摘要
第一部分甘草多糖的提取及质量分析
目的：从甘草生药中提取精甘草多糖。并对提取的甘草多糖进行质量分析。
方法：将甘草生药粉碎成粉末状,利用离心法除去水中不溶物、Sevag法除去游离蛋白质、活性炭吸附色素。并从已提取的精甘草多糖中检测残余蛋白质含量,计算换算因子、回收率、甘草生药中多糖的含量,对已提取的甘草多糖进行质量分析鉴定。
结果：甘草多糖提取品经Sevag法除蛋白质后,已提取精甘草多糖中游离蛋白质成分已基本除尽。计算甘草多糖回收率为98.59%,换算因子f=1.584,样品中多糖平均含量为2.33%
结论：所提取甘草多糖最终产物为灰白色固体,Sevag法可将甘草多糖中的游离蛋白质成分基本除尽。本实验提取甘草多糖方法可靠。
第二部分甘草多糖对离体大鼠软骨细胞GAG合成和GlcAT-1mRNA表达的影响
目的：研究不同浓度甘草多糖(Glycyrrhiza polysaccharides, GP)对大鼠退变软骨细胞糖胺多糖(Glycosaminoglycan, GAG)合成和细胞内葡萄糖醛酸转移酶Ⅰ(galactose-β-1,3-glucuronosyltransferase Ⅰ, GlcAT-1) mRNA表达的影响及其可能的作用机制。
方法：分离大鼠关节软骨细胞进行传代培养并加入IL-1p造模,分别将浓度为0.1mg/L、l mg/L、10mg/L、100mg/L甘草多糖作用于该软骨细胞,以4.5mg/L的硫酸氨基葡萄糖(Glucosamine)作为阳性对照。倒置显微镜下观察各组细胞形态学变化,其后进行细胞增殖、细胞内GAG含量、GlcAT-lmRNA表达的检测。
结果：各浓度的甘草多糖对正常大鼠软骨细胞的增殖活性均有促进作用；不同浓度甘草多糖组均呈现出促进GAG合成的趋势,且甘草多糖100mg/L浓度组表现出最强的促进GAG合成能力(P<0.01)；各浓度的甘草多糖未能显著增加GlcAT-lmRNA的表达,但与模型对照组比较有显著差异。
结论：甘草多糖对离体大鼠软骨细胞增殖和GAG合成均有促进作用。甘草多糖对离体大鼠软骨细胞增殖和GAG合成、GlcAT-1mRNA表达水平的影响的机制可能是通过提供底物,增加底物浓度,直接促进GAG的合成；通过促进关键酶GlcAT-I的表达,提高GAG的生成效率来实现的。
第三部分关节腔内注射甘草多糖对大鼠骨关节炎软骨的保护作用及其作用机制
目的：研究关节腔内注射不同浓度甘草多糖(Glycyrrhiza polysaccharides)对大鼠骨关节炎软骨细胞的保护作用及其可能的作用机制。
方法：将70只Wistar大鼠随机分成A、B、C1、C2、C3、C4、D七组。B、C1、C2、C3、C4、D六组大鼠分别于实验第1、4、7日向双膝关节腔内注射4%的木瓜蛋白酶溶液0.2ml,建立OA模型,A组大鼠双膝于上述时间点注射等量生理盐水,作为正常对照。于实验的第10日开始向C1、C2、C3、C4四组大鼠的双膝关节腔内分别注射0.1mg/L、1mg/L、10mg/L、100mg/L的甘草多糖溶液,D组注射4.5mg/L硫酸氨基葡萄糖,每周注射两次,连续5周；A、B两组大鼠的双膝关节腔内于上述时间点注射等量生理盐水。试验后第6周后处死所有大鼠,根据大体观察评分标准在肉眼及显微镜观察关节软骨形态学变化并评分；将关节软骨制作石蜡切片并分别用甲苯胺蓝染色和HE染色,依据关节软骨Mankin's评分标准评分；然后检测血清和关节滑膜中MDA含量,血清中SOD活性；测定关节软骨中GAG含量。
结果：大体观察及Mankin's评分结果提示甘草多糖对骨关节炎关节软骨的形态结构有明显改善,且lOmg/L甘草多糖溶液组改善更为明显,与阳性对照组相当。各浓度甘草多糖组的SOD活性均较模型对照组显著增加,甘草多糖不同浓度组与阳性对照组比较无显著差异。各浓度甘草多糖组关节滑膜中MDA含量均较模型对照组显著降低,且0.1mg/L、1mg/L甘草多糖组关节滑膜中MDA含量与阳性对照组仍存在差异(p<0.01),10mg/L、100mg/L甘草多糖组关节滑膜中MDA含量与阳性对照相比较无显著差别。各浓度甘草多糖组均能增加关节软骨中GAG的合成(均P<0.01),且0.1mg/L、1mg/L甘草多糖组促GAG合成作用与阳性对照组仍存在差异(p<0.01),lOmg/L、100mg/L甘草多糖组促GAG合成作用与阳性对照相比较无显著差别。
结论：大鼠关节腔内注射木瓜蛋白酶建立OA模型简便快捷,可重复性和稳定性好,相关并发症少,可导致类似于临床骨关节炎的病理改变。甘草多糖对骨关节炎软骨具有一定的保护作用,且10mg/L的甘草多糖溶液对软骨保护作用最强。甘草多糖对骨关节炎软骨的保护作用机制可能是通过抑制氧自由基对软骨细胞的损害；促进软骨细胞外GAG的合成,促进软骨基质的生成来实现的。
Part1:The extraction and quality analysis of Glycyrrhiza polysaccharides
Objective:To extact the polysaccharides form crude glycyrrhiza, and take quality analysis for the Glycyrrhiza polysaccharides.
Methods:Crude glycyrrhiza was crumbled into powder, centrifugation method was used to remove the insoluble material, Sevag method was used to remove free protein, pigment in glycyrrhiza polysaccharides was adsorpted by activated carbon. And then detected residual protein content of the glycyrrhiza polysaccharides that has been extracted, calculated the conversion factor, recovery rate, and the content of glycyrrhiza polysaccharides in the crude glycyrrhiza, analysis the quality of glycyrrhiza polysaccharides that has been extracted.
Results:The conversion factor was1.584, recovery rate was98.59%, the content of glycyrrhiza polysaccharides in the crude glycyrrhiza was2.33%.
Conclusion:The extracted glycyrrhiza polysaccharides was hoar solid. The protein in glycyrrhiza was almost removed by Sevag method.
Part2:The effects and mechanism of glycyrrhiza polysaccharides on GAG synthesis and expression of GIcAT-1mRNA in rat chondrocytes in vitro
Objective:To investigate the effect and mechanism of different concentration of glycyrrhiza polysaccharides on GAG synthesis and expression of GlcAT-1mRNA in rat osteoarthritic chondrocytes in vitro.
Methods:Chondrocytes isolated from knee and shoulder joints cartilage of rats were cultured. After monolayers were established, chondrocytes induced by IL-1β were treated with glycyrrhiza polysaccharides of0.1、1、10、100mg/L respectively.4.5mg/L Glucosamine was used as the positive control. The effects on chondrocytes were analyzed using MTT assay for chondrocyte proliferation and1,9-dimethylmethylene blue assay with purified shark chondroitin sulphate (CS) as a standard for GAG content, mRNA expression of GlcAT-1by rat chondrocytes was analyzed using a quantitative multistandard RT-PCR.
Results:Glycyrrhiza polysaccharides promoted normal chondrocyte proliferation in all observed concentrations. Glycyrrhiza polysaccharides up-regulated GAG synthesis in all observed concentrations, with the concentration of0.1mg/L was the most effective (P<0.01). All concentrations of glycyrrhiza polysaccharides didn't enhance the expression of GlcAT-1, but we observed all concentrations of glycyrrhiza polysaccharides groups had a significantly different compared with model control group.
Conclusion:The effects and mechanism of glycyrrhiza polysaccharides that promoted normal chondrocyte proliferation, up-regulated GAG synthesis of rat chondrocytes induced by IL-1β in vitro was probably because of the increase of substrate concentration. And also the enhancing of the expression of GlcAT-1probably improve the efficiency of the GAG synthesis.
Part3:The protective effect and mechanism of glycyrrhiza polysaccharides in rat osteoarthritic articular cartilage in vivo
Objective:To investigate The protective effect and mechanism of glycyrrhiza polysaccharides in rat osteoarthritic articular cartilage in vivo.
Methods:70Wistar rats were randomly divided into A、B、C1、C2、C3、C4、D groups. Group A was normal control group. Group D was4.5mg/L Glucosamine used as the positive control. OA models of rats were established by0.2ml intra-articular injections of4%papain solution into both knees in group B、C1、C2、C3、C4、D at the1st,4th and7th day. The knees of rats in group C1、C2、C3、C4respectively received 0.2ml intra-articular injections of glycyrrhiza polysaccharides at concentrations of0.1、1、10、100mg/L at the10th day, group D was given4.5mg/L Glucosamine, group A and B were given physiological saline at the same time, twice weekly for five weeks. Six weeks later, all rats were sacrificed. Observed the morphological changes of articular cartilage both in naked eye and under microscopy, graded according to the general observed score standard. Made articular cartilage into paraffins sections, and deal with toluidine blue staining and HE staining, graded according to the Mankin's score standard. And detected the content of MDA in serum and synovial, the activity of SOD in serum, and the content of GAG in articular cartilage.
Results:The results of both general observed score and Mankin's score shown that the morphological of articular cartilage in glycyrrhiza polysaccharides groups were obviously improved, particularly in l0mg/L group which has no diffrernce compare with group D. The activity of SOD in serum in all glycyrrhiza polysaccharides groups and group D were obviously increased compare with the model group. The content of MDA in all glycyrrhiza polysaccharides groups and group D were obviously reduced compare with the model group. But the effects in C1、C2still has difference compare with group D(p<0.01). The content of GAG in all glycyrrhiza polysaccharides groups and group D were obviously increased compare with the model group, and the effects in C3、C4has no difference compare with group D.
Conclusion:Intra-articular injection of papain in rats to establish OA model was simple and quick, repeatability and stability,with less related complications, which could lead to pathological changes similar to clinical OA. The glycyrrhiza polysaccharides had a protective effect, and10mg/L group shown the strongest protective effect on articular cartilage. The mechanism of protective effect on OA cartilage was probably because of the glycyrrhiza polysaccharides could promote the GAG synthesis, and then promote the generation of cartilage matrix by inhibiting oxygen free radical damage to the cartilage cells.
引文
[1]Jean-Noel Gouze, Karim Bordji, Sandrine Gulberti. Interleukin-lbate Down Regulates the Expression of Glucuronosyltransferase Ⅰ, a key Enzyme Priming Glycosaminoglycan Biosynthesis. Arthritis Rheum.2001, February, Vol.44, NO.2 pp.351-360.
[2]刘满红,王如阳.植物活性多糖清除羟自由基的抗氧化性能研究.云南中医中药杂志,2009,30(6)：57—58.
[3]李晓明.百药的调解员：甘草.健康天地,2007,2：29.
[4]邓伟.甘草附子汤治疗膝骨关节炎的临床研究.中药材,2008,31(7)：1107-1110.
[5]方积年.多糖的分离纯化及其纯度鉴别与分子量测定.药学通报,1984,19(10)：46-49.
[6]田庚元,冯宇澄.多糖类免疫调节剂的研究和应用.化学进展,1994,6(2)：114-124.
[7]张翼伸.有关糖复合物的分级纯化结构确定、生物活性的几个问题.生命的化学,1994,14(6)：42-44.
[8]徐惠,于志洁,苏富强,等.螺旋藻粘多糖的分离及其免疫学作用[J].中国生化药物杂志,1997,18(2)：72-75.
[9]程安玮,金征宇,万发春.甘草多糖对小鼠腹腔巨噬细胞化学成分及胞内酶的影响.食品与生物技术学报,2008,27(1)：76—79.
[10]刘霞,谢建新,李艳等.甘草多糖免疫功能研究.中国公共卫生,2004,20(5)：572—573.
[11]郑尧,何景华,高建华等.甘草多糖对小鼠巨噬细胞吞噬功能的影响.中医药学刊,2003,21(2)：254—255.
[12]李发胜,赵珏,池晓峰等.甘草多糖对小鼠免疫调节作用的影响.中国中医药信息杂志,2009,16(6)：35—36.
[13]王忱,谢广茹,史玉荣等.甘草多糖的体内抑瘤作用及其机制的研究.临床肿瘤学杂志,2003,8(2)：85—87.
[14]王岳五,史玉荣,张海波,等.甘草残渣中多糖GPS抗肿瘤作用的研究.南开大学学报,2000,33(4)：46-48.
[15]常雅萍,毕无邪,杨贵贞.甘草多糖抗病毒作用研究.中国中药杂志,1989,14(4)：44-46.
[16]王岳五,史玉荣,张海波等.甘草多糖GPS对病毒的抑制作用.南开大学学报, 2001,34(2)：126-128.
[17]杨玲,汪河滨,罗锋.甘草多糖清除自由基活性研究,塔里木大学学报,2007,19(1)：1-3.
[1]Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum.1998, Aug,41(8):1343-55.
[2]Rob inson D, A sh H, Yayon A, e t al. Characteristics of cartilageb iopsies used for autologou s chondrocytes transp Ian tat ion.Cell Tran sp lant,2001,10:203-208.
[3]Gouze JN, Bianchi A. Glucosamine modulates IL-l-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-κB pathway. FEBS Lett.2002 Jan 16; 510(3):166-70.
[4]Jean Noel Gouze, Karim Bordji, Sandrine Gulberti. Interleukin-l beta Down Regulates the Expression of Glucuronosyltransferase I,a key Enzyme Priming Glycosaminoglycan Biosynthesis. Arthritis Rheum.2001, February, Vol.44, NO.2 pp.351-360.
[5]刘满红,王如阳.植物活性多糖清除羟自由基的抗氧化性能研究.云南中医中药杂志,2009,30(6)：57—58.
[6]Kit ag aw a H, Ujikaw a M, Sug ahara K. Developmental changes in serum UDP-GlcA: chondroitin glucuronyl transferase activity. The Jour nal of Biological Chemistry,1996,271: 6583-6585.
[7]孙润广,张静.甘草多糖螺旋结构的原子力显微镜研究.化学学报,2006,64：2467-72.
[8]谭杨,陈廖斌,汪晖,胡爱心,谷锐.黄芪多糖对体外培养大鼠软骨细胞内糖胺多糖合成的影响及其机制.武汉大学学报(医学版),2008,29(2)：153-156.
[9]王昭佩,杨仁轩,许树柴,郭玉海,邓晋丰.人参皂甙Rbl对体外软骨细胞Ⅱ型胶原mRNA表达的影响.中国骨伤,2005,18(9)：543-545.
[10]刘军,陈廖斌,汪晖,商亮,刘岩松,张觅.当归多糖对大鼠骨性关节炎形态改变的影响.武汉大学学报(医学版),2010,31(5)：608-612.
[11]Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum.1998, Aug,41(8):1343-55.
[12]Sanshiro Hashimoto, Robert L. Chondrocyte-derived apoptotic bodies and calcification of articular cartilage. Proc. Natl. Acad.1998(95):3094-3099.
[13]Ouzzine M, Gulberti S, Netter P. Structure function of human Galbl,3-glucuronosyl transferase [J]. J Biol Chem,2000,275(36):28 254-28 260.
[14]邱贵兴.骨关节炎流行病学和病因学进展.继续教育,2004,9(12)：22.
[15]谈超,魏伟.骨关节炎的病因研究和治疗的进展.颈腰痛杂志,2002,23(2)：166.
[16]Busija L, Bridgett L, Williams SR, Osborne RH, Buchbinder R, March L, Fransen M.Osteoarthritis. Best Pract Res Clin Rheumatol.2010 Dec;24(6):757-68.
[17]Fernandes JC, Martel Pelletier J. The role of cytokines in osteoarthritis pathophysiology.Bio rheology,2002,39(1-2):237-246.
[18]邓康复,柴本甫,杨庆铭.肿瘤坏死因子-α对骨关节炎滑膜细胞增殖和RNA表达的影响.中国药理学通报,1998,11(6)：506-508.
[19]李忆农.细胞因子与关节炎.中华风湿病杂志,2000,4(1)：56-58.
[20]丽放,范振毕,屠丹云.骨关节炎与胰岛素样生长因子.国外医学.物理医学与康复学手册,1998,18(2)：1-4.
[21]许彭,郭雄.一氧化氮对骨关节软骨退变的研究进展.中国矫形外科杂志.1999,9(6)：705-706.
[22]谈超,魏伟.骨关节炎的病因研究和治疗的进展.颈腰痛杂志,2002,23(2)：166.
[23]赵庆华,亓建洪,孙巧玲等.白细胞介素1对骨关节炎软骨细胞基质金属蛋白酶阻滞剂1基因表达的影响.中国临床康复.2004,8(20)：4020-4022.
[24]刘世清,邱波,彭吴等.透明质酸钠对兔骨关节炎软骨和滑膜中MMP-1-3及其组织抑制物mRNA表达的影响.中华风湿病学杂志,2004,8(10)：587-590.
[25]Schaefer JF, Millham ML, de Crombrugghe B,et al.FGF signaling antagonizes cytokine-mediated repression of Sox9 in SW1353 chondrosarcoma cells. Osteoarthritis Cartilage,2003, 11:233-41.
[26]Michael A. Pratta, Wenqing Yao, Carl Decicco, et al. Aggrecan Protects Cartilage Collagen from Proteolytic Cleavage. The Journal of Biological Chemistry,2003,278(46):45539-45545.
[27]Kristian Prydz, Knut Tomas Dalen. Synthesis and sorting of proteoglycans. Journal of Cell Science.2000,113:193-205.
[28]Pottenger LA, Webb JE, Lyon NB. Kinetics of extraction of PG from human cartilage. Arthritis Rheum,1985,28:323-330.
[1]邱贵兴.骨关节炎流行病学和病因学进展.中华医学信息导报,2004,12：22.
[2]谈超,魏伟.骨关节炎的病因研究和治疗的进展.颈腰痛杂志,2002,(23)：166-169.
[3]Michael A. Pratta, Wenqing Yao, Carl Decicco, et al. Aggrecan Protects Cartilage Collagen from Proteolytic Cleavage. The Journal of Biological Chemistry,2003,278(46):45539-45545.
[4]Kristian Prydz, Knut Tomas Dalen. Synthesis and sorting of proteoglycans. Journal of Cell Science.2000,113:193-205.
[5]Pottenger LA, Webb JE, Lyon NB. Kinetics of extraction of PG from human cartilage. Arthritis Rheum,1985,28:323-330.
[6]李晓明.百药的调解员：甘草.健康天地,2007,2：29.
[7]邓伟.甘草附子汤治疗膝骨关节炎的临床研究.中药材,2008,31(7)：1107-1110.
[8]王淑华.芦荟多糖的分离纯化与分析.江苏化工,2007,35(5)：15-18.
[9]Lin YS,Huang MH,Chai CY. Effects of heliumne on laser on the mucopolysa ccharide induction in experimental osteoarthritic cartilage.Osteoarthritis Cartilage,2005,12:339-346.
[10]Hayami T,Pickarski M,Zhuo Y,et al.Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis.Bone,2006,2:234-243.
[11]Arner EC,Harris RR,Dimeo TM,et al.Interleukin-1 receptor antagonist inhibits proteoglycan breakdown in antigen induced but not polycation induced arthritis in the rabbit. Rheumatol, 1995,22:1338-1346.
[12]Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum.1998, Aug,41(8):1343-55.
[13]吕厚山.骨关节炎的基本治疗与药物治疗.中国临床医生杂志,2007,35(9)：3-5.
[14]黄枫,谢国平.中医药治疗膝骨关节炎实验研究进展.中国中医药信息杂志,2005,12(8)：109-110.
[15]Havdrup T,Telbag H.Papain-induced change in the knee joints of adult rabbits.Acta Orthop Scand,1977,48:143-9.
[16]钟汉馨,黄涛.不同运动方式引起关节软骨改变的病理研究.现代康复,2001,5(1)：36.
[17]Papaioannou N,Krallis N,Triantafillopoulos I.Optimal timing of research after anterior cruciate ligamint resection in rabbits Contemporary topics in laboratory animal science.Contemp Top Lab Anim Sci,2004,43(6),22.
[18]胡阿威,张磊,张功礼.改良Hulth(?)去骨关节炎动物模型的建立.中华医学丛刊,2003,9(3)：5.
[19]Marijmissen AC,Van Roermund PM,TeKoppele JM.The canine'groove'model, compared with the ACLT modle of osteoarthritis.Osteoarthritis and cartilage,2002,10(2):145.
[20]Wancket LM,Baragi V,Bove S.Anatomical localization of cartilage degradation markers in a surgically induced rat osteoarthritis model.Toxicologic pathology,2005,33(4):484.
[21]Lahm A,Uhl M,Edlich M.An experimental canine model for subchondral lesions of the knee.Joint Knee,2005,12(1):51.
[22]Marcelon G,Cros J,Guiraud R.Experimental modle of osteoarthritis.Agents Actions,1976,6: 191-194.
[23]Kobayashi K, Imaizumi R, Sumichika H. Sodium iodoacetate induced experimental osteoarthritis and associated pain model in rats.The Journal of veterinary medical science.J Vet Med Sci,2003,65.
[24]Kikuchi T,Sakuta T,Yanguchi T.Inter articular injection of collagenase induces experimental osteoarthritis in mature rabbits.Osteoarthritis and cartilage,1998,6(3):177.
[25]Lapadula G,Nico B,Cantatore FP.Early ultrastructural changes of articular cartilage and synovial membrane in experimental vitamin A-induced osteoarthritis. Rheuma-tol,1995,22(10):1913.
[26]Moaikow itiz RW.Experiment models of osteoarthritis Inosteoarthritis Saunders. Philadelphia, 1984,109.
[27]邓宇,伍筱梅,任医民等.关节腔内注射不同蛋白酶建立兔膝骨关节炎模型的对比研究,中华关节外科杂志,2009,20(10)：330.
[28]黄义星,陈廖斌,汪晖,易先宏,汪鹏.脱氢表雄酮对大鼠骨关节炎影响的实验研究.中国临床药理学与治疗学,2005,10(10)：1181-1185.
[29]Yamamaoto K,Shishido T,Masaoka T.Morphologicalstudies on the ageing and osteoarthritis of the articular cartilage in C57 black mice.Journal of Orthopaedic Sugery,2005,13(1):8.
[30]Yamamoto H, Iwase N,Kohmo M.Histopathological characterization of spontaneously developing osteoarthropathy in the BCBC/Y mouse established newly from B6C3F1 mice Experimental and toxicologic pathology.official Jonrnal of the Gesellschaft F Toxikologische Pathologic,1999,51(1):15.
[31]Debri E,Reinholt FP,Svensson O.Primary osteoarthrosis in guinea Pigs:a stereologi-cal study.Orthop Rea,1995,13(5):769-776.
[32]钟秀倩.氧自由基与疾病.韶关学院学报.2006,27(6)：87-90.
[33]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. Osteoarthritis Cartilage,2006,2:139-145.
[34]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.中华骨科杂志,1992,12：433.
[35]卢敏,谢心军,严可,龚志贤,左亚杰,王林华,谭旭仪.伤速康巴布膏对兔膝骨性关节炎模型关节液中SOD、NO水平变化影响的实验研究.中国中医骨伤科杂志,2011,19(10)：1-3.
[36]Gouze JN, Bianchi A. Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-κB pathway. FEBS Lett.2002,510(3):166-70.
[37]Jean-Noel Gouze, Karim Bordji, Sandrine Gulberti. Interleukin-1β Down Regulates the Expression of Glucuronosyltransferase Ⅰ,a key Enzyme Priming Glycosaminoglycan Biosynthesis. Arthritis Rheum.2001,44(2):351-360.
[38]刘军,陈廖斌,汗晖,商亮,刘岩松,张觅.当归多糖对大鼠骨性关节炎形态改变的影响.武汉大学学报(医学版),2010,31(5)：608-612.
[39]王昭佩,杨仁轩,许树柴,郭玉海,邓晋丰.人参皂甙Rbl对体外软骨细胞Ⅱ型胶原mRNA表达的影响.中国骨伤,2005,18(9)：543-545.
[40]胡爱心,陈廖斌,汪晖,谷锐,谭杨.黄芪多糖对大鼠骨关节炎的影响.武汉大学学报(医学版),2008,29(2)：157-161.
[41]Black C, Clar C, Henderson R, MacEachern C, McNamee P, Quayyum Z, Royle P, Thomas S.The clinical effectiveness of glucosamine and chondroitin supplements in slowing or arresting progression of osteoarthritis of the knee:a systematic review and economic evaluation.Health Technol Assess.2009 Nov;13(52):Ⅰ-148.
[1]吕厚山.骨关节炎的基本治疗与药物治疗.中国临床医生杂志,2007,35(9)：3-5.
[2]王裕民,李欣,王敬强,郭术勇.全髋关节置换术后假体松动的预防研究.天津医药,2005,33(8)：492-494.
[3]Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum.1998, Aug,41(8):1343-55.
[4]彭成忠,曹建民,肖涛,彭程,杨红波,陈湘,方建珍.骨关节炎关节滑液中IL-18与PGE_2含量的测定及意义.中南大学学报(医学版),2001,31(6)：862-865.
[5]Fernandes JC, Martel Pelletier J. The role of cytokines in osteoarthritis pathophysiology. Bio rheolog y,2002,39(1-2):237-246.
[6]庆华,亓建洪,孙巧玲等.白细胞介素1对骨关节炎软骨细胞基质金属蛋白酶阻滞剂1基因表达的影响.中国临床康复.2004,8(20)：4020-4022.
[7]刘世清,邱波,彭吴等.透明质酸钠对兔骨关节炎软骨和滑膜中MMP-1-3及其组织抑制物mRNA表达的影响.中华风湿病学杂志,2004,8(10)：587-90.
[8]Kobayashi M, Sato M, Li Y, Soh N, Nakano K, Toko K, Miura N, Matsumoto K, Hemmi A, Asano Y, Imato T.Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate.Talanta. 2005,68(2):198-206.
[9]Flannery CR, Little CB, Caterson B, Hughes CE.Effects of culture conditions and exposure to catabolic stimulators (IL-1 and retinoic acid) on the expression of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs) by articular cartilage chondrocytes. Matrix Biol.1999,18(3):225-37.
[10]Alaaeddine N, Di Battista JA, Pelletier JP, Kiansa K, Cloutier JM, Martel-Pelletier J.Differential effects of IL-8, LIF (pro-inflammatory) and IL-11 (anti-inflammatory) on TNF-alpha-induced PGE(2)release and on signalling pathways in human OA synovial fibroblasts.Cytokine. 1999,11(12):1020-30.
[11]Koshy PJ, Henderson N, Logan C, Life PF, Cawston TE, Rowan AD.Interleukin 17 induces cartilage collagen breakdown:novel synergistic effects in combination with proinflammatory cytokines.Ann Rheum Dis.2002,61 (8):704-13.
[12]Saha N, Moldovan F, Tardif G, Pelletier JP, Cloutier JM, Martel-Pelletier J.Interleukin-1 beta-converting enzyme/caspase-1 in human osteoarthritic tissues:localization and role in the maturation of interleukin-lbeta and interleukin-18.Arthritis Rheum.1999,42(8):1577-87.
[13]Futani H, Okayama A, Matsui K, Kashiwamura S, Sasaki T, Hada T, Nakanishi K, Tateishi H, Maruo S, Okamura H.Relation between interleukin-18 and PGE2 in synovial fluid of osteoarthritis:a potential therapeutic.J Immunother.2002 Mar-Apr;25 Suppl 1:S61-4.
[14]Malemud CJ. Fundamental pathways in osteoarthritis:an overview.Front Biosci. 1999;4:D659-61. Review.
[15]Aizawa T.Diode-Laser Wavelength-Modulation Absorption Spectroscopy for Quantitative in situ Measurements of Temperature and OH Radical Concentration in Combustion Gases.Appl Opt.2001;40(27):4894-903.
[16]Vuolteenaho K, Moilanen T, Al-Saffar N, Knowles RG, Moilanen E.Regulation of the nitric oxide production resulting from the glucocorticoid-insensitive expression of iNOS in human osteoarthritic cartilage.Osteoarthritis Cartilage.2001,9(7):597-605.
[17]Greenwel P, Dominguez-Rosales JA, Mavi G, Rivas-Estilla AM, Rojkind M.Hydrogen peroxide: a link between acetaldehyde-elicited alphal(I) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells.Hepatology.2000,31(1):109-16.
[18]Henrotin YE, De Groote DD, Labasse AH, Gaspar SE, Zheng SX, Geenen VG, Reginster JY.Effects of exogenous lL-1 beta, TNF alpha,IL-6,IL-8 and LIF on cytokine production by human articular chondrocytes.Osteoarthritis Cartilage.1996,4(3):163-73.
[19]Westacott CI, Whicher JT, Hutton CW, Dieppe PA.Increased spontaneous production of interleukin-1 together with inhibitory activity in systemic sclerosis.Clin Sci (Lond). 1988,75(6):561-7.
[20]Jikko A, Wakisaka T, Iwamoto M, Hiranuma H, Kato Y, Maeda T, Fujishita M, Fuchihata H.Effects of interleukin-6 on proliferation and proteoglycan metabolism in articular chondrocyte cultures.Cell Biol Int.1998;22(9-10):615-21.
[21]Silacci P, Dayer JM, Desgeorges A, Peter R, Manueddu C, Guerne PA.Interleukin (IL)-6 and its soluble receptor induce TIMP-1 expression in synoviocytes and chondrocytes, and block IL-1-induced collagenolytic activity.J Biol Chem.1998273(22):13625-9.
[22]Alaaeddine N, Di Battista JA, Pelletier JP, Kiansa K, Cloutier JM, Martel-Pelletier J.Differential effects of IL-8, LIF (pro-inflammatory) and IL-11 (anti-inflammatory) on TNF-alpha-induced PGE(2)release and on signalling pathways in human OA synovial fibroblasts.Cytokine. 1999,11 (12):1020-30.
[23]Lubberts E, Joosten LA, van de Loo FA, van den Gersselaar LA, van den Berg WB.Reduction of interleukin-17-induced inhibition of chondrocyte proteoglycan synthesis in intact murine articular cartilage by interleukin-4. Arthritis Rheum.2000,43(6):1300-6.
[24]Fernandes JC, Martel-Pelletier J, Pelletier JP.The role of cytokines in osteoarthritis pathophysiology.Biorheology.2002;39(1-2):237-46.
[25]Lubberts E, Joosten LA, van Den Bersselaar L, Helsen MM, Bakker AC, van Meurs JB, Graham FL, Richards CD, van Den Berg WB.Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.J Immunol.1999,163(8):4546-56.
[26]Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB.Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor beta/bone morphogenetic protein.Arthritis Rheum.2003,48(12):3442-51.inhibitors during experimental osteoarthritis.
[27]Glansbeek HL, van Beuningen HM, Vitters EL, van der Kraan PM, van den Berg WB.Expression of recombinant human soluble type Ⅱ transforming growth factor-beta receptor in Pichia pastoris and Escherichia coli:two powerful systems to express a potent inhibitor of transforming growth factor-beta.Protein Expr Purif.1998,12(2):201-7.
[28]Sunic D, McNeil JD, Rayner TE, Andress DL, Belford DA.Regulation of insulin-like growth factor-binding protein-5 by insulin-like growth factor Ⅰ and interleukin-1 alpha in ovine articular chondrocytes.Endocrinology.1998,139(5):2356-62.
[29]Loeser RF, Pacione CA, Chubinskaya S.The combination of insulin-like growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes.Arthritis Rheum.2003,48(8):2188-96.
[30]Colotta F, Re F, Polentarutti N, Sozzani S, Mantovani A.Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products.Blood.1992 Oct 15;80(8):2012-20.
[31]Gowen M, Wood DD, Russell RG.Stimulation of the proliferation of human bone cells in vitro by human monocyte products with interleukin-1 activity.J Clin Invest.1985 Apr;75(4):1223-9.
[32]Krakauer T.A macrophage-derived factor that inhibits the production and action of interleukin2.J Leukoc Bio1.1985,38(3)：429-39.
[33]Le Maitre CL, Hoyland JA, Freemont AJ.Interleukin-1 receptor antagonist delivered directly and by gene therapy inhibits matrix degradation in the intact degenerate human intervertebral disc:an in situ zymographic and gene therapy study.Arthritis Res Ther.2007;9(4):R83.
[34]Martel-Pelletier J, McCollum R, DiBattista J, Faure MP, Chin JA, Fournier S, Sarfati M, Pelletier JP.The interleukin-1 receptor in normal and osteoarthritic human articular chondrocytes. Identification as the type I receptor and analysis of binding kinetics and biologic function.Arthritis Rheum.1992 May;35(5):530-40.
[35]van der Zee E, Everts V, Beertsen W.Cytokines modulate routes of collagen breakdown. Review with special emphasis on mechanisms of collagen degradation in the periodontium and the burst hypothesis of periodontal disease progression.J Clin Periodontol.1997 May;24(5):297-305.Review.
[36]Kuhn K, Steinmetzer K, Brantl S.Transcriptional repressor CopR:the structured acidic C terminus is important for protein stability.J Mol Biol.2000 Jul 28;300(5):1021-31.
[37]Sanchez C, Mathy-Hartert M, Deberg MA, Ficheux H, Reginster JY, Henrotin YE.Effects of rhein on human articular chondrocytes in alginate beads.Biochem Pharmacol.2003 Feb1；65(3)：377-88.
[38]Kay MH, Wyllie R, Steffen RM.The endoscopic appearance of eosinophilic gastroenteritis in infancy.Am J Gastroenterol.1995 Aug;90(8):1361-2. No abstract available.
[39]David F, Farley J, Huang H, Lavoie JP, Laverty S.Cytokine and chemokine gene expression of IL-lbeta stimulated equine articular chondrocytes.Vet Surg.2007 Apr;36(3):221-7.
[40]Gouze JN, Bianchi A. Becuwe P. Dau?a M, Netter P. Magdalou J, Terlain B, Bordji K.Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-kappa B pathway.FEBS Lett.2002 Jan 16;510(3):166-70.
[41]Scheller M, Zimmermann B, Bernimoulin JP, Scholz P.Induction of metalloproteinase activity, cartilage matrix degradation and inhibition of endochondral mineralization in vitro by E. coli lipopolysaccharide is mediated by interleukin 1 alpha.Cytokine.1995 May;7(4):331-7.
[42]Roman-Bias JA, Stokes DG, Jimenez SA.Modulation of TGF-beta signaling by proinflamm-atory cytokines in articular chondrocytes.Osteoarthritis Cartilage.2007 Dec; 15(12):1367-77. Epub 2007 Jun 29.
[43]Bauge C, Legendre F, Leclercq S, Elissalde JM, Pujol JP, Galera P, Boumediene K.Interleukin-lbeta impairment of transforming growth factor betal signaling by down-regulation of transforming growth factor beta receptor type Ⅱ and up-regulation of Smad7 in human articular chondrocytes.Arthritis Rheum.2007 Sep;56(9):3020-32.
[44]Shakibaei M, John T, Schulze-Tanzil G, Lehmann Ⅰ, Mobasheri A.Suppression of NF-kappaB activation by curcumin leads to inhibition of expression of cyclo-oxygenase-2 and matrix metalloproteinase-9 in human articular chondrocytes:Implications for the treatment of osteoarthritis.Biochem Pharmacol.2007 May 1;73(9):1434-45. Epub 2007 Jan 7.
[45]Goldring MB.The role of cytokines as inflammatory mediators in osteoarthritis:lessons from animal models.Connect Tissue Res.1999;40(1):1-11. Review.
[46]Blanco FJ, Serrano L, Forman-Kay JD.High populations of non-native structures in the denatured state are compatible with the formation of the native folded state.J Mol Biol.1998 Dec 11;284(4):1153-64.
[47]van't Hof RJ, Armour KJ, Smith LM, Armour KE, Wei XQ, Liew FY, Ralston SH.Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption. Proc Natl Acad Sci USA.2000 Jul 5;97(14):7993-8.
[48]Pelletier JP, Mineau F, Fernandes JC, Duval N, Martel-Pelletier J.Diacerhein and rhein reduce the interleukin lbeta stimulated inducible nitric oxide synthesis level and activity while stimulating cyclooxygenase-2 synthesis in human osteoarthritic chondrocytes.J Rheumatol. 1998 Dec:25(12):2417-24.
[49]Terkeltaub R, Johnson K, Murphy A, Ghosh S.Invited review:the mitochondrion in osteoarthritis. Mitochondrion.2002 Feb;1(4):301-19.
[50]Boyault S, Simonin MA, Bianchi A, Compe E, Liagre B, Mainard D, Becuwe P, Daua M, Netter P, Terlain B, Bordji K.15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-lbeta effects in human chondrocytes by inhibiting NF-kappaB and AP-1 activation pathways.FEBS Lett.2001 Jul 13;501(1):24-30.
[51]Gouze JN, Gouze E, Palmer GD, Kaneto H, Ghivizzani SC, Grodzinsky AJ, Evans CH.Adenovirus-mediated gene transfer of glutamine:fructose-6-phosphate amidotransferase antagonizes the effects of interleukin-lbeta on rat chondrocytes.Osteoarthritis Cartilage.2004 Mar; 12(3):217-24.
[52]Fernandes J, Tardif G, Martel-Pelletier J, Lascau-Coman V, Dupuis M, Moldovan F, Sheppard M, Krishnan BR, Pelletier JP.In vivo transfer of interleukin-1 receptor antagonist gene in osteoarthritic rabbit knee joints:prevention of osteoarthritis progression.Am J Pathol.1999 Apr; 154(4):1159-69.
[53]Guo WS, Ma L, Li ZR.Suppression of articular cartilage breakdown in osteoarthritis by interleukin-1 receptor antagonist using ex vivo gene therapy. Zhonghua Yi Xue Za Zhi.2004 May 17;84(10):853-6. Chinese.
[54]Wang HJ, Zhai FY, Du SF, Wang ZH, He YN, Yu WT.Trend of distribution of body mass index among Chinese adults in some provinces from 1989 to 2000.Wei Sheng Yan Jiu.2006 Nov;35(6):794-7. Chinese.
[55]GHaudenschild DR, Curtiss SB, Moseley TA, Reddi AH.eneration of interleukin-17 receptor-like protein (IL-17RL) in prostate by alternative splicing of RNA.Prostate.2006 Sep 1;66(12):1268-74.
[56]Cornell CN, Lane JM.Newest factors in fracture healing.Clin Orthop Relat Res.1992 Apr;(277): 297-311. Review.
[57]Tsukazaki T, Usa T, Matsumoto T, Enomoto H, Ohtsuru A. Namba H, Iwasaki K, Yamashita S.Effect of transforming growth factor-beta on the insulin-like growth factor-I autocrine/ paracrine axis in cultured rat articular chondrocytes.Exp Cell Res.1994 Nov;215(1):9-16.
[58]Dodds RA, Merry K, Littlewood A, Gowen M.Expression of mRNA for IL1 beta, IL6 and TGF beta 1 in developing human bone and cartilage.J Histochem Cytochem.1994 Jun;42(6):733-44.
[59]Blaney Davidson EN, Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB.Reduced transforming growth factor-beta signaling in cartilage of old mice:role in impaired repair capacity.Arthritis Res Ther.2005;7(6):R1338-47.
[60]Tchetina EV, Antoniou J, Tanzer M, Zukor DJ, Poole AR.Transforming growth factor-beta2 suppresses collagen cleavage in cultured human osteoarthritic cartilage, reduces expression of genes associated with chondrocyte hypertrophy and degradation, and increases prostaglandin E(2) production.Am J Pathol.2006 Jan;168(1):131-40.
[61]Roman-Blas JA, Stokes DG, Jimenez SA.Modulation of TGF-beta signaling by proinflamma-tory cytokines in articular chondrocytes.Osteoarthritis Cartilage.2007 Dec; 15(12):1367-77. Epub 2007 Jun 29.
[62]Goldring SR, Goldring MB.The role of cytokines in cartilage matrix degeneration in osteoarthritis.Clin Orthop Relat Res.2004 Oct;(427 Suppl):S27-36. Review.
[63]Kraus VB, Li YJ, Martin ER, Jordan JM, Renner JB, Doherty M, Wilson AG, Moskowitz R, Hochberg M, Loeser R, Hooper M, Sundseth S.Articular hypermobility is a protective factor for hand osteoarthritis.Arthritis Rheum.2004 Jul;50(7):2178-83.
[64]Grimaud E, Heymann D, Redini F. Recent advances in TGF-be2 ta effects on chondrocyte metabolism. Potential therapeutic roles of TGF-beta in cartilage disorders. Cytokine Growth Factor Rev,2002,13 (3):241-257.
[65]van Beuningen HM, Glansbeek HL, van der Kraan PM, et al. Osteoarthritis-like changes in the murine knee joint resulting from intraarticular transforming growth factor-beta injections. Osteoarthritis Cartilage.2000,8(1):25-33.
[66]Itayem R, Mengarelli-Widholm S, Hulth A, et al. Ultrastructural studies on the effect of transforming growth factor-beta 1 on rat articular cartilage. APMIS,1997,105 (3):221-228.
[67]Van Osch GJ,Vander Veen SW,Buma P,et al.Matrix Biol 1998,17 (6):413-2
[68]Worster AA, Nixon AJ, Brower-Toland BD, Williams J.Effect of transforming growth factor betal on chondrogenic differentiation of cultured equine mesenchymal stem cells.Am J Vet Res. 2000 Sep;61(9):1003-10.
[69]Goto H, Shuler FD, Niyibizi C, et al. Gene therapy formeniscal injury:enhanced synthesis of p roteoglycan and collagen by meniscal cells transduced with a TGFbeta (1) gene. Osteo Cart, 2000,8 (4):266-271.
[70]Doherty PJ, Zhang H, Trenblay I. Resurfacing of articular cartilage exp lants with genetically-modified human chondrocytes in vitro. Osteo Car,1998,6 (3):153-159.
[71]Song XY, GuM, J inWW, et al. Plasmid DNA encoding transforming growth factor-beta 1 supp resses chronic disease in a streptococcal cell wall-induced arthritis model. J Clin Invest,1998, 101 (12):2615-2621.