摘要
研究背景
关节软骨的损伤和退变是临床最常见的疾病之一。而关节软骨的自
我修复作用非常有限。因此，怎样促进关节软骨修复重建一直是骨科工
作者不断追求，渴望解决的难题。然而，无论是自体或异体软骨移植、
软骨细胞移植，或者三维立体细胞培养及组织工程技术的利用，都仅能
在早期生成透明和类透明软骨组织，且随后不可避免会发生退变。而这
些再生的软骨组织，无论是其结构、生物化学或者生物力学特性都与正
常透明软骨不同。因此怎样提高再生软骨质量和防止其发生退变就成为
急需解决的问题。同时，系列研究证实诱导型一氧化氮合酶抑制剂的应
用有助于炎性软骨代谢的恢复，而目前研究均着重于寻找使关节软骨缺
损修复的方法，很少去研究如何提高修复组织的质量以及如何防止发生
退变，更少探讨其原因。既然NO对软骨形成和基质合成有抑制作用，
能促进软骨的退变，那么进一步弄清NO作用于再生软骨退变过程的机
理，并且采用iNOS抑制剂等干涉手段，可望使修复组织不发生退变或
减漫退变，提高修复组织的质量，维护关节的功能。
目的和意义
（1）探讨各种一氧化氮合酶抑制剂对软骨细胞和软骨生物学特性的
影响，为动物试验提供理论基础；（2）建立关节软骨缺损模型，探讨一
氧化氮合酶抑制剂SMT对再生软骨的影响，为临床应用提供理论基础；
（3）探讨一氧化氮合酶抑制剂SMT对骨性关节炎的潜在治疗意义。
材料与方法
门）从成年新西兰大白兔膝关节无菌分离软骨细胞和软骨片，通过
MTT法检测 ILj 和 LPS以及 NOS抑制剂对软骨细胞毒性反应，确定
ILq和 LPS对软骨细胞合适的作用浓度。然后，以合适浓度 ILl 十LPS，
IL－16 ＋ LPS＋SMT，IL－16 ＋LPS＋L－NIL，IL－10＋LPS＋L－NMA分别作
用于软骨细胞和软骨，采用重氮化反应法测定NO释放量，NOS活性；
3咕掺入法检测蛋白多糖合成；原位杂交检测软骨细胞 iNOSmRNA表达；
RT－PCR检测软骨组织 eOS和 MMPgmRNA的表达以评价不同 NOS抑
制剂对软骨细胞和软骨代谢的影响。
（2）ZI只雄性新西兰大白兔随机均分为正常组（不使用任何干预
药物人对照组（皮下注射生理盐水后给予膝关节内注射 IL刁 卜g·ml”‘
和 LPS 20ug·ml一各 0．lml）；SMT组（将 SMT sing·kgJ，l次·12h1经皮
下注射，持续 24小时后，膝关节内注射相同浓度 IL1时0 LPS混合液人
8小时后检测关节软骨、滑膜和滑液NO释放量和NOS活性，48小时后
测定软骨蛋白多糖合成和oOSInRNA表达。初步确定NOS抑制剂SMT
对炎性因子刺激的关节软骨代谢的影响，以及给药的浓度和方式。通过
聚丙烯酷胺凝胶电泳鉴定AsMPZ以及用肌袋埋植法检测其成骨活性。
（3）将66只雄性新西兰兔，随机均分为正常对照组（双侧膝关节
软骨缺损不充填任何物质及用药物干涉）；BMP组（缺损应用BMP纤维
蛋白凝胶复合物充填）；SMT组（术前给予sing上g－’·12h”’皮下注射SMT，
缺损应用BMP纤维蛋白凝胶复合物充填）。根据观察时间不同，治疗时
间为8周、16周和1年。按观察时间处死动物，分别作组织形态学、组
织化学、原位杂交和RTPCR观察SMT对修复组织质量的影响；3咕掺
入检测修复组织蛋白多糖合成。
（4）无菌获取OA患者的关节软骨和滑膜，分为3组。对照组不
加药物干预；L一NIL组力入 1。l，L”’L一NIL干预；SW组力入 1。OI·L－‘
SMT干预。孵育刀小时后，通过检测硝酸盐和亚硝酸盐的含量来观察
软骨NO的释放量以及WOS的活性；原位杂交检测软骨iNOSmRNA和
MMPglnRNA的表达。孵育 10天后，化学比色法观察软骨蛋臼多糖含量
和胶原释放量的变化。
．4．
结果分析
门）＊TI’法显示，软骨细胞分别与2＊g·m＊uS走lllg、Ml1卜lp
以及0《 浓度的NOS抑制剂共同培养72小时，未造成明显的毒
性反应。…gthl”‘外源性ILl和0上屹·m1“‘L陀可明显促进培养软骨细
胞和软骨 NO释放，诱导 iNOSmANA表达，增加 NOS活性以及抑制软
骨基质蛋白多糖的合成。当 LPS浓度为 5－20pg·ml”时 NO释放量最为显
著：而加入100《000叩＊＊OS抑制剂均能剂量依赖性抑制炎性因子诱
发的软骨细胞和软骨NO释放和NOS活性的增加；lmmol浓度的NOS
抑制剂可显著逆转软骨细胞和软骨蛋白多糖合成抑制以及抑制oOS和
MMPg基因的表达。
Q）动物实验显示lpg·ml－‘1卜lp和10叱·m卜＊＊S关节内注射后8
小时，能显著增加在体软骨、滑膜和滑液中硝酸盐和亚硝酸盐的含量，
增高NOS活性，以软骨对刺激因子的反应最敏感。注射后48小时，软
骨 3咕 摄入量被显著抑制，yOSmRNA 表达明显上调，而
sing·kg－‘·12h－‘SMT皮下注射能明显逆转其对软骨蛋白多糖合成的抑制，
下调ffoOSlllJLrvA的表达。聚丙烯酞胺凝胶电泳显示thBMPZ获得表达；
成骨活性表明thBMPZ植入1周后，出现软骨组织；2周后，新骨组织形
成；4周后，新骨成熟。
（3）软骨缺损动物模型显示；术后8周、16周和1年后，形态?
BACKGROUND
Articular cartilage damage and degradation are the most common diseases in clinic, but the self-repaired ability of articular cartilage is limited. How could restore cartilage defects is still the problem of orthopedic doctors. No matter what applying to autograft and allograft, or chondrocytes graft and tissue engineer culture, the defect could be filled with para-hyaline cartilage or hyaline cartilage in the early stage that subsequently could be fibrous degeneration ineluctability. It is important to promote repair tissues quality and retard its degeneration; because these repair tissues have different structure, biochemical and biomechanics characteristic. A serial of studies showed NOS inhibitors were benefit for arthritis. At present, Studies emphasize to the skill of cartilage defect repair, so that it is inadequate to promote the quality of repair tissues and retard its degeneration. Now that nitric oxide could increase cartilage matrix synthesis, NOS inhibitors were applied for cartilage repair would mediator repair tissue degradation and ameliorate its quality.
OBJECTIVE
( 1 ) To explore the effects of NOS inhibitors for chondrocytes and cartilage entities, and offer bases of animal model study. (2) To discuss the effects of NOS inhibitors for the cartilage repair tissue. (3) To study the
clinical potential effects of NOS inhibitors in OA.
METHODS
( 1) Articular entities and chondrocytes taken from mature New Zealand white rabbits, the toxicity of IL-lp and LPS and NOS inhibitor on chondrocytes were measured through MTT. Then the experiment were performed in five groups: control group; IL-lp+LPS group; IL-lp+LPS + SMT group; IL-lp+LPS + L-NIL group; IL-lp + LPS + L-NMA group. The release of NO and the activity of NOS in rabbit chondrocytes and cartilage entities were measured by Griess reaction and spectrophotometric methods. The proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate Na235S04. iNOS and MMP9mRNA expression were measured by in situ hybridization and RT-PCR.
(2) Twenty-one New Zealand white rabbits were divided into normal group, the control and SMT group. The normal group did not use any drug interfere, the control and SMT groups were given normal saline and SMT respectively by a single hypodermic injection after IL-lp and LPS were administered by interarticular injection. The NO release and the activity of NOS were measured by Griess reaction and spectrophotometric methods in 8h. The proteoglycan synthesis and iNOSmRNA expression was assessed by incorporation of radiolabelled sodium sulphate Na235SO4 and in situ hybridization respectively after 48h. Through polyacrylamide and mouse muscle bag model, the bioactivity of rhBMP-2 protein was identified.
(3) Full-thickness defects of cartilage were created in the trochlearn groove of sixty-six adult New Zealand White rabbits. Forty-four defects were empty, Forty-four were filled with a fibrin glue impregnated with rhBMP, and Forty-four were filled with a fibrin glue impregnated with rhBMP and hypodermic injection with Smg-kg'Mltf1 SMT. The animals were killed at eight, sixteen weeks and one-year postoperatively, and the gross appearance of the healed defect was assessed. The repair tissue was examined histologically and was evaluated according to a grading scale. The tissue
sections were immunostained with antibodies against type-II collagen and type- I collagen, RT-PCR examines the expreesion of iNOSniRNA, MMPsmRNA and type- II collagen.
(4) 17 specimens of articular cartilage were taken from osteoarthrosis patients. The experiments were performed in three groups: control group and L-NIL.and SMT intervenient group. The release of NO and the activity of NOS on OA cartilage were measured by Griess reaction and spectrophotometric methods after 72h culture. iNOSmRNA and MMPg expression was measured by in situ hybridization. Proteoglycan and hydroxyproline in OA cartilage was measured after lOd culture.
RESULTS
( 1 ) chondrocytes incubated in vitro for 72h in presen
引文
1． Wambach BA, Cheung H, Josephson GD, et al. artilage tissue engineering using thyroid chondrocytes on a type I collagen matrix. Laryngoscope, 2000; 110:2008-2011
2． Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials, 2000; 21:2589-2598
3． Ochi M, Uchio Y, Tobita M, et al. Current concepts in tissue engineering technique for repair of cartilage defect. Artif Organs, 2001; 25:172-179
4． Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone and Joint Surg, 1994; 76-A: 579-592
5． Stefanovic-Racic M, Stadler J, Evans CH. Nitric oxide and arthritis. Arthr Rherm, 1993; 36:1036-1044
6． Park CS, Krishna G, Ahn MS, et al. Differential and constitutive expression of neuronal, inducible, and endothelial nitric oxide synthase mRNAs and proteins in pathologically normal human tissues. Nitric Oxide, 2000; 4:459-471
7． Lotz M. The role of nitric oxide in articular cartilage damage. Rheum Dis Clin North Am, 1999; 25:269-282
8． Jang D, Murrekk, GAC. Nitric oxide in arthritis. Free Radic Biol Med, 1998;4: 1511-1519
9． Homandberg GA, Davis G, Maniglia C, et al. Cartilage chondrolysis by fibronectin fragments carses cleavage of aggrecan at the same site as found in osteoarthritic cartilage. Osteoarthritis Cartilage, 1997; 5:450-453
10． Lee DA, Noguchi T, Frean SP, et al. The influence of mechanical loading on isolated chondrocytes seeded in agarose constructs. Biorheology, 2000; 37: 149-161
11． 钟慈声，孙安阳主编．一氧化氮的生物医学．第一版．上海：上海医 科大学出版社，1997；784-285
12． Ramirez MM, Quardt SM, Kim D, et al. Platelet activating factor modulates microvascular permeability through nitric oxide synthesis. Microvasc Res, 1995; 50:223-234
13． Amin AR, Abramson SB. The role of nitric oxide in articular cartilage breakdown in osteoarthritis. Curr Opin Rheumatol, 1998; 10:263-268
14． Lo YY, Conquer JA, Grinstein S, et al. Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species. J Cell Biochem, 1998; 69: 19-29
15． Attur MG, Dave M, Cipolletta C, et al. Reversal of autocrine and paracrine effects of interleukin 1 (IL-1) in human arthritis by type II IL-1 decoy receptor. Potential for pharmacological intervention. J Biol Chem, 2000; 275:40307-40315．
16． Studer RK, Georgescu HI, Miller LA, et al. Inhibition of transformation growth factor βp production by nitric oxide-treated chondrocytes: Implication for matrix synthesis. Arthr Rheum, 1999; 42:248-257
17． Hauselmann HJ, Stefanovic-Racic M, Michel BA, et al. Differences in nitric oxide production by superficial and deep human articular chondrocytes: implications for proteoglycan turnover in inflammatory joint diseases. J Immunol, 1998;160:1444-1448
18． Cao M, Larson AW, Niyibizi C, et al. Nitric oxide inhibits the synthesis of type-II collagen without altering CoI2A1 mRNA abundance: prolyl hydroxylase as a possible target. Biochem J, 1997; 324:305-310
19． Callejas NA, Casado M, Diaz-Guerra MJ, et al. Expression of cyclooxygenase-2 promotes the release of matrix metalloproteinase-2 and-9 in fetal rat hepatocytes. Hepatology. 2001; 33: 860-7．
20． Pelletier JP, Lascau CV, Jovanovic D, et al. Selective inhibition of inducible nitric oxide synthase in experimental osteoarthritis is associated with reduction in tissue levels of catabolic factors. J Rheumatol, 1999;
26:2002-2014
21． Lane Smith R, Trindade MC, Ikenoue T, et al. Effects of shear stress on articular chondrocyte metabolism. Biorheology, 2000; 37: 95-107
22． Vant Hof RJ, Hocking L, Wright PK, et al. Nitric oxide is a mediator of apopto is in the rheumatoid joint. Rheumatology (Oxford), 2000; 39:1004-1008
23． Kondo S, Ishiguro N, Iwata H, et al. The effects of nitric oxide on chondrocytes and lymphocytes. Biochem Biophys Res Commun, 1993; 97:1431-1437
24． Hashimoto S, Ochs RL, Lotz M. The linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rhium, 1998; 41:1266-1274
25． Stefanovic-Racic M, Morales TL, Taskiran D, et al. The role of nitric oxide in proteoglycan turnover by bovine articular cartilge organ cultures. J Immunol, 1996; 137:3729-3737
26． McCartney-Franncis N, Allen JB, Mizel SB, et al. Suppprission of arthritis by an inhibitor of nitric oxide synthase. J Exp Med, 1993; 178:749-754
27． Presle N, Cipolletta C, Jouzeau JY, et al. Cartilage protection by nitric oxide synthase inhibitors after intraarticular injection of interleukin-β in rats. Arthr Rheum, 1999; 42:2094-2102
28． Moore WM, Webber RK, Jerome GM, et al. L-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase. J Med Chem, 1994; 37:3886-3888
29． Pelletier J, Jovanovic D, Fernandes JC, et al. Reduction in the structural changes of experimental osteoarthritis by a nitric oxide inhibitor. Osteoarthritis Cartilage, 1999; 7:416-418
30． Van den Berg WB, Van de Loo F, Joosten LA, et al. Animal models of arthritis in NOS2-deficient mice. Osteoarthritis Cartilage, 1999; 7:413-415
31． Consgance RC, Miyasaka N., Hirata Y. Nitric oxide and inflammatory
arthritides. Life-Sci, 1997; 61: 2073-2081
32、 Grabowski PS; Macpherson H; Ralston SH. Nitric oxide production in cells derived from the human joint. Br J Rheumatol, 1996; 35:207-212
33、 Jouzeau JY, Cipolletta C, Preste N, et al. Modulation of IL-1 effects on cartilage by NO synthase inhibitors: pharmacological studies in rat. Osteoarthritis Cartilage, 1999; 7:382-385
34、 Cipolletta C, Jouzeau JY, Gegout-Pottie P, Presle N, et al. Modulation of IL-1 induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities. Br J Pharmacol, 1998; 124:1719-1727
35、 Konturek JW, Fischer H, Gromotka PM, et al. Endogenous nitric oxide in the regulation of gastrc secretory and motor activity in humans. Aliment Pharmacol Ther, 1999; 13:1683-1691
36、 Sato H, Zgao ZQ, Vinten-Johansen J. L-arginine inhibits neutrophil adherence and coronary artery dysfunsion. Cardiovasc Res, 1996; 31:63-72
37、 Heusch G, Post H, Michel MC, et al. Endogenous nitric oxide and myocardial adaptation to ischemia. Circ Res, 2000; 87:146-152
38、 Studer RK, Levicoff E, Georgescu H, et al. Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation. Am J Physiol Cell Physiol, 2000; 279:C961-9
39、 刘建国，王宪，陈明哲。一氧化氮和细胞因子之间的相互调节作用。 生理科学进展，2000；31：61-64
40、 Wink DA, Mitchell JB. Chemical biology of nitric oxide:insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biology & medicine, 1998; 25:434-456
41、 Studer R, Jaffurs D, Stefanovic RM, et al. Nitric oxide in osteoarthritis. Osteoarthritis Cartilage, 1999; 7:377-379
42、 Jauernig S, Schweighauser A, Reist M, et al. The effects of doxycycline on nitric oxide and stromelysin production in dogs with cranial cruciate ligament rupture. Vet Surg, 2001; 30:132-9
43、 McCarty MF, Russell AL. Niacinamide therapy for osteoarthritis-does it inhibit nitric oxide synthase induction by interleukin 1 in chondrocytes? Med Hypotheses, 1999; 53:350-60
44、 徐云根，罗穗，华维一。诱生型一氧化氮合酶抑制剂的研究。药学 进展，2000；24：137-141
45、 Frey C, Narayanan K, Mcmillan K, et al. L-thiocitrulline. A stereospicific, heme-binding inhibitor of nitric oxide synthases. J Biol Chem, 1994; 269:26083-26091
46、 Cohen RI, Shapir Y, Davis A, et al. Comparison between selective and nonselective nitric oxide synthase inhibition and phenylephrine in normal and endotoxic swine. Crit Care Med, 2000; 28:3257-3267
47、 Patel RN, Attur MG, Dave MN, et al. A novel mechanism of action of chemically modified tetracyclines: inhibition of COX-2-mediated prostaglandin E2 production. J Immunol, 1999; 163:3459-3467
48、 Johnson K; Jung A; Murphy A, et al. Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization. Arthritis-Rheum, 2000; 43:1560-70
49、 Perka C, Settinger M, Schultz O, et al. Tissue engineered cartilage repair using cryoproserved ans noncryopriserved chondrocytes. Clin Orthop, 2000; 378:245-254
50、 Mixon AJ, Fortier LA, Williams J, et al. Enhanced repair of extensive articular defects by insulin-like growth facter-I-Iaden fibrin composites. J Orthop Res, 1999; 17:475-487
51、 Fujimoto E, Ochi M, Kato Y, et al. Beneficial effect of basic fibroblast growth factor on the repair of full thickness defects in rabbit articular cartlage. Arch Orthop Trauma Surg, 1999; 119:139-145
52、 Nakajima H, Goto T, Horikawa O, et al. Characterization of the cells in the repair tissue of full-thickness articular cartilage defects. Histochem Cell Biol, 1998; 109:331-338
53、 Sellers RS, Peluso D, Morris EA, et al. The effect of recombinant
human bone morphogenetic protein 2(rhBMP2) on the healing of full-thickness defects of artivular cartilage. J Bone Jont Surg Am, 1997; 79:1452-1463
54、 Maddoc E, Zhan M, Mundy GR, et al. Optimizing human demineralized bone matrix for clinical application. Tissue Eng, 2000; 6:441-448
55、 Blanquaert F, Barritault D, Caruelle JP. Effects of heparin-like polymers associate with growth factors on osteoblast proliferation and phenotype expression. J Biomed Mater Res, 1999; 44:63-72
56、 Okubo Y, Bessho K, Fujimura K, et al. Effect of Elcatonin on osteoinduction by recombinant human bone morphogenetic protein-2． Biochem Biophus Res Commun, 2000; 269:317-321
57、 Kessler S, Kastler S, Mayr-wohlfart U, et al. Stimulation of primary osteoblast cultures with rh-TGF-beta, rh-bFGF, rh-BMP2 and rx-BMP4 in an in vitro model. Orthopade, 2000; 29:107-111
58、 Barboza E, Caula A, Machado F, et al. Potential of recombinant human bone morphogenetic protein-2 in bone regeneration. Implant Dent, 1999; 8:360-367
59、 Tatsuyama K, Maezawa Y, Baba H, et al. Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone. Eur J Histochem, 2000; 44:269-278．
60、 Winn SR, Uludag H, Hollinger JO. Carrier systems for bone morphogenetic proteins. Clin Orthop, 1999; 367suppl:s95-106
61、 Peretti GM, Randolph MA, Villa MT, et al. Cell-based tissue-engineered allogeneic implant for cartilage repair. Tissue Eng, 2000; 6:567-676
62、 Silverman RP, Bonasser L, Passaretti D, et al. Adhesion of tissue-engineered cartilate to native cartilage. Plast Reconstr Surg, 2000; 105:1393-1398
63、 Shirazi I, Yaron I, Wollman Y, et al. Down regulation of interleukin 1beta production in human osteoarthritic synovial tissue and cartilage
cultures by aminoguanidine. Ann Rheum Dis, 2001; 60:391-394．
64 McCartney-Francis NL, Song X, Mizel DE, et al. Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease. J Immunol,2001; 166:2734-2740
65、 Peretti GM, Bonassar LJ, Caruso EM, et al. Biomechanical analysis of a chondrocyte-based repair model of articular cartilage. Tissue Eng, 1999; 5:317-326
66、 Van der Kraan PM, Vitters EL, van Beuningen HM, et al. Role of nitric oxide in the inhibition of BMP-2-mediated stimulation of proteoglycan synthesis in articular cartilage. Osteoarthritis Cartilage, 2000; 8:82-86
67、 Spreng D, Sigrist N, Jungi T, et al. Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture. Am J Vet Res, 2000; 61:530-536
68、 Moos V, Rudwaleit M, Herzog V, et al. Association of genotypes affecting the expression of interleukin-lbeta or interleukin-1 receptor antagonist with osteoarthritis. Arthritis Rheum, 2000; 43:2417-2422．
69、 Schiff MH. Role of interleukin 1 and interleukin 1 receptor antagonist in the mediation of rheumatoid arthritis. Ann Rheum Dis, 2000; 59 Suppl 1:i103-108．
70、 Shapiro F, Koide S, Glimeger MJ, et al. Cell origin and differeatiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg(Am), 1993 ;75:532-533
71、 Gilbert JE. Current treatment option for the restoration of articular cartilage. Am J Knee Surg, 1998; 11:42-46
72、 Brittberg M, Nisson A, Lindabl A, et al. Rabbit articular cartilage defects treated with artologous cultured chondrocytes. Clin Orthop, 1996; 326:270-283
73、 Sellers RS, Zhang R, Glasson SS, et al. Repair of articular cartilage defects one year after treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2) . J Bone Joint Surg Am, 2000;
82:151-60.
74、 Sellers RS, Peluso D, Morris EA. The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage. J Bone Joint Surg Am, 1997; 79:1452-63.
75、 Peretti GM, Randolph MA, Villa MT, et al. Cell-based tissue-engineered allogeneic implant for cartilage repair. Tissue Eng, 2000; 6:567-76
76、董启榕，郑祖根，周海斌等。兔膝关节软骨缺损修复的初步研究。苏州医学院学报，2000；20：335-337
77、 Duda GN, Haisch A, Endres M, et al. Mechanical quality of tissue engineered cartilage: results after 6 and 12 weeks in vivo. J Biomed Mater Res, 2000; 53:673-677
78、 Jin G, Sah RL, Li YS, et al. Biomechanical regulation of matrix metalloproteinase-9 in cultured chondrocytes. J Orthop Res, 2000; 18: 899-908.
79、 Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes. Arthritis Rheum, 2001; 44:585-94.
80、褚大由。骨关节炎的治疗现状。中国矫形外科杂志，2000；7：693-695
81、 Keisu KS, Orozco F, Sharkey PF, et al. Primary cementless total hip arthroplasty in octogenarians. Two to eleven-year follow-up. J Bone Joint Surg Am, 2001; 83-A: 359-363
82、 Robertsson O, Knutson K, Lewold S, et al. The routine of surgical management reduces failure after unicompartmental knee arthroplasty. J Bone Joint Surg Br, 2001; 83:45-49.
83、 Honda S, Migita K, Hirai Y, et al. Induction of COX-2 expression by nitric oxide in rheumatoid synovial cells. Biochem Biophys Res Commun, 2000; 268:928-931.
84、 Crabowski PS; Macpherson H; Ralston SH. Nitric oxide production in cells derived from the human joint. Br J Rheumatol 1996; 35:207-212
85、Spreng D, Sigrist N, Jungi T, et al. Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture. Am J Vet Res, 2000; 61:530-536
86、Shirazi I, Yaron I, Wollman Y, et al. Down regulation of interleukin lbeta production in human osteoarthritic synovial tissue and cartilage cultures by aminoguanidine. Ann Rheum Dis, 2001; 60:391-4
87、方福德等。现代医学实验技巧全书(上册)。北京医科大学，协和医科大学联合出版社，1995；第一版，551页。
88、Miyasaka N, Hirata H. Nitric oxide and inflammatory arthritides. Life Sci, 1997;61:2073-2076
89、许鹏，郭雄。一氧化氮对骨关节病软骨退变影响的研究进展。中国矫形外科杂志，1999；6：705-706
90、Pugliese G, Pricci F, Leto G, et al. The diabetic milieu modulates the advanced glycation end product-receptor complex in the mesangium by inducing or upregulating galectin-3 expression. Diabetes, 2000; 49:1249-1257
91、Bahrami S, Fitzal F, Peichl G, et al. Protection against endotoxemia in rats by a novel tetrahydrobiopterin analogue. Shock, 2000; 13:386-391