TWEAK诱导体外培养的骨关节炎软骨细胞合成MMP-9的实验研究
摘要
背景:肿瘤坏死因子样弱凋亡诱导因子(TNF-like weak inducer of apop- ptosis,TWEAK)是肿瘤坏死因子配体超家族的新成员,按被发现的时间先后也称为第十二成员(TNF-superfamily member 12,TNFSF12),现发现其具有广泛的生物学活性,在多个系统的疾病发病机制中起着重要作用。骨关节炎(Osteoarthritis,OA)是一种最常见的关节疾病和分布最广泛的慢性疾病之一,而关节软骨的破坏是OA的标志性特征。虽然确切的发病机制目前尚不明确,但目前已清楚的是,OA关节软骨的降解是通过多种的细胞因子介导的,如多种基质金属蛋白酶(MMPs)以及其他一些细胞激活后的产物。研究发现,TWEAK能诱导多种细胞分泌促炎症细胞因子和趋化因子及高表达MMPs和RANTES。目前国内外有关TWEAK对OA软骨细胞的作用及其信号传导研究较少。本研究旨在通过对骨关节炎软骨细胞的体外培养及其相关研究,探讨TWEAK参与骨关节炎关节软骨破坏的可能机制,进而寻求治疗OA的新方法。
目的:
1建立软骨细胞体外培养体系;
2观察TWEAK分子在软骨细胞的表达情况;
3研究TWEAK在不同浓度下诱导体外培养的骨关节炎软骨细胞合成MMP-9的影响;
4观察TNF-α和IL-1β对TWEAK诱导软骨细胞合成MMP-9的影响。
1收集行关节置换的骨关节炎患者关节软骨组织,用胰蛋白酶-Ⅱ型胶原酶顺序消化法分离培养原代软骨细胞;
2制作细胞爬片,应用免疫荧光法检测TWEAK分子的表达;
3将rhTWEAK与软骨细胞共培育(其中协同作用组分别加入TNF-α和IL-1β),应用ELISA法检测刺激后培养液中MMP-9的水平;用逆转录-聚合酶链反应(RT-PCR)检测软骨细胞中MMP-9 mRNA的表达水平。
结果:
1总共收集到42例符合标准的骨关节炎关节软骨标本,成功培养并传代的有30例,总成活率为71.4%;
2 TWEAK在OA软骨细胞中广泛表达,且主要定位于细胞浆;
3当TWEAK终浓度为50、100μg/L时,其诱导OA软骨细胞合成MMP-9的水平明显高于对照组,具有显著的统计学意义(p<0.05);TWEAK终浓度为100μg/L时,其诱导OA软骨细胞MMP-9 mRNA的表达水平为对照组的1.28倍,具有显著的统计学意义(p<0.05)。
4与单用TWEAK组比较,TNF-α和IL-1β协同作用组诱导软骨细胞合成MMP-9的水平明显高于对照组,具有显著的统计学意义(p<0.05),而协同作用组之间无统计学意义。
结论:
1应用胰蛋白酶-Ⅱ型胶原酶顺序消化法分离培养软骨细胞是一种可行的软骨细胞原代培养方法,与国内外的报道相一致;
2 TWEAK在OA软骨细胞中广泛表达且主要定位于细胞浆中;
3 TWEAK通过诱导OA软骨细胞合成MMP-9,直接参与OA关节软骨的破坏,从而在OA的发病中起作用。方法:
4 TNF-α和/或IL-1β在TWEAK诱导OA软骨细胞合成MMP-9的过程中起协同作用。
Background:TWEAK(TNF-like weak inducer of apoptosis), is a novel member of the TNF superfamilies, also known as TNFSF12(TNF-superfamily member 12). TWEAK is a multifunctional cytokine which has extensive biological activities, plays an important role in pathogenesis of many diseases , especially in autoimmune diseases. Osteoarthritis(OA)is the most common disease of the joints, and one of the most widespread of all chronic diseases. Deterioration of articular cartilage is a hallmark of OA pathogenesis. Although the precise aetiology of this disease is still unknown,but it is clear that the degradation of articular cartilage is mediated by various factors, such as the production of metalloproteinases(MMPs)and other products resulting from cellular activity.Findings, TWEAK can induce a variety of cells secreting proinflammatory, cytokines and chemokines and high expression of MMPs and RANTES.The objective of our study was to investigate the mechanism of how TWEAK involves in deterioration of articular cartilage through OA chondrocytes culture in vitro, and then to found the new therapy of OA.
Objective:
1 To establish a cultivation method for human osteoarthritic chondrocytes in vitro;
2 To observe the expression of TWEAK in human osteoarthritic chondrocytes;
3 To investigate the effects of TWEAK on the synthesis of MMP-9 in chondrocytes of osteoarthritis at different concentrations and to discuss the relative mechanism of how TWEAK involves in the destruction of cartilage. 4 To investigate the effects of TNF-αand IL-1βon the activity of MMP-9 that stimulated with TWEAK.
Methods:
1 Human articular cartilage was obtained from OA patients who underwent total hip or total knee replacement.Cells were isolated by sequential digestion with 0.25% proteinase and 0.2% collagenaseⅡwith H-DMEM.
2 To prepare cell coverslips and detect expression of TWEAK in chondrocytes by immunofluorescence.
3 Chondrocytes were co-cultured and stimulated with TWEAK at different concentration. ELISA was used to detect concentration of MMP-9 in cell-cultured supernatant fluid. The gene mRNA level of MMP-9 was measuured by RT-PCR.
Results:
1 Samples selected from 42 osteoarthritic patients ,of which 30 specimens succeed to obtain cells and serial subcultivation.
2 Immunofluorescence revealed that the TWEAK expressed in osteoarthritic chondrocytes widespreadly and mainly located in cytoplasm.
3 The level of MMP-9 induced by TWEAK at 50μg/L and 100μg/L was higher than in control group, which had significant statistic difference(P<0.05). the expression level of MMP-9 mRNA induced by TWEAK at 100 ug/L was 1.28 times higher than that in the control group, which had significant statistic difference(P<0.05).
4 TNF-αand IL-1βhad synergetic effect on the synthesis and mRNA expression of MMP-9.The level in the synergetic group was significantly higher than that in the simple TWEAK group , which had significant statistic difference (P<0.05).
Conclusion:
1 Our study confirmed that chondrocyte isolation by sequential digestion with 0.25% proteinase and 0.2% collagenaseⅡwas good for human osteoarthritic chondrocytes.
2 Immunofluorescence revealed that the TWEAK widespreadly expressed in osteoarthritic chondrocytes and mainly located in cytoplasm.
3 TWEAK can induce osteoarthritic chondrocytes to synthesize MMP-9 and damage the articular cartilage directly and play a partin in pathogenesy of OA.
4 TNF-α,IL-1βand TWEAK had synergetic effects during the synthesis of MMP-9 in human osteoarthritic chondrocytes.
目的:
1建立软骨细胞体外培养体系;
2观察TWEAK分子在软骨细胞的表达情况;
3研究TWEAK在不同浓度下诱导体外培养的骨关节炎软骨细胞合成MMP-9的影响;
4观察TNF-α和IL-1β对TWEAK诱导软骨细胞合成MMP-9的影响。
1收集行关节置换的骨关节炎患者关节软骨组织,用胰蛋白酶-Ⅱ型胶原酶顺序消化法分离培养原代软骨细胞;
2制作细胞爬片,应用免疫荧光法检测TWEAK分子的表达;
3将rhTWEAK与软骨细胞共培育(其中协同作用组分别加入TNF-α和IL-1β),应用ELISA法检测刺激后培养液中MMP-9的水平;用逆转录-聚合酶链反应(RT-PCR)检测软骨细胞中MMP-9 mRNA的表达水平。
结果:
1总共收集到42例符合标准的骨关节炎关节软骨标本,成功培养并传代的有30例,总成活率为71.4%;
2 TWEAK在OA软骨细胞中广泛表达,且主要定位于细胞浆;
3当TWEAK终浓度为50、100μg/L时,其诱导OA软骨细胞合成MMP-9的水平明显高于对照组,具有显著的统计学意义(p<0.05);TWEAK终浓度为100μg/L时,其诱导OA软骨细胞MMP-9 mRNA的表达水平为对照组的1.28倍,具有显著的统计学意义(p<0.05)。
4与单用TWEAK组比较,TNF-α和IL-1β协同作用组诱导软骨细胞合成MMP-9的水平明显高于对照组,具有显著的统计学意义(p<0.05),而协同作用组之间无统计学意义。
结论:
1应用胰蛋白酶-Ⅱ型胶原酶顺序消化法分离培养软骨细胞是一种可行的软骨细胞原代培养方法,与国内外的报道相一致;
2 TWEAK在OA软骨细胞中广泛表达且主要定位于细胞浆中;
3 TWEAK通过诱导OA软骨细胞合成MMP-9,直接参与OA关节软骨的破坏,从而在OA的发病中起作用。方法:
4 TNF-α和/或IL-1β在TWEAK诱导OA软骨细胞合成MMP-9的过程中起协同作用。
Background:TWEAK(TNF-like weak inducer of apoptosis), is a novel member of the TNF superfamilies, also known as TNFSF12(TNF-superfamily member 12). TWEAK is a multifunctional cytokine which has extensive biological activities, plays an important role in pathogenesis of many diseases , especially in autoimmune diseases. Osteoarthritis(OA)is the most common disease of the joints, and one of the most widespread of all chronic diseases. Deterioration of articular cartilage is a hallmark of OA pathogenesis. Although the precise aetiology of this disease is still unknown,but it is clear that the degradation of articular cartilage is mediated by various factors, such as the production of metalloproteinases(MMPs)and other products resulting from cellular activity.Findings, TWEAK can induce a variety of cells secreting proinflammatory, cytokines and chemokines and high expression of MMPs and RANTES.The objective of our study was to investigate the mechanism of how TWEAK involves in deterioration of articular cartilage through OA chondrocytes culture in vitro, and then to found the new therapy of OA.
Objective:
1 To establish a cultivation method for human osteoarthritic chondrocytes in vitro;
2 To observe the expression of TWEAK in human osteoarthritic chondrocytes;
3 To investigate the effects of TWEAK on the synthesis of MMP-9 in chondrocytes of osteoarthritis at different concentrations and to discuss the relative mechanism of how TWEAK involves in the destruction of cartilage. 4 To investigate the effects of TNF-αand IL-1βon the activity of MMP-9 that stimulated with TWEAK.
Methods:
1 Human articular cartilage was obtained from OA patients who underwent total hip or total knee replacement.Cells were isolated by sequential digestion with 0.25% proteinase and 0.2% collagenaseⅡwith H-DMEM.
2 To prepare cell coverslips and detect expression of TWEAK in chondrocytes by immunofluorescence.
3 Chondrocytes were co-cultured and stimulated with TWEAK at different concentration. ELISA was used to detect concentration of MMP-9 in cell-cultured supernatant fluid. The gene mRNA level of MMP-9 was measuured by RT-PCR.
Results:
1 Samples selected from 42 osteoarthritic patients ,of which 30 specimens succeed to obtain cells and serial subcultivation.
2 Immunofluorescence revealed that the TWEAK expressed in osteoarthritic chondrocytes widespreadly and mainly located in cytoplasm.
3 The level of MMP-9 induced by TWEAK at 50μg/L and 100μg/L was higher than in control group, which had significant statistic difference(P<0.05). the expression level of MMP-9 mRNA induced by TWEAK at 100 ug/L was 1.28 times higher than that in the control group, which had significant statistic difference(P<0.05).
4 TNF-αand IL-1βhad synergetic effect on the synthesis and mRNA expression of MMP-9.The level in the synergetic group was significantly higher than that in the simple TWEAK group , which had significant statistic difference (P<0.05).
Conclusion:
1 Our study confirmed that chondrocyte isolation by sequential digestion with 0.25% proteinase and 0.2% collagenaseⅡwas good for human osteoarthritic chondrocytes.
2 Immunofluorescence revealed that the TWEAK widespreadly expressed in osteoarthritic chondrocytes and mainly located in cytoplasm.
3 TWEAK can induce osteoarthritic chondrocytes to synthesize MMP-9 and damage the articular cartilage directly and play a partin in pathogenesy of OA.
4 TNF-α,IL-1βand TWEAK had synergetic effects during the synthesis of MMP-9 in human osteoarthritic chondrocytes.
引文
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