【作者】 周惠琼；

【导师】 张奉春； Rob K Will； 徐家科；

【作者基本信息】 中国协和医科大学 ， 风湿病学， 2007， 博士

【摘要】 研究背景:骨关节炎(osteoarthritis,OA)是一种关节软骨进行性消失,骨质过度增生,临床表现为活动后加重的慢性关节疼痛、关节僵硬、肥大及活动受限的常见风湿病。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的失衡。既往的研究认为OA是一种非炎症性的关节病变,但近年来大量的研究证实异常炎症反应在OA的发病中起着重要的作用。多种细胞因子均参与OA的发病,如TNF-α和IL-1β都在其中扮演着重要的角色。对OA发病机制的重新认识将给OA的治疗提供更多的选择。尽管目前有多种药物治疗OA,包括控制症状的药物和另一大类改善病情的药物及软骨保护剂,但即使这些药物能够控制炎症与疼痛,内在的软骨组织和骨组织仍不能受到很好的保护和修复,OA的预后还不尽人意,仍有不少的患者最终需行关节置换。目前的研究认为蛋白聚糖的异常降解在OA的发病及进展中都起着重要的作用,而前炎症因子导致一些蛋白酶活性的增高,从而促进蛋白聚糖的降解是OA发病的主要原因之一。所以近年来治疗关节炎的发展趋势是寻找降解软骨组织的蛋白酶的抑制剂,保护基质完整,从根本上治疗OA。OA的炎症发病机制给我们寻找新的治疗OA的靶位提供了理论依据。菊蒿是一种草本类植物,它的主要药物活性成分为小白菊内酯(parthenolide,PAR)。大量的研究证实PAR对多种细胞具有较强的抗炎免疫抑制作用,促使我们想通过有关实验了解PAR是否对OA的关节软骨细胞也有免疫抑制作用,是否能保护OA软骨细胞的蛋白聚糖免受异常降解,为OA的治疗提供新的药物选择。目的:利用体外培养的人OA关节软骨细胞体系,通过不同的测定方法观察三种细胞因子(TNF-α、IL-1β和LPS)对体外培养的人OA关节软骨细胞的蛋白聚糖的影响;并通过相同的实验体系初步了解PAR是否对人OA软骨细胞的蛋白聚糖代谢具有保护作用。方法:本研究分为三部分:①软骨细胞的体外培养和细胞活性的鉴定;②细胞因子TNF-α、IL-1β及LPS对软骨细胞蛋白聚糖代谢的调节作用;③PAR对TNF-α、IL-1β及LPS诱导的软骨细胞蛋白聚糖代谢异常的保护作用。具体方法如下:1.软骨细胞取自人OA膝关节置换术后的股骨髁。采用第一代体外培养的软骨细胞,用MTT法检测细胞的存活率,以细胞存活率大于95%为标准,选择合适的细胞因子及PAR的作用浓度。2.将软骨细胞分为不同条件的培养组,分别为:对照组、PAR组、IL-1β组、TNF-α组、LPS组、PAR+IL-1β组、PAR+TNF-α组及PAR+LPS组。3.用二甲基亚甲兰(1,9-dimethylmethylene blue,DMMB)分光光度法测定不同组软骨细胞及培养液中的GAG含量,计算GAG释放入培养液中的百分率。此为蛋白聚糖降解的标记物,比较各组间的差别。4.应用抗蛋白聚糖单克隆抗体(Mab)-5D4及3B3,分别采用ELISA法及WesternBlotting法,检测并比较不同条件培养液中的两种蛋白聚糖代谢片段的含量。其中Mab-5D4可检测蛋白聚糖核心蛋白的KS链,Mab-3B3可检测蛋白聚糖核心蛋白的CS链。5.应用半定量RT-PCR方法,测定不同条件培养的软骨细胞中8种细胞成分的mRNA表达。8种细胞成分分别为:TNF-α、IL-1β、蛋白聚糖、ADAMTS-4、ADAMTS-5、TIMP-1、COX-Ⅱ及MAPK-1。结果:1.在一定作用浓度的各种细胞因子及PAR作用下,除了LPS组的部分细胞形态略有梭形变外,其余各组细胞形态在光镜下无明显差别;各组细胞的存活率均大于95%。2.TNF-α、IL-1β和LPS组软骨细胞GAG释放的百分率均明显高于对照组,同时培养液中的5D4片段也明显高于对照组;LPS组培养液中3B3片段明显低于对照组,而TNF-α、IL-1β组培养液中的3B3片段与对照组比较无差别。3.TNF-α、IL-1β和LPS可不同程度地影响软骨细胞内不同细胞因子的mRNA表达:①三种细胞因子均可上调软骨细胞内的ADAMTS-4、TNF-α、IL-1β及COX-Ⅱ的mRNA表达;同时下调蛋白聚糖的mRNA表达。②TNF-α和LPS可促进培养的软骨细胞内ADAMTS-5及MAPK-1的mRNA表达:而IL-1β组此两种细胞成分的mRNA表达与对照组无差别。③三种细胞因子对软骨细胞的TIMP-1mRNA表达均无影响。4.PAR+IL-1β组、PAR+TNF-α及PAR+LPS组的软骨细胞GAG的释放率均低于其相应的单独细胞因子组。说明PAR可分别抑制TNF-α、IL-1β及LPS所导致的体外培养的人OA关节软骨细胞GAG的释放。5.PAR+IL-1β组、PAR+TNF-α及PAR+LPS组的培养液中5D4片段均低于其相应的单独细胞因子组,即PAR可抑制这三种细胞因子所导致的软骨细胞蛋白聚糖核心蛋白区硫酸角质素链的降解。6.RT-PCR的结果显示:①PAR可下调TNF-α、IL-1β及LPS所导致的体外培养的人OA关节软骨细胞内ADAMTS-5及TNF-α的mRNA过度表达;并同时上调蛋白聚糖mRNA表达。②PAR可下调IL-1β所致的ADAMTS-4的mRNA过度表达;③PAR可下调TNF-α及IL-1β所致的IL-1β的mRNA过度表达:④PAR可下调TNF-α及LPS所致的COX-Ⅱ及MAPK-1的mRNA过度表达。结论:1.在不改变细胞存活率的情况下,TNF-α、IL-1β及LPS可促进体外培养的人OA关节软骨细胞蛋白聚糖的降解,尤其是核心蛋白区硫酸角质素链的降解。推断三种细胞因子在OA的发病中起着一定的作用;而PAR可不同程度地减轻以上三种细胞因子所导致的蛋白聚糖降解。2.TNF-α、IL-1β和LPS可不同程度地上调软骨细胞内多种与炎症相关的细胞因子的mRNA表达,同时下调蛋白聚糖的mRNA表达,说明三种细胞因子可能在不同的细胞功能水平作用于软骨细胞,导致软骨细胞功能的受损。3.PAR可不同程度地抑制TNF-α、IL-1β和LPS所导致的软骨细胞多种炎症细胞因子的基因过度表达,而同时上调软骨细胞蛋白聚糖的mRNA表达,推测PAR可能具有保护软骨细胞功能的潜在作用。4.PAR可望成为治疗OA药物的新选择,值得进一步做相关的研究。更多还原

【Abstract】 BackgroundOsteoarthritis(OA) is a slowly progressive degenerative disease characterized by gradual loss of articular cartilage,but the mechanism of OA pathology is still unknown.Biochemical and genetic factors contribute to the OA lesions in cartilage by disrupting chondrocyte-matrix associations and altering metabolic responses in the chondrocyte.OA is characterized by a progressive depletion of aggrecan from articular cartilage,with subsequent impairment of tissue function.The most abundant cartilage proteoglycan is aggrecan,comprising a protein core to which are attached many chondroitin sulphate(CS) and keratin sulphate(KS) chains.Monoclonal antibodies (Mab)which specifically against structural carbohydrate epitopes on the KS and CS side chains of aggrecan have been produced.The antibody 5D4 recognises KS neo-epitopes and has been used to estimate KS in body fluids as well as cultured media in vitro.Many studies found that the level KS epitopes are increased in OA patients.Certain neo-epitopes on CS chains recognised by the Mab 3B3 were also found to increase in OA patients.Although OA has been regarded primarily as a noninflammatory arthropathy, symptoms of local inflammation and synovitis are present in many patients and have been observed in animal models of OA.Proinflammatory cytokines,such as interleukin-1(IL-1),IL-17,IL-18 and tumor necrosis factor-α(TNF-α),are suspected of causing damage to OA cartilage and degradation of matrix components.The balance of synthesis and degradation of matrix components is disturbed,with both degradation and synthesis usually enhanced.The new insight of OA aetiology will be leading some new potential therapeutic applications.Extracts from plants of sesquiterpene lactones have been used as folk remedies for migraine,inflammatory arthritis,and tumors.Parthenolide(PAR),one of the major products from sesquiterpene lactones is an active ingredient responsible for the anti-inflammatory properies.Because of the key role of cytokines in the pathegenesis of OA,our interest in this field prompted us to hypothesize that PAR,which has been reported to be anti-inflammatory agent on several kinds of cells,might have anti-inflammatory ability to OA chondrocyte and protect degradation of aggrecan.Objective1.To evaluate the effects of TNF-α,IL-1βand LPS on aggrecan metabolism in cultured human chondrocytes with OA.2.To investigate whether these three cytokines can regulate the gene expresstions of 8 different cell ingredients.3.Further to study the possible inhibition of PAR in cartilage aggrecan depletion induced by three cytokines(TNF-α,IL-1βand LPS) and PAR may have potential therapeutic effect in OA.Methods1.Human Chondrocytes were cultured with different stimulated subjects for up to 8 days.The cytotoxic effect of the experimental substances was evaluated by a cell viability test based on the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) by mitochondrial dehydrogenases of metabolically active cells.2.Cultured chondrocytes were classified into 8 groups;they are control,TNF-α, IL-1β,LPS,PAR,PAR+TNF-α,PAR+IL-1βand PAR+LPS.3.The content and release of aggrecan in chondrocyte and conditional culture media as sulfated glycosaminoglycan(GAG),which is primarily a measure of aggrecan content.GAG content was measured by using a modification of a 1,9-dimethylmethylene blue spectrophotometric assay(DMMB).GAG release into medium was represented as a percentage of the total cumulative GAG in the chondrocyte plus cumulative GAG release into medium.4.Aggrecan catabolic fragments in medium were measured by methods of ELISA and Western Blotting using anti-aggrecan monoclonal antibodies 3B3 and 5D4.5.RNA was isolated from chondrocyte with different culture condition and analyzed for expression of aggrecan,A disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS-4),ADAMTS-5,TIMP-1,COX-Ⅱ,MAPK-1, TNF-α,IL-1βand 18S by reverse transcription-polymerase chain reaction (RT-PCR).Results1.The phenotypes of different conditional cultured chondrocyte were no difference in light microscope except LPS group and the cell viability rate were all more than 95%in 8 groups.2.Levels of GAG in medium samples of three groups(TNF-α,IL-1βand LPS) increased significantly comparing with control group.Fragments of 5D4 in medium of three groups also experienced significantly increases at the same time. There was significant decrease of 3B3 level in group LPS than control,whereas 3B3 fragments had no difference among TNF-α,IL-1βand control groups.3.TNF-α,IL-1βand LPS can alter gene expression of some cytokines in cultured chondrocyte.①Levels of aggrecan mRNA in three groups were lower than control,and the gene expression of ADAMTS-4、TNF-a、IL-1βand COX-Ⅱwas up-regulated in three groups comparing the control.②TNF-a and LPS up-regulated ADAMTS-5 and MAPK-1 gene expression while there was no effect of IL-1βon these two kinds of gene expression.③There was no difference of TIMP-1 gene expression among four groups.4.Levels of GAG in medium of PAR+TNF-α,PAR+IL-1βand PAR+LPS group decreased significantly comparing to TNF-α,IL-1βand LPS groups,respectively. PAR produced dose-dependent decreases of GAG release in OA chondrocyte induced by three studied cytokines.5.Fragments of 5D4 in medium of PAR+TNF-α,PAR+IL-1βand PAR+LPS group decreased significantly comparing to TNF-α,IL-1βand LPS groups,respectively. PAR can inhibit the degradation of KS chains in aggrecan stimulated by three cytokines.6.RT-PCR results showed:①PAR down-regulated gene expression of ADAMTS-5 and TNF-αin cultured chondrocyte stimulated by TNF-α,IL-1βand LPS,and up-regulated aggrecan gene expression at the same time.②PAR inhibited gene expression of ADAMTS-4 in chondrocyte induced by IL-1β,whereas there was no difference of ADAMTS-4 gene expression in chondrocyte when stimulated by combination of TNF-αand PAR,LPS and PAR,respectively.③IL-1βgene expression induced by TNF-αand IL-1βwas suppressed by PAR.④PAR inhibited COX-Ⅱand MAPK-1 gene expression of chondrocyte induced by TNF-αand LPS.Conclusion1.TNF-α,IL-1βand LPS can stimulate aggrecan catabolism in cultured human OA chondrocyte;especially the degradation of KS chains in core protein of aggrecan. These three cytokines may play an important role in pathogenesis of OA.2.TNF-a,IL-1βand LPS can up-regulate different kinds of pro-inflammatory cytokines to some degree,and suppress aggrecan gene expression.3.PAR can inhibit aggrecan catabolism of chondrocyte induced by TNF-a,IL-1βand LPS. 4.PAR can down-regulate gene expression of proinflammatory cytokines and aggrecanase,such as TNF-αand ADAMTS-5,in chondrocyte,and up-regulate aggrecan gene expression,indicating PAR may have chondrocyte protection in OA.5.PAR may have potential therapeutic effect in OA.更多还原