Objective: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD),the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. Methods: Chondroc...Objective: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD),the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. Methods: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA),soluble CD44 (sCD44),IL-1β and TNF-α levels in super-natants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was deter-mined by flow cytometry (FCM). CD44,hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13,3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. Results: T-2 toxin inhibited CD44,HAS-2,and aggrecan mRNA expressions,but promoted aggrecanase-2 mRNA expression. Meanwhile,CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition,ELISA results indicated that there were higher sCD44,IL-1β and TNF-α levels in T-2 toxin group. Similarly,higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore,using monoclonal antibodies BC-13,3-B-3 and 2-B-6,strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin,whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. Conclusion: T-2 toxin could inhibit aggrecan synthesis,promote aggrecanases and pro-inflammatory cytokines production,and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage,inducing aggrecan loss in the end,which may be the initiation of the cartilage degradation.展开更多
The ADAMTS(a disintegrin and metalloproteinase with thrombospondin motifs)family consists of 19 proteases.These enzymes are known to play important roles in development,angiogenesis and coagulation;dysregulation and m...The ADAMTS(a disintegrin and metalloproteinase with thrombospondin motifs)family consists of 19 proteases.These enzymes are known to play important roles in development,angiogenesis and coagulation;dysregulation and mutation of these enzymes have been implicated in many disease processes,such as inflammation,cancer,arthritis and atherosclerosis.This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions,focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis,including the aggrecanases(ADAMTS-4 and ADAMTS-5),ADAMTS-7 and ADAMTS-12,the latter two are associated with cartilage oligomeric matrix protein(COMP),a component of the cartilage extracellular matrix(ECM).We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels,including their interaction with substrates,induction by pro-inflammatory cytokines,protein processing,inhibition(e.g.,TIMP-3,alpha-2-macroglobulin,GEP),and activation(e.g.,syndecan-4,PACE-4).展开更多
目的:观察独活寄生汤血清对膝骨性关节炎退变软骨细胞Aggrecan和Collagen X mRNA表达的影响。方法:选择6例患者标本均来源于云南省中医医院骨科膝骨性关节炎住院患者,行手术截取的新鲜退变软骨组织,采用组织块法培养原代细胞;MTT法检测...目的:观察独活寄生汤血清对膝骨性关节炎退变软骨细胞Aggrecan和Collagen X mRNA表达的影响。方法:选择6例患者标本均来源于云南省中医医院骨科膝骨性关节炎住院患者,行手术截取的新鲜退变软骨组织,采用组织块法培养原代细胞;MTT法检测传代第一代细胞生长情况,并绘制生长曲线;制备独活寄生汤大鼠含药血清;均取传代第一代的退变软骨细胞,配制5%、10%、20%独活寄生汤含药血清及相应体积比浓度的生理盐水血清作为对照;MTT法检测不同浓度的独活寄生汤血清对退变软骨细胞增殖的影响;实时荧光定量PCR检测不同给药时间点各组退变软骨细胞Aggrecan和Collagen Ⅹ mRNA的表达。结果:退变软骨细胞传代第一代生长曲线见传代第8天时细胞增殖达到顶峰,故选择细胞传代第8天为给药干预时间点;与含同体积比的生理盐水血清组比较,不同浓度的独活寄生汤血清可对退变软骨细胞增殖产生促进作用(P<0.01),尤以10%独活寄生汤血清更为明显(P<0.05);与含同体积比生理盐水血清组比较,不同时间点的5%、10%、20%独活寄生汤血清干预后的退变软骨细胞Aggrecan mRNA的表达均上调(P<0.01),Collagen Ⅹ mRNA的表达均下调(P<0.01),且以10%独活寄生汤血清组的作用最为明显(P<0.05)。结论:独活寄生汤可能通过调节退变软骨组织的胶原及其蛋白多糖的表达而产生延缓膝骨性关节炎退变软骨组织的部分作用。展开更多
目的通过观察针刀干预对膝骨关节炎(KOA)兔行为学、髌韧带(PT)力学特性及膝关节软骨白介素4(IL-4)、基质金属蛋白酶-3(MMP-3)、聚集蛋白聚糖(Aggrecan)表达的影响,探讨针刀"调筋治骨"法治疗KOA的作用机制。方法将40只新西兰...目的通过观察针刀干预对膝骨关节炎(KOA)兔行为学、髌韧带(PT)力学特性及膝关节软骨白介素4(IL-4)、基质金属蛋白酶-3(MMP-3)、聚集蛋白聚糖(Aggrecan)表达的影响,探讨针刀"调筋治骨"法治疗KOA的作用机制。方法将40只新西兰兔随机分为空白组、模型组、针刀组和电针组,每组10只。模型组、针刀组和电针组采用改良后Videman伸直位固定法建立兔KOA模型。针刀组和电针组造模后分别给予针刀、电针治疗。造模后及治疗后采用改良Lequesne MG的膝关节级别评估法对各组进行行为学评价。治疗后取材,应用Bose Electro Force 3300疲劳试验机对PT进行拉伸、应力松弛和蠕变力学测试,应用ELISA方法检查软骨细胞IL-4表达,应用Real-time PCR法检测MMP-3、Aggrecan m RNA表达水平。结果造模后,模型组Lequesne MG评分与空白组比较,差异具有统计学意义(P<0.01);针刀组、电针组Lequesne MG评分与模型组比较,差异均无统计学意义(P>0.05)。针刀组和电针组治疗后Lequesne MG评分与模型组比较,差异均有统计学意义(P<0.01,P<0.05);针刀组治疗后Lequesne MG评分与电针组比较,差异具有统计学意义(P<0.05)。模型组PT最大应力、最大位移、弹性模量及应力松弛率、蠕变率与空白组比较,差异均具有统计学意义(P<0.01,P<0.05)。针刀组治疗后PT最大应力、最大位移、弹性模量及应力松弛率、蠕变率与模型组比较,差异均有统计学意义(P<0.01,P<0.05);电针组治疗后弹性模量与模型组比较,差异有统计学意义(P<0.01)。模型组造模后IL-4含量、Aggrecan m RNA表达均显著下降,MMP-3 m RNA表达显著上升,与空白组比较差异均有统计学意义(P<0.01,P<0.05);针刀组及电针组治疗后IL-4含量较模型组明显升高(P<0.01,P<0.05),两组Aggrecan m RNA表达均有上升趋势,MMP-3 m RNA表达均有下降趋势,针刀组在调整Aggrecan、MMP-3 m RNA表达方面优于电针组,但与模型组比较差异均展开更多
基金Project supported by the National Natural Science Foundation of China (Nos. 30471499 and 30170831)the Ministry of Education of China (No.Key 03152)the Science Foundation of Shaanxi Province of China (No.2004KW-20)
文摘Objective: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD),the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro. Methods: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA),soluble CD44 (sCD44),IL-1β and TNF-α levels in super-natants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was deter-mined by flow cytometry (FCM). CD44,hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13,3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro. Results: T-2 toxin inhibited CD44,HAS-2,and aggrecan mRNA expressions,but promoted aggrecanase-2 mRNA expression. Meanwhile,CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition,ELISA results indicated that there were higher sCD44,IL-1β and TNF-α levels in T-2 toxin group. Similarly,higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore,using monoclonal antibodies BC-13,3-B-3 and 2-B-6,strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin,whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above. Conclusion: T-2 toxin could inhibit aggrecan synthesis,promote aggrecanases and pro-inflammatory cytokines production,and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage,inducing aggrecan loss in the end,which may be the initiation of the cartilage degradation.
文摘The ADAMTS(a disintegrin and metalloproteinase with thrombospondin motifs)family consists of 19 proteases.These enzymes are known to play important roles in development,angiogenesis and coagulation;dysregulation and mutation of these enzymes have been implicated in many disease processes,such as inflammation,cancer,arthritis and atherosclerosis.This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions,focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis,including the aggrecanases(ADAMTS-4 and ADAMTS-5),ADAMTS-7 and ADAMTS-12,the latter two are associated with cartilage oligomeric matrix protein(COMP),a component of the cartilage extracellular matrix(ECM).We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels,including their interaction with substrates,induction by pro-inflammatory cytokines,protein processing,inhibition(e.g.,TIMP-3,alpha-2-macroglobulin,GEP),and activation(e.g.,syndecan-4,PACE-4).
文摘目的通过观察针刀干预对膝骨关节炎(KOA)兔行为学、髌韧带(PT)力学特性及膝关节软骨白介素4(IL-4)、基质金属蛋白酶-3(MMP-3)、聚集蛋白聚糖(Aggrecan)表达的影响,探讨针刀"调筋治骨"法治疗KOA的作用机制。方法将40只新西兰兔随机分为空白组、模型组、针刀组和电针组,每组10只。模型组、针刀组和电针组采用改良后Videman伸直位固定法建立兔KOA模型。针刀组和电针组造模后分别给予针刀、电针治疗。造模后及治疗后采用改良Lequesne MG的膝关节级别评估法对各组进行行为学评价。治疗后取材,应用Bose Electro Force 3300疲劳试验机对PT进行拉伸、应力松弛和蠕变力学测试,应用ELISA方法检查软骨细胞IL-4表达,应用Real-time PCR法检测MMP-3、Aggrecan m RNA表达水平。结果造模后,模型组Lequesne MG评分与空白组比较,差异具有统计学意义(P<0.01);针刀组、电针组Lequesne MG评分与模型组比较,差异均无统计学意义(P>0.05)。针刀组和电针组治疗后Lequesne MG评分与模型组比较,差异均有统计学意义(P<0.01,P<0.05);针刀组治疗后Lequesne MG评分与电针组比较,差异具有统计学意义(P<0.05)。模型组PT最大应力、最大位移、弹性模量及应力松弛率、蠕变率与空白组比较,差异均具有统计学意义(P<0.01,P<0.05)。针刀组治疗后PT最大应力、最大位移、弹性模量及应力松弛率、蠕变率与模型组比较,差异均有统计学意义(P<0.01,P<0.05);电针组治疗后弹性模量与模型组比较,差异有统计学意义(P<0.01)。模型组造模后IL-4含量、Aggrecan m RNA表达均显著下降,MMP-3 m RNA表达显著上升,与空白组比较差异均有统计学意义(P<0.01,P<0.05);针刀组及电针组治疗后IL-4含量较模型组明显升高(P<0.01,P<0.05),两组Aggrecan m RNA表达均有上升趋势,MMP-3 m RNA表达均有下降趋势,针刀组在调整Aggrecan、MMP-3 m RNA表达方面优于电针组,但与模型组比较差异均
文摘目的探讨人胰岛素样生长因子(human insulin-like growth factor,hIGF-1)基因对软骨细胞分泌聚集蛋白多糖(aggrecan)、Ⅱ型胶原的影响.方法构建并鉴定携带hIGF-1基因的重组腺病毒(Ad/CMV-hIGF-1),采用1、10、100及500感染复数单位(multiplicity of infection,MOI)的Ad/CMV-hIGF-1转染软骨细胞,PBS为阴性对照,100μg/L hIGF-1生长因子为阳性对照,转染后4 d进行aggrecan、Ⅱ型胶原免疫组化,参照文献分析免疫组化阳性单位.结果在1～100MOI范围内,随着Ad/CMV-hIGF-1滴度增加,aggercan、Ⅱ型胶原表达递增,500 MOI Ad/CMV-hIGF-1转染后,aggrecan表达骤减;Ⅱ型胶原表达下降不明显.结论 hIGF-1基因对软骨细胞aggrecan、Ⅱ型胶原的表达有影响;100 MOI Ad/CMV-hIGF-1优于100 μg/L hIGF-1生长因子的作用.
