂对膝关节退行性骨关节病患者氧自由基代谢的影响[J]．中国中医骨伤科．1996，4(2)：81．
60. Stadler J, Stefanovic RacicM, billiard TR. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide[J]. J Immunol,1991, 147:3915-3920.
61. Grabowski PS, Wright PK. Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis[j]. Br J Rheumatol, 1997,36(6):651-655.
62．秦霞南，李金荣，龙星．一氧化氮合成酶抑制剂治疗骨关节病研究进展[J]．国外医学口腔医学分册，2004，3：188-190．
63. Pelletier JP, Martel-Pelletier J, Tardif G, et al. The increased synthesis of inducible nitric oxide inhibits IL-I rasynthesis by human articular chondrocytes: possible role in osteoarthritie cartilage degradation [J]. Ostoarthritis Cartilage,1996,4 (1):77-84.
64. Cao M, Evans CH, Stefanovic-Racia M,et al. Nitric oxide inhibits the synthesis of theⅡcollagen without alteringCoL2A1 mRNA abundance prolyl hydroxylase as a possible target[J]. Biochem J, 1997,324:305-310.
65. Task iran D, Stefanovic-RacicM, Georgescu H, et al. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1[J]. Biochem Biophys Res Commun,1994; 200:142-148.
66. Lotz M ,Blanco FJ ,von Kempis J ,et al. Cytokine regulation of chondrocyte function[J]. J Rheumatol, 1995, 43(1): 104-108.
67. George ACM, Daniel J, and Williams RJ. Nitric oxide activates metallopro tease enzymes in articular cartilage[J]. Biochem Biophys Res Commun, 1995,206:15-21.
68. Stadler J, Stefanovic-RacicM, Billiar TR, et al. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopo lysaccharide[J]. J Immunol, 1991, 147:3915-3920.
69. Loeser RF, Carlson CS,Del CarloM, et al. Detection ofnitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin—1β and with chondrocyte resistance to insulin—like growth factor—1 [J]. Arthritis Rheum, 2002, 46: 2349-2357.
70. Blanco F, Lotz M. IL-1 induced nitric oxide inhibits chondrocytes prolixferation via PGE2 [J]. FASEB J 1994, 8(1):365-371.
71. Stefanovic-Racic M, Stadlter J, Gerogesc H, et al. Nitricoxide and energy production in arthicular chondrocytes[J]. Cell Physiol, 1994, 159:274-280.
72. Kim H ,Lo M ,Pillarisetty R. Chondrocyte apopotosis following Intra articular fracture in humans[J]. Osteo Cart,2002,10 (9): 747-753.
73．许鹏，靳卫章，郭雄．骨关节病关节软骨细胞凋亡的研究进展[J]．实用老年医学．1999，13(6)：312-313．
74. Hashimoto S,Takahashi K, Amiel D,et al. Chondrocyte apoptosis and nitic oxide production during experimentlly induced osteoarthristis[J]. Arthritis Rheum, 1998,41(7): 1266-1274.
75. Blanco F J, Ochs RL, Schwarz H, et al. Chondrocyte apoptosis induced by nitix oxide[J] .Am JPathol, 1995,146(1):75-85.
76. Hashimoto S, Lotz M, Ochs RL, et al. Fas/FasL expression and induction of apoptosis in chondrocytes[J]. Arthritis Rheum, 1997,40(10): 1749-1755.
2. Creamer P, HochbergMC. Osteoarthritis[R]. Lancet. 1997. 350: 503-508.
3. Anderson J, Felson D. Factors associated with Knee osteoarthritis (OA) in a national survey [J]. The Framingham study Arthritis Rheum, 1986,29:256
4. Blot L, Marcelis A, Derogelaer JP, et al. Effects diclofenac, aceclofenac and meloxicam on the metabolism ofproteoglycans and hyaluronan in osteoarthritic human cartilage [J]. Br J Pharmacol, Dec 2000,131(7):14-21.
5．黄枫，谢国平．中医药治疗膝骨关节炎实验研究进展[J]．中国中医药信息志，2005，12(8)：109-111．
6. Pelletier JP.[J]. osteoarthritis Cartilage, 1999,7(3):308-309.
7．黄涛，徐传毅，邹季．活血止痛汤治疗早期骨性关节炎的实验研究[J]．中医正骨，1999，11(3)：3-5．
8．徐国钧，何宏贤，徐珞珊．中国药材学[M]．北京：中国医药科技出版社，1996．332-338．
9．郑红。明清医家论治骨痹(骨关节炎)用药规律研究[J]．山东中医药大学学报，2003，27(2)：95-97．
10．方洪钜．当归成分的研究[J]．药学学报，1997，(14)：617-621．
11．夏泉，张平，李绍平．当归的药理作用进展[J]．时珍国医国药，2004，15：164-166．
12．靖会，姚凌云，李教社．国内阿魏酸钠的药理研究进展[J]．西北药学杂志，2002．17：236-238．
13．陶学勤．当归对超氧化物歧化酶的作用[J]．南京中医学院学报，1992，8：103-106．
14．胡永欣，陈红，瞿美英．几种天然药物清除超氧阴离子自由基的作用[J]．第二军医大学学报，1993，14(1)：48-50．
15．吴建龙，王大元．阿魏酸钠的药理研究进展[J]．中国药学杂志，1993， 28：267．
16．欧阳静萍，王雄，Muller S，Stoltz J F．TNF—α导致细胞骨架重排和当归的逆转作用[J]．中国病理生理杂志，2000，16(10)：981-982．
17．刘保新，陈廖斌，汪晖．当归、阿魏酸钠对骨关节炎软骨退变与自由基的影响[J]．武汉大学学报(医学版)，2006，(05)：626-628．
18．宋手中，蒋清涛，赵大庆．关节内注射当归注射液治疗膝关节骨性关节炎40例观察[J]．中国中西医结合杂志，1993，13(7)：425．
19．司徒镇强，吴军正．细胞培养[M]．西安：世界图书出版公司，1996年4月，第一版．
20. Plumb MS, Aspden RM. The response of elderly human articular cartilage to mechanical stimuli in vitro [J]. Osteoarthritis Cartilage, 2005,13(12):1084-1091
21. 李保林，王海彬，周正新．兔软骨细胞的分离与培养[J]．广州医药，2005，(1)：2-4．
22．张志光，郑卫平，苏凯．兔关节软骨细胞的分离培养和形态学特征[J]．中山大学报：医学科学版，2004，25(1)：63．
23. Ishizaki Y, Julia FB, Martin CR. Autocrine signals enable chondrocytes to survive in culture[J].J Cell Biolol, 1994,26 (4): 1069.
24. Kart sogiannis V, Ng KW. Cell lines and primary cell cultures in the study of bone cell biology [J].Mol Ceil Endocrinol, 2004, 228 (122) : 79.
25. Garnero P, Rousseau JC, Delmas PD. Molecular basis and clinical use of biochemical markers of bone, cartilage, and synovium in joint diseases [J]. Arthritis Rheum, 2000,43(5):953-968.
26. Archer CW, Francis-West P. The chondrocyte [J]. Int J Biochem Cell Biol,2003,35(4) :401-404.
27. Terry DE, Chopra RK, Ovenden J, et al. Differential use of alcian blue and toluidine blue dyes for quantification and isolation of anionic glycoconjugates from cell culture :application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes [J]. Analytical Biochemistry, 2000, 285:211-219.
28. Gueme P A, Sublet A, Lotz M. Growth factor responsiveness of human articular chondrocytes: distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts[J]. J Cell Phsiol, 1994,158 (3):476-484.
29. Marlovits S, Zeller P, Singer P, et al. Cartilage repair:generations of autologous chondrocyte transplantation [J]. Eur J Radiol, 2006,57(1):24-31.
30. Tallheden T, Bengtsson C, Brantsing C, et al.Proliferation and differentiation potential of chondrocytes from osteoarthritic patients [J]. Arth Res Ther, 2005, 7(3):560-568.
31. Tamamura Y, Iwamoto M. Rapid phenotypic changes in passaged articular chondrocyte subpopulations[J]. Clin Calcium, 2004,14(7): 15.
32. Davis MA, Ettinger WH, Neuhans JM, Hauck WW. The association of knee injury and obesity with unilateral and bilateral osteoarthritis of the knee. Am J Epidemiol, 1999, 130:278-288.
33．冯伟，石印玉，沈培芝．MTT法分析中药含药血清对体外软骨细胞增殖影响的研究[J]．上海中医药大学学报．2000，14(1)：43-45．
34．吕存贤，王维佳．膝骨关节炎病因病理研究及中医治疗概况[J]．浙江中医学院学报，2005，29(1)：82-84．
35．林木南，刘献祥．骨性关节炎中细胞因子的协同效应[J]．福建中医学院学报，2006，16(2)：69-71．
36．陈百强，张洪斌，张汉杰．生长因子对软骨细胞的作用[J]．中华骨科杂志．1999，19(2)：746-747．
37. Arner EC, Tortorella MD. Signal transduction through chondrocyte integrin receptors induces matrix metalloproteinase synthesis and synergizes with interleukin-1 [J]. Arthritis Rheum, 1995,38:1304.
38. Kanyama M, Kuboki T,Kojima S, et al. Matrix metalloproteinases andtessue inhibitors of metalloproteinases in synovial fluids of patients with temporomandibular joint osteoarthritis[J]. Orofac Pain,2000,14(1):20.
39．解志杰，许建中．关节软骨的结构和代谢与年龄相关的变化[J]．中华老年医学杂志．2000：19(5)：394-396．
40. Salvatierra J, Escames G, Hernandez P, et al. Cartilageand serum levels of nitric oxide inpatients with hip osteoarthritis[J]. Rheumatol,1999, 26(9):2015.
41．薛延．骨质疏松症诊断与治疗指南[M]．北京：科学出版社，1999年9月第一版，314-315．
42．邓廉夫，柴本甫，杨庆铭．骨关节炎滑膜细胞分泌肿瘤坏死因子生物学特征研究[J]．中华骨科杂志1999，19(12)：726-728．
43. Westacott TI, Atkins RM, Dieppe PA, et al. Tumor necrosis factor-α receptor expression on chondrocytes isolated from human articular cartilage. J Rheumatol, 1994, 21:1710-1715.
44. Homandberg GA, Umadi V, Kang H, et al. High molecular weight hyaluronan promotes of IL-1 beta damaged explants from both young and old bovines[J].Osteoarthritis Cartilage,2003,11(3): 177-186.
45．田华，娄思权，党耕町．肿瘤坏死因子-α导致软骨损伤的机制探讨[J]．中华外科杂志，2000，38(1)：76-77．
46. Arend WP, Dayer JM.Inhibition of the production and effects of IL-1 and TNF-α in rheumatoid arthritis [J]. Arthritis Rheum, 1995, 38:151-160.
47．吴蠡荪．自由基与临床医学[J]．微循环学杂志，1998，8：24-26．
48. Frederiks WM, Bosch KS. Localization of superoxide dismutase activity in rat tissue[J]. Free Radic Biol Med, 1997, 22:241-248.
49．孙材江，王慧，杨锡兰．退行性膝关节炎患者氧自由基代谢的观察[J]．中华骨科杂志．1992，12(6)：433-435．
50．许鹏，蔡乾坤．氧自由基对软骨代谢的影响[J]．中国矫形外科杂志，2000，7(10)：1008-1010．
51. Regan E, Flannelly J,Bowler R, et al.Ext racellular superoxide dismutase and oxidant damage in osteoarthritis [J].Arthritis Rheum, 2005,11:3479-3491.
52. Ostalowska A, Birkner E, Wiecha M, et al. Lipid peroxidation and antioxidant enzymes in synovial fluid of patients with primary and secondary osteoarthritis of the knee joint [J]. Osteoarthritis Cartilage, 2006, 2:139-145.
53．李松，曹峻岭，郝家骥．在培养的人胚软骨细胞中硒抗氧自由基损伤作 用观察[J]．中国国矫形外科杂志2000，7(4)：362-365．
54. Martin JA, Brown TD, Heiner AD, et al, Chondrocyte senescence,joint loading and osteoarthritis[J]. Olin Orthop Relat Res, 2004, 427(Suppl): 96-103.
55. Yudoh K, Nguyen T, Nakamura H, et al. Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function[J]. Arthritis Res Ther, 2005, 7: 380-391.
56．李松，黄石登，曹峻岭．自由基对体外培养人胚关节软骨细胞的影响[J]．中华骨科杂志．1998，18(7)：426-428．
57．马建兵，刘德玉，李堪印．中药对家兔实验性膝关节骨性关节炎氧自由基代谢的影响[J]．中医正骨．2000，12(1)：8-9．
58．王耶，刘建宇，宋艳．丹参和透明质酸钠注射液对骨性关节炎治疗作用的实验研究[J]．中国生化药物杂志．1998，19(5)：248-225．
59．谢林，周尊谦，孙达武．丹紫康冲剂对膝关节退行性骨关节病患者氧自由基代谢的影响[J]．中国中医骨伤科．1996，4(2)：81．
60. Stadler J, Stefanovic RacicM, billiard TR. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide[J]. J Immunol,1991, 147:3915-3920.
61. Grabowski PS, Wright PK. Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis[j]. Br J Rheumatol, 1997,36(6):651-655.
62．秦霞南，李金荣，龙星．一氧化氮合成酶抑制剂治疗骨关节病研究进展[J]．国外医学口腔医学分册，2004，3：188-190．
63. Pelletier JP, Martel-Pelletier J, Tardif G, et al. The increased synthesis of inducible nitric oxide inhibits IL-I rasynthesis by human articular chondrocytes: possible role in osteoarthritie cartilage degradation [J]. Ostoarthritis Cartilage,1996,4 (1):77-84.
64. Cao M, Evans CH, Stefanovic-Racia M,et al. Nitric oxide inhibits the synthesis of theⅡcollagen without alteringCoL2A1 mRNA abundance prolyl hydroxylase as a possible target[J]. Biochem J, 1997,324:305-310.
65. Task iran D, Stefanovic-RacicM, Georgescu H, et al. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1[J]. Biochem Biophys Res Commun,1994; 200:142-148.
66. Lotz M ,Blanco FJ ,von Kempis J ,et al. Cytokine regulation of chondrocyte function[J]. J Rheumatol, 1995, 43(1): 104-108.
67. George ACM, Daniel J, and Williams RJ. Nitric oxide activates metallopro tease enzymes in articular cartilage[J]. Biochem Biophys Res Commun, 1995,206:15-21.
68. Stadler J, Stefanovic-RacicM, Billiar TR, et al. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopo lysaccharide[J]. J Immunol, 1991, 147:3915-3920.
69. Loeser RF, Carlson CS,Del CarloM, et al. Detection ofnitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin—1β and with chondrocyte resistance to insulin—like growth factor—1 [J]. Arthritis Rheum, 2002, 46: 2349-2357.
70. Blanco F, Lotz M. IL-1 induced nitric oxide inhibits chondrocytes prolixferation via PGE2 [J]. FASEB J 1994, 8(1):365-371.
71. Stefanovic-Racic M, Stadlter J, Gerogesc H, et al. Nitricoxide and energy production in arthicular chondrocytes[J]. Cell Physiol, 1994, 159:274-280.
72. Kim H ,Lo M ,Pillarisetty R. Chondrocyte apopotosis following Intra articular fracture in humans[J]. Osteo Cart,2002,10 (9): 747-753.
73．许鹏，靳卫章，郭雄．骨关节病关节软骨细胞凋亡的研究进展[J]．实用老年医学．1999，13(6)：312-313．
74. Hashimoto S,Takahashi K, Amiel D,et al. Chondrocyte apoptosis and nitic oxide production during experimentlly induced osteoarthristis[J]. Arthritis Rheum, 1998,41(7): 1266-1274.
75. Blanco F J, Ochs RL, Schwarz H, et al. Chondrocyte apoptosis induced by nitix oxide[J] .Am JPathol, 1995,146(1):75-85.
76. Hashimoto S, Lotz M, Ochs RL, et al. Fas/FasL expression and induction of apoptosis in chondrocytes[J]. Arthritis Rheum, 1997,40(10): 1749-1755.