雷奈酸锶、hEGF、NPY和WISP3对体外人关节软骨细胞的作用
摘要
骨性关节炎(Osteoarthritis, OA)是一种以关节软骨进行性消失与骨质过度增生,以及关节疼痛、关节僵硬、关节肿胀及活动受限为主要特征的的慢性骨关节疾病。OA是一种最常见的关节病变,骨性关节炎的名称极多,如肥大性骨性关节炎、退行性关节炎、变性性关节炎、增生性骨性关节炎或骨关节病,国内一般称为骨性关节炎。其患病率随着年龄而增加,女性比男性多发。OA以手的远端和近端指间关节,膝、肘和肩关节以及脊柱关节更容易受累,而腕、踝关节则相对较少发生。
骨性关节炎的主要病理改变为软骨的退行性变性和消失,以及关节边缘韧带附着处和软骨下骨质反应性增生与骨赘,并由此引起关节疼痛、僵直畸形和功能障碍。
OA的确切发病原因不明,可能由多种因素诱发,其本质为关节软骨基质的分解代谢和合成代谢失衡。既往的研究认为,OA是一种非炎症性关节病变,但近年来大量的研究证实,异常的炎症反在OA的发病中起了重要作用。近年的研究发现,多种关节代谢酶的功能异常是OA的重要发病因素,如基质金属蛋白酶(Matrix metalloproteinases, MMPs)和含凝血酶敏感蛋白的解聚素与金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs, ADAMTs)对OA发病机制的重新认识给OA提供了更多的治疗靶点与选择。
晚发型脊柱骨髓发育不良伴进行性骨关节病(spondyloepiphyseal sdysplasia tarda with progressivearthropathy, SEDT-PA)是一种主要累及软骨组织的常染色体隐性遗传性骨关节病,其主要临床特征是进行性关节僵硬、疼痛、活动受限、软组织肿胀以及关节畸形。本病又称为儿童进行性假类风湿性关节病(arthropathy progressive pseudo-rheumatoid of childhood, APPRC)或进行性假类风湿性骨发育不良(progressive pseudorheumatoid dysplasia, SEDT-PA, OMIM 208230),男女发病率类似。致病基因为TWnt-1诱导的信号蛋白3(Wnt-l-induced signaling protein 3, WISP3),该基因定位于6q22-q23,编码含354个氨基酸残基且富含半胱氨酸的分泌型蛋白,WISP3蛋白产物的完整性是维持正常软骨代谢的关键因素,最近对SEDT-PA基因突变的功能表达研究表明WISP3基因可能通过调节Ⅱ型胶原和aggrecan在软骨基质中的表达,维持软骨的完整性,而SEDT-PA相关基因突变可引起这种功能的丧失,导致持续性软骨丢失,可能部分解释SEDT-PA的发病机制。
OA及SEDT-PA的软骨组织病变是其主要病理改变,以前对软骨基质退变研究较多的是基质金属蛋白酶系(MMPs)。一般认为,MMPs是降解基质中软骨蛋白聚糖和胶原的主要酶类,尤其以MMP-1、MMP-3和MMP-13等的作用研究得较多。但是近年来发现,软骨蛋白聚糖酶(aggrecanase),特别是含凝血酶敏感蛋白的解聚素与金属蛋白酶(ADAMTS)在软骨的退变中起了更重要的作用。现认为,ADAMTS-5是软骨基质蛋白多糖的主要降解酶。WISP3通过调节Ⅱ型胶原和蛋白聚糖的合成维持软骨稳定性,WISP3基因突变是SEDT-PA发病的主要原因。
研究发现,雷奈酸锶、人表皮生长因子(human epidermal growthfactor,hEGF).神经肽Y(neuropeptide Y,NPY)和WISP3对骨形成及骨的动态平衡起了重要作用。本研究主要包括以下三个部分：①观察骨性关节炎患者的关节软骨ADAMTS-5、WISP3.MMP-1、-3和-13的表达,探讨其与骨性关节炎软骨退变的关系；结果表明,在骨性关节炎中,ADAMTS-5、WISP3.MMP-1、-3和-13的表达增多,由其引起的细胞外基质异常降解是导致关节软骨破坏与退变的原因之一。②在含鼠尾I型胶原蛋白的包埋板中,观察培养人关节软骨细胞的生长情况；我们首次发现,鼠尾I型胶原蛋白包埋板培养是一种有效的软骨细胞培养方法。在体外,分离培养的OA软骨细胞符合OA软骨细胞退变表型,因此为OA等软骨疾病的研究提供了更好的实验技术。③用特定浓度的雷奈酸锶、hEGF.NPY和WISP3干预体外培养的OA及SEDT-PA软骨细胞,比较各种酶类的表达差异,以探讨它们与OA的可能关系和发病特点；结果表明,在OA及SEDT-PA的发病过程中,雷奈酸锶、WISP3.hEGF和NPY通过抑制ADAMTS-5.MMP-3.MMP-13而阻止软骨退变。
第一部分ADAMTS-5、WISP3.MMP-1、-3和-13在人关节软骨中的表达目的：观察骨性关节炎患者的关节软骨ADAMTS-5、WISP3、MMP-1、-3和-13的表达,探讨其与骨性关节炎软骨退变的关系。方法：选取14例因骨性关节炎行关节置换的软骨组织,常规HE染色,观察组织学形态,并应用SP法观察关节软骨ADAMTS-5、WISP3、MMP-1、-3和-13的表达情况；另取4例外伤后截肢患者及股骨颈骨折的正常关节软骨作为本研究的对照。
结果：骨性关节炎的关节软骨出现裂隙、纤维化,软骨细胞数目减少,排列紊乱,并出现肥大软骨细胞,细胞凋亡,细胞外基质减少。免疫组化发现,ADAMTS-5、WISP3、MMP-1、-3和-13等在OA组中的表达均高于正常对照组。
结论：在骨性关节炎中,ADAMTS-5、WISP3、MMP-1、-3和-13的表达增多,由其引起的细胞外基质异常降解是导致关节软骨破坏与退变的原因之一。
第二部分人关节软骨细胞在含鼠尾I型胶原蛋白包埋板中的体外培养
目的：在含鼠尾Ⅰ型胶原蛋白的包埋板中,观察培养人关节软骨细胞的生长情况。
方法：选取14例因骨性关节炎行关节置换的软骨组织及4例因外伤而截肢患者及股骨颈骨折的正常关节软骨,体外分离培养软骨细胞。用晚发型脊柱骨骺发育不良伴进行性骨关节病(spondyloepiphyseal dysplasia tarda with progressive arthropathy, SEDT-PA)患者关节软骨传代细胞,单层培养传代后再在含鼠尾Ⅰ型胶原蛋白的包埋板上继续培养,显微镜下观察细胞形态,并应用MTT法和甲苯胺蓝染色法分别测量细胞增殖及细胞分泌蛋白的表达。
结果：在鼠尾Ⅰ型胶原蛋白包埋板上,培养的软骨细胞表型维持良好,甲苯胺蓝阳染率较高。SEDT-PA患者的关节软骨细胞增殖速度最快(OD值为0.075),OA患者的关节软骨细胞增殖速度最慢(OD值为0.048),而正常关节软骨细胞的增殖速度中等。在甲苯胺蓝染色中,SEDT-PA患者的软骨细胞阳染率最高,正常软骨细胞其次,而OA软骨细胞阳染率最低。
结论：在体外,分离培养的OA软骨细胞符合人OA软骨细胞退变的表型,而鼠尾Ⅰ型胶原蛋白包埋板培养是一种有效的软骨细胞培养方法,为OA等软骨疾病的研究提供了更好的实验技术。
第三部分雷奈酸锶、WISP3、hEGF和NPY对人关节软骨细胞功能的作用
目的：在体外培养条件下,探讨雷奈酸锶、WISP3、hEGF和NPY对不同关节软骨细胞功能的影响。
方法：体外培养正常关节软骨细胞、OA软骨细胞及SEDT-PA软骨细胞,用合适浓度的雷奈酸锶、WISP3、hEGF及NPY加入培养液中培养,MTT测定细胞增殖变化,提取软骨细胞裂解液及上清液进行Western-blot与ELISA分析。
结果：MTT结果显示,WISP3和雷奈酸锶能促进OA软骨细胞增殖,但对SEDT-PA软骨细胞无明显影响。Western-blot结果示,雷奈酸锶、WISP3、hEGF和NPY使OA软骨细胞的ADAMTS-5表达水平降低而WISP3表达升高。雷奈酸锶和hEGF能提高OA软骨细胞的JNK的表达,而NPY和hEGF可增加OA软骨细胞ERK的表达。ELISA结果示,雷奈酸锶能降低OA软骨细胞MMP-3表达,NPY、hEGF、雷奈酸锶和WISP3提高OA软骨细胞MMP-8的表达,NPY、hEGF和雷奈酸锶降低SEDT-PA软骨细胞MMP-8表达,NPY、hEGF、雷奈酸锶和WISP3增加正常软骨细胞MMP-13表达,降低OA软骨细胞MMP-13表达,同时降低SEDT-PA软骨细胞MMP-13的表达。
结论：在OA及SEDT-PA的发病过程中,雷奈酸锶、WISP3、hEGF和NPY通过抑制ADAMTS-5、MMP-3、MMP-13能阻止软骨退变。
Background
Osteoarthritis (OA) is a slowly progressive degenerative disease characterized by gradual loss of articular cartilage, but its mechanism is still unknown. Biochemical and genetic factors contribute to cartilage lesions in OA by disrupting chondrocyte-matrix associations and altering metabolic responses in the chondrocyte. Spondyloepiphyseal dysplasial tarda with progressive arthropathy (SEDT-PA) is an autosomal-recessive hereditary disorder of cartilage homeostasis and characterized by progressive joint stiffness, pain, limited mobility, soft tissue swelling and joint deformities. The pathogenesis of SEDT-PA is unkown though it has been demonstrated that WISP3 is the causing gene.
The degradation of articular cartilage is the main pathological changes in OA and SEDT-PA. Matrix metalloproteinases (MMPs) are long considered to play a central role in the degradation of cartilage aggrecan and collagens, especially for MMP-1, MMP-3 and MMP-13. Recent biochemical studies demonstrated that one of the aggrecanases, the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families play a significant role in the progression of OA. However, ADAMTS-5 (aggrecanase 2) has generally been considered to be the most important aggrecanase in OA, while Wnt-1-induced signaling protein 3(WISP3), an essential protein for mainting cartilage intergrity by regulating the expression of collagen II and aggrecan, was recognized to be the causative factor related to SEDT-PA.
Some studies have demonstrated that Strontium Ranelate, hEGF, NPY and WISP3 played siginificant roles in the formation and metabolic homeostasis of bone, whether they have functions in the progression of OA is not clear. Aim of the study is to understand the effects of these factors on articular chondrocytes and three conclusions could be drawn from the present investigation:(1) articular chondrocytes cultured in embedded board containing rat tail collagen type I is an excellent differentiation technique for chondrocytes study in vitro and we have found that the proliferation of SEDT-PA chondrocytes is the fastest while OA chondrocytes proliferate slowestly; (2) increased expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 is likely to be linked to the cartilage degeneration in many disordered conditions; (3) SR, NPY, WISP3 and hEGF can prevent the cartilage from degradation in OA and SEDT-PA by inhibiting the expression of ADAMTS-5, MMP-3 and MMP-8.
PART ONE
Immunohistochemial Expression and Significance of ADAMTS-5,WISP3,MMP-1,MMP-3 and MMP-13 in human articular cartilage
Objective:To ivestigate the relationship between cartilage degeneration and the expression of aggrecanase 2 (ADAMTS-5), Wnt-1 inducible secreted protein 3 (WISP3), matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3) and matrix metalloproteinase (MMP-13) in osteoarthritis.
Methods:The histological changes of cartilages by hematoxyllin-eosin staining and Immunohistochemical expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 were studied in 14 osteoarthritis cases and 4 normal controls.
Results:Articular tissue became predominantly fibroplastic complicated by a decrease in the number of chondrocytes and with the appearance of clustered and hypertrophic ones which subjected to apoptosis, as well as a reduction of extracellular matrix. The immunohistochemical expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 were higher in OA articular cartilage compared to that of the normal subjects.
Conclusions:The increased expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 was likely linked to cartilage degeneration.
PART TWO
Culture of human articular chondrocytes in embedded board containing rat tail collagen type I in vitro
Objective:To investigate the proliferation of chondrocytes cultured in embedded board containing rat tail collagen type I.
Methods:Chondrocytes were isolated by enzymic digestion from the articular cartilage from patients with knee replacement surgery, and cultured in monolayer system until the second generation, then cells were cultured in embedded board containing rat tail collagen type I. The cells were used for evaluation cell growth by phase-contrast microscope, toluidine blue method, and cell proliferation by MTT method.
Results:The chondrocytes cultured in embedded board containing rat tail collagen type I grew much better than cultured in monolayer system. The SEDT-PA chondrocytes proliferated fastestly and showed the strongest heterochromia to toluidine blue staining,while the OA chondrocytes proliferated slowestly and showed the weakest heterochromia when compared to normal chondrocytes which proliferated with a medium speed and showed middle heterochromia.
Conclusions:OA chondrocytes cultured in embedded board containing rat tail collagen type I presented the nature phenotypes characterized by human OA articular chondrocytes and can be regarded as the better techniques for experimental approach of OA.
PART THREE
The Effects of Strontium Ranelate, WISP3, hEGF and NPY on Human Articular Chondrocytes in Vitro
Objective:To investigate the effects of Strontium Ranelate(SR), WISP3, hEGF and NPY in human articular chondrocytes in vitro.
Methods:Normal, OA, and SEDT-PA articular chondrocytes were cultured in vitro. Appropriate concentrations of SR, WISP3, hEGF and NPY were added to these cells culture medium. The effects of these factors on cultured cells were evaluated by MTT, Western-blot and ELISA methods.
Results:A promoted proliferation of SR and WISP3 on OA chondrocytes but not for SEDT-PA cells was repeatedly observed with MTT. The results of ELISA and Western blotting were supported by the following findings:①SR, WISP3, hEGF and NPY could reduce the expression of ADAMTS-5,and WISP3 increased the expression of ADAMTS-5；②SR, hEGF could increase the expression of singalling molecules JNK in OA, and hEGFas well as NPY showed to increase the expression of singalling molecules ERK in OA;③SR reduced the expression of MMP-3, NPY, hEGF, SR, WISP3 increased the expression of MMP-8 in OA, but NPY, hEGF, SR reduced the expression of MMP-8 in SEDT-PA, NPY, hEGF, SR, WISP3 reduced the expression of MMP-13 in OA and SEDT-PA.
Conclutions:SR, NPY, WISP3 and hEGF could prevent the OA and SEDT-PA cartilage from degradation by inhibiting the expression of ADAMTS-5, MMP-3 and MMP-8.
骨性关节炎的主要病理改变为软骨的退行性变性和消失,以及关节边缘韧带附着处和软骨下骨质反应性增生与骨赘,并由此引起关节疼痛、僵直畸形和功能障碍。
OA的确切发病原因不明,可能由多种因素诱发,其本质为关节软骨基质的分解代谢和合成代谢失衡。既往的研究认为,OA是一种非炎症性关节病变,但近年来大量的研究证实,异常的炎症反在OA的发病中起了重要作用。近年的研究发现,多种关节代谢酶的功能异常是OA的重要发病因素,如基质金属蛋白酶(Matrix metalloproteinases, MMPs)和含凝血酶敏感蛋白的解聚素与金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs, ADAMTs)对OA发病机制的重新认识给OA提供了更多的治疗靶点与选择。
晚发型脊柱骨髓发育不良伴进行性骨关节病(spondyloepiphyseal sdysplasia tarda with progressivearthropathy, SEDT-PA)是一种主要累及软骨组织的常染色体隐性遗传性骨关节病,其主要临床特征是进行性关节僵硬、疼痛、活动受限、软组织肿胀以及关节畸形。本病又称为儿童进行性假类风湿性关节病(arthropathy progressive pseudo-rheumatoid of childhood, APPRC)或进行性假类风湿性骨发育不良(progressive pseudorheumatoid dysplasia, SEDT-PA, OMIM 208230),男女发病率类似。致病基因为TWnt-1诱导的信号蛋白3(Wnt-l-induced signaling protein 3, WISP3),该基因定位于6q22-q23,编码含354个氨基酸残基且富含半胱氨酸的分泌型蛋白,WISP3蛋白产物的完整性是维持正常软骨代谢的关键因素,最近对SEDT-PA基因突变的功能表达研究表明WISP3基因可能通过调节Ⅱ型胶原和aggrecan在软骨基质中的表达,维持软骨的完整性,而SEDT-PA相关基因突变可引起这种功能的丧失,导致持续性软骨丢失,可能部分解释SEDT-PA的发病机制。
OA及SEDT-PA的软骨组织病变是其主要病理改变,以前对软骨基质退变研究较多的是基质金属蛋白酶系(MMPs)。一般认为,MMPs是降解基质中软骨蛋白聚糖和胶原的主要酶类,尤其以MMP-1、MMP-3和MMP-13等的作用研究得较多。但是近年来发现,软骨蛋白聚糖酶(aggrecanase),特别是含凝血酶敏感蛋白的解聚素与金属蛋白酶(ADAMTS)在软骨的退变中起了更重要的作用。现认为,ADAMTS-5是软骨基质蛋白多糖的主要降解酶。WISP3通过调节Ⅱ型胶原和蛋白聚糖的合成维持软骨稳定性,WISP3基因突变是SEDT-PA发病的主要原因。
研究发现,雷奈酸锶、人表皮生长因子(human epidermal growthfactor,hEGF).神经肽Y(neuropeptide Y,NPY)和WISP3对骨形成及骨的动态平衡起了重要作用。本研究主要包括以下三个部分：①观察骨性关节炎患者的关节软骨ADAMTS-5、WISP3.MMP-1、-3和-13的表达,探讨其与骨性关节炎软骨退变的关系；结果表明,在骨性关节炎中,ADAMTS-5、WISP3.MMP-1、-3和-13的表达增多,由其引起的细胞外基质异常降解是导致关节软骨破坏与退变的原因之一。②在含鼠尾I型胶原蛋白的包埋板中,观察培养人关节软骨细胞的生长情况；我们首次发现,鼠尾I型胶原蛋白包埋板培养是一种有效的软骨细胞培养方法。在体外,分离培养的OA软骨细胞符合OA软骨细胞退变表型,因此为OA等软骨疾病的研究提供了更好的实验技术。③用特定浓度的雷奈酸锶、hEGF.NPY和WISP3干预体外培养的OA及SEDT-PA软骨细胞,比较各种酶类的表达差异,以探讨它们与OA的可能关系和发病特点；结果表明,在OA及SEDT-PA的发病过程中,雷奈酸锶、WISP3.hEGF和NPY通过抑制ADAMTS-5.MMP-3.MMP-13而阻止软骨退变。
第一部分ADAMTS-5、WISP3.MMP-1、-3和-13在人关节软骨中的表达目的：观察骨性关节炎患者的关节软骨ADAMTS-5、WISP3、MMP-1、-3和-13的表达,探讨其与骨性关节炎软骨退变的关系。方法：选取14例因骨性关节炎行关节置换的软骨组织,常规HE染色,观察组织学形态,并应用SP法观察关节软骨ADAMTS-5、WISP3、MMP-1、-3和-13的表达情况；另取4例外伤后截肢患者及股骨颈骨折的正常关节软骨作为本研究的对照。
结果：骨性关节炎的关节软骨出现裂隙、纤维化,软骨细胞数目减少,排列紊乱,并出现肥大软骨细胞,细胞凋亡,细胞外基质减少。免疫组化发现,ADAMTS-5、WISP3、MMP-1、-3和-13等在OA组中的表达均高于正常对照组。
结论：在骨性关节炎中,ADAMTS-5、WISP3、MMP-1、-3和-13的表达增多,由其引起的细胞外基质异常降解是导致关节软骨破坏与退变的原因之一。
第二部分人关节软骨细胞在含鼠尾I型胶原蛋白包埋板中的体外培养
目的：在含鼠尾Ⅰ型胶原蛋白的包埋板中,观察培养人关节软骨细胞的生长情况。
方法：选取14例因骨性关节炎行关节置换的软骨组织及4例因外伤而截肢患者及股骨颈骨折的正常关节软骨,体外分离培养软骨细胞。用晚发型脊柱骨骺发育不良伴进行性骨关节病(spondyloepiphyseal dysplasia tarda with progressive arthropathy, SEDT-PA)患者关节软骨传代细胞,单层培养传代后再在含鼠尾Ⅰ型胶原蛋白的包埋板上继续培养,显微镜下观察细胞形态,并应用MTT法和甲苯胺蓝染色法分别测量细胞增殖及细胞分泌蛋白的表达。
结果：在鼠尾Ⅰ型胶原蛋白包埋板上,培养的软骨细胞表型维持良好,甲苯胺蓝阳染率较高。SEDT-PA患者的关节软骨细胞增殖速度最快(OD值为0.075),OA患者的关节软骨细胞增殖速度最慢(OD值为0.048),而正常关节软骨细胞的增殖速度中等。在甲苯胺蓝染色中,SEDT-PA患者的软骨细胞阳染率最高,正常软骨细胞其次,而OA软骨细胞阳染率最低。
结论：在体外,分离培养的OA软骨细胞符合人OA软骨细胞退变的表型,而鼠尾Ⅰ型胶原蛋白包埋板培养是一种有效的软骨细胞培养方法,为OA等软骨疾病的研究提供了更好的实验技术。
第三部分雷奈酸锶、WISP3、hEGF和NPY对人关节软骨细胞功能的作用
目的：在体外培养条件下,探讨雷奈酸锶、WISP3、hEGF和NPY对不同关节软骨细胞功能的影响。
方法：体外培养正常关节软骨细胞、OA软骨细胞及SEDT-PA软骨细胞,用合适浓度的雷奈酸锶、WISP3、hEGF及NPY加入培养液中培养,MTT测定细胞增殖变化,提取软骨细胞裂解液及上清液进行Western-blot与ELISA分析。
结果：MTT结果显示,WISP3和雷奈酸锶能促进OA软骨细胞增殖,但对SEDT-PA软骨细胞无明显影响。Western-blot结果示,雷奈酸锶、WISP3、hEGF和NPY使OA软骨细胞的ADAMTS-5表达水平降低而WISP3表达升高。雷奈酸锶和hEGF能提高OA软骨细胞的JNK的表达,而NPY和hEGF可增加OA软骨细胞ERK的表达。ELISA结果示,雷奈酸锶能降低OA软骨细胞MMP-3表达,NPY、hEGF、雷奈酸锶和WISP3提高OA软骨细胞MMP-8的表达,NPY、hEGF和雷奈酸锶降低SEDT-PA软骨细胞MMP-8表达,NPY、hEGF、雷奈酸锶和WISP3增加正常软骨细胞MMP-13表达,降低OA软骨细胞MMP-13表达,同时降低SEDT-PA软骨细胞MMP-13的表达。
结论：在OA及SEDT-PA的发病过程中,雷奈酸锶、WISP3、hEGF和NPY通过抑制ADAMTS-5、MMP-3、MMP-13能阻止软骨退变。
Background
Osteoarthritis (OA) is a slowly progressive degenerative disease characterized by gradual loss of articular cartilage, but its mechanism is still unknown. Biochemical and genetic factors contribute to cartilage lesions in OA by disrupting chondrocyte-matrix associations and altering metabolic responses in the chondrocyte. Spondyloepiphyseal dysplasial tarda with progressive arthropathy (SEDT-PA) is an autosomal-recessive hereditary disorder of cartilage homeostasis and characterized by progressive joint stiffness, pain, limited mobility, soft tissue swelling and joint deformities. The pathogenesis of SEDT-PA is unkown though it has been demonstrated that WISP3 is the causing gene.
The degradation of articular cartilage is the main pathological changes in OA and SEDT-PA. Matrix metalloproteinases (MMPs) are long considered to play a central role in the degradation of cartilage aggrecan and collagens, especially for MMP-1, MMP-3 and MMP-13. Recent biochemical studies demonstrated that one of the aggrecanases, the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families play a significant role in the progression of OA. However, ADAMTS-5 (aggrecanase 2) has generally been considered to be the most important aggrecanase in OA, while Wnt-1-induced signaling protein 3(WISP3), an essential protein for mainting cartilage intergrity by regulating the expression of collagen II and aggrecan, was recognized to be the causative factor related to SEDT-PA.
Some studies have demonstrated that Strontium Ranelate, hEGF, NPY and WISP3 played siginificant roles in the formation and metabolic homeostasis of bone, whether they have functions in the progression of OA is not clear. Aim of the study is to understand the effects of these factors on articular chondrocytes and three conclusions could be drawn from the present investigation:(1) articular chondrocytes cultured in embedded board containing rat tail collagen type I is an excellent differentiation technique for chondrocytes study in vitro and we have found that the proliferation of SEDT-PA chondrocytes is the fastest while OA chondrocytes proliferate slowestly; (2) increased expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 is likely to be linked to the cartilage degeneration in many disordered conditions; (3) SR, NPY, WISP3 and hEGF can prevent the cartilage from degradation in OA and SEDT-PA by inhibiting the expression of ADAMTS-5, MMP-3 and MMP-8.
PART ONE
Immunohistochemial Expression and Significance of ADAMTS-5,WISP3,MMP-1,MMP-3 and MMP-13 in human articular cartilage
Objective:To ivestigate the relationship between cartilage degeneration and the expression of aggrecanase 2 (ADAMTS-5), Wnt-1 inducible secreted protein 3 (WISP3), matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3) and matrix metalloproteinase (MMP-13) in osteoarthritis.
Methods:The histological changes of cartilages by hematoxyllin-eosin staining and Immunohistochemical expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 were studied in 14 osteoarthritis cases and 4 normal controls.
Results:Articular tissue became predominantly fibroplastic complicated by a decrease in the number of chondrocytes and with the appearance of clustered and hypertrophic ones which subjected to apoptosis, as well as a reduction of extracellular matrix. The immunohistochemical expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 were higher in OA articular cartilage compared to that of the normal subjects.
Conclusions:The increased expression of ADAMTS-5, WISP3, MMP-1, MMP-3 and MMP-13 was likely linked to cartilage degeneration.
PART TWO
Culture of human articular chondrocytes in embedded board containing rat tail collagen type I in vitro
Objective:To investigate the proliferation of chondrocytes cultured in embedded board containing rat tail collagen type I.
Methods:Chondrocytes were isolated by enzymic digestion from the articular cartilage from patients with knee replacement surgery, and cultured in monolayer system until the second generation, then cells were cultured in embedded board containing rat tail collagen type I. The cells were used for evaluation cell growth by phase-contrast microscope, toluidine blue method, and cell proliferation by MTT method.
Results:The chondrocytes cultured in embedded board containing rat tail collagen type I grew much better than cultured in monolayer system. The SEDT-PA chondrocytes proliferated fastestly and showed the strongest heterochromia to toluidine blue staining,while the OA chondrocytes proliferated slowestly and showed the weakest heterochromia when compared to normal chondrocytes which proliferated with a medium speed and showed middle heterochromia.
Conclusions:OA chondrocytes cultured in embedded board containing rat tail collagen type I presented the nature phenotypes characterized by human OA articular chondrocytes and can be regarded as the better techniques for experimental approach of OA.
PART THREE
The Effects of Strontium Ranelate, WISP3, hEGF and NPY on Human Articular Chondrocytes in Vitro
Objective:To investigate the effects of Strontium Ranelate(SR), WISP3, hEGF and NPY in human articular chondrocytes in vitro.
Methods:Normal, OA, and SEDT-PA articular chondrocytes were cultured in vitro. Appropriate concentrations of SR, WISP3, hEGF and NPY were added to these cells culture medium. The effects of these factors on cultured cells were evaluated by MTT, Western-blot and ELISA methods.
Results:A promoted proliferation of SR and WISP3 on OA chondrocytes but not for SEDT-PA cells was repeatedly observed with MTT. The results of ELISA and Western blotting were supported by the following findings:①SR, WISP3, hEGF and NPY could reduce the expression of ADAMTS-5,and WISP3 increased the expression of ADAMTS-5；②SR, hEGF could increase the expression of singalling molecules JNK in OA, and hEGFas well as NPY showed to increase the expression of singalling molecules ERK in OA;③SR reduced the expression of MMP-3, NPY, hEGF, SR, WISP3 increased the expression of MMP-8 in OA, but NPY, hEGF, SR reduced the expression of MMP-8 in SEDT-PA, NPY, hEGF, SR, WISP3 reduced the expression of MMP-13 in OA and SEDT-PA.
Conclutions:SR, NPY, WISP3 and hEGF could prevent the OA and SEDT-PA cartilage from degradation by inhibiting the expression of ADAMTS-5, MMP-3 and MMP-8.
引文
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