【作者】 夏军；

【导师】 黄煌渊； 俞永林；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 背景骨关节炎(osteoarthritis OA)是一种慢性、渐进性、退行性关节病变,是临床上最常见的关节疾病。据统计,在50岁以上人群中,OA的发病率仅次于心脏病列第二位。作为高发病率、高致残率的常见关节疾病,一直受到国内外学者的重视。但是由于其细胞信号转导以及各信号通路之间的交叉对话极其纷繁复杂,迄今为止,其具体发病机制仍然无法完全清楚。目前研究认为,软骨局部基质MMPs的异常增高可能是导致软骨细胞外基质合成与降解失衡的重要原因。OA是力学因素和生物学因素作用下软骨合成和降解偶联失衡的结果,细胞因子学说目前并不能解释OA软骨破坏的全部原因,但是不论软骨的哪个部位以何种形式发生退变,都不能脱离IL-1等炎症介质的作用。近年来,针对IL-1诱导MMPs增高机制方面的研究发现包括MAPK,NF-κB等信号通路在内的信号转导系统在此过程中发挥重要作用。本研究在目前国内外诸多研究的基础上,制作膝关节OA动物模型。检测造模手术后不同时间点关节软骨内MMP-1/-13和MAPKs的mRNA以及蛋白表达,初步探讨上述蛋白的时相规律和相互关系。并使用阻滞剂阻断ERK1/2信号转导通路,评价该信号通路的作用。通过上述研究,探讨MMPs与MAPKs表达的时相差异,分析二者之间的规律性,从信号通路水平为OA的防治提供理论基础。第一部分兔骨关节炎模型的建立以及造模后不同时间点关节软骨中MMP-1/-13 mRNA和蛋白表达的研究目的评价家兔膝关节前交叉韧带切断(ACLT)及内侧半月板部分切除制作骨关节炎(OA)模型方法的可行性,同时探索关节炎发病的不同时期关节软骨中MMP-1,MMP-13的基因表达、蛋白表达水平的变化情况。材料与方法实验组家兔30只行ACLT及内侧半月板前1/3切除术造模,分别于术后4周、8周、12周处死10只,假手术对照组家兔10只于术后12周处死。进行股骨髁关节软骨退变的大体评分;取退变的软骨组织,用实时定量PCR(real-time PCR)及Western blot印记杂交方法测定软骨组织中MMP-1和MMP-13的mRNA和蛋白表达水平。结果ACLT及内侧半月板前1/3切除术后4周即可见软骨退变,8周和12周时退变进一步加重,对照组无明显软骨退变,不同组动物关节软骨评分有统计学差异。MMP-1的mRNA表达和蛋白表达在正常对照组水平均较低,造模4周开始升高,到第8周及第12周持续升高,且mRNA表达和蛋白表达升高的趋势相同;MMP-13在造模4周时表达开始升高,mRNA表达和蛋白表达趋势相同,造模第8周时持续升高,但在造模第12周时MMP-13 mRNA表达和蛋白表达下降。结论家兔膝关节ACLT及内侧半月板前1/3切除制作OA模型的方法简便可行,MMP-1和MMP-13在骨关节炎的发病过程中有不同程度的升高,可以作为OA研究的评价指标。本实验结果提示,在OA发展的过程中,MMP-1和MMP-13的表达不平行,二者调控通路可能存在差异。MMP-13的mRNA表达的变化趋势与其蛋白水平的变化趋势相符合,说明引起MMP-13在OA病程中晚期下降的调控发生在翻译前水平。第二部分兔OA模型不同时间点关节软骨中MAPKs信号通路激酶表达以及活化状况的研究目的检测兔OA模型关节软骨组织中MAPKs信号通路激酶表达以及活化的时相变化。材料与方法40只家兔制作OA模型,分别于术后4周、8周、12周处死10只,另外10只作为对照。取关节软骨,用real-time RT-PCR测定MAPKs mRNA表达状况;用Western blot方法测定MAPKs总蛋白以及磷酸化状态蛋白表达的状况。结果造模术后几个时间点MAPKs(包括ERK1/2,P38 MAPK,JNK)的基因表达与蛋白表达均无明显变化;与正常对照相比,磷酸化ERK1/2在术后4、8、12周均有高水平表达,磷酸化P38 MAPK术后4周高水平表达,磷酸化JNK术后4周、8周高水平表达,其他时间点差异无统计学意义。结论骨关节炎软骨组织中MAPKs的活化主要依赖于信号通路蛋白的磷酸化,而非总蛋白表达的上调。信号通路蛋白的活化存在时相差异。第三部分ERK-1/2激酶通路抑制剂PD98059对家兔关节软骨保护作用以及机制的初步观察目的观察MAPK信号转导通路中ERK激酶1/2(MEK-1/2)选择性阻滞剂PD98059在骨关节炎家兔模型活体使用对骨关节炎关节软骨的保护作用,测定该阻滞剂对关节软骨中ERK1/2及其活化状态磷酸化ERK1/2和基质金属蛋白酶-1/-13(MMP-1/-13)的作用,评价ERK1/2信号转导通路在骨关节炎发病过程中的作用。材料与方法30只家兔制作OA模型后随机分3组,关节腔内分别注射100μM PD98059,40μM PD98059和PBS安慰剂进行干预,另取10只家兔作为正常对照。8周后处死家兔,观察关节软骨破坏情况,用real-time RT-PCR测定MMP-1/-13 mRNA表达状况;用Western blot方法测定MMP-1/-13和ERK1/2总蛋白以及磷酸化状态蛋白表达的状况。结果与使用PBS安慰剂的对照组相比,使用PD98059的干预组关节软骨破坏明显减轻,评分后不同剂量的阻滞剂组和安慰剂对照组差别有统计学意义。不同组总ERK1/2表达无差异,磷酸化的ERK1/2在100μM PD98059干预组明显低于40μM干预组和安慰剂对照组。而MMP-1和MMP-13的mRNA表达则在不同浓度的干预组均低于安慰剂对照组。结论MAPK信号转导通路参与了骨关节炎关节软骨破坏的病理过程。MEK-1/2选择性阻滞剂PD98059可以在关节炎动物活体内有效抑制关节炎软骨破坏的程度,该作用可能与其有效抑制ERK-1/2的磷酸化激活有关。更多还原

【Abstract】 BackgroundOsteoarthritis(OA) is chronic and degenerative disease of joint and is one of the most common diseases.According to statistics,the incidence of OA is the second in the people above 50 years old,only lower than that of heart disease.And the incidence is higher in the people suffering from limbs deformity or joint trauma.Previous researches in the field of OA has characterized that OA is the result of disequilibrium between cartilage degeneration and synthesis controlled by the mechanic and biologic factors.The effect of inflammation mediators,such as interleukin-1(IL-1),is necessary in the cartilage degeneration although cytokines is not enough.It has been certificated that mitogen-activated protein kinases(MAPKs)and nuclear factor-κB(NF-κB) signaling pathways participate in the process IL-1β-induced MMP-1 express and chondrocytes apoptosis.The primary goal of the present study was to establish rabbit OA model and to detect the mRNA and protein expressions of MMP-1/13 and MAPKs.The second goal was to evaluate the effect of ERK1/2 signaling pathway through block it with PD98059.On the base of above results,we discussed the phase difference and relationships between MMPs and MAPKs.PartⅠEstablishment of rabbit osteoarthritis model and expression of MMP-1/-13 in osteoarthritic joint cartilage.Objectives To evaluate the feasibility of animal model of osteoarthritis(OA) by anterior cruciate ligament transaction(ACLT) and excision of partial medial meniscus,and to explore the mRNA and protein expression of matrix metalloproteinase(MMP)-1 and MMP-13 in joint cartilage of rabbit knee at different time after ACLT and partial excision of medial meniscus.Material and methods Thirty rabbits in experimental groups received unilateral ACLT and partial excision of medial meniscus,another 10 rabbits received unilateral arthrotomy as pseudo-operation control.Ten experimental rabbits were sacrificed at 4,8 and 12 weeks after surgery respectively,and 10 pseudo-operation rabbits were sacrificed at 12 weeks after surgery.Knees were harvested to observe the macropathologic changes of joint cartilage.Cartilage near the lesion were obtained,and mRNA and protein ofMMP-1 and MMP-13 were detected by real-time PCR and western blot respectively.Results Cartilage degeneration was obvious at 4 weeks and became more severe at 8 and 12 weeks after operation,while cartilage still remained normal at 12weeks after pseudo-operation.The mRNA and protein expression of MMP-1 and MMP-13 were all lower in normal cartilage,while the mRNA and protein expression of those two proteins increased in 4 and 8 weeks after operation.At 12 weeks after operation,mRNA and protein expression ofMMP-1 still remained high,while mRNA and protein expression of MMP-13 decreased respectively.Conclusions Anterior cruciate ligament transaction(ACLT) with partial excision of medial meniscus is a good method to make osteoarthritis model.MMP-1 and MMP-13,especially MMP-1,are good markers in osteoarthritis study.MMP-13 expression was very different from that of MMP-1 at 12 weeks after operation,which suggest that their upstream signaling pathways are different,and the signaling pathway difference may be before translation because the mRNA and protein expression change of MMP-13 are in parallel.PartⅡExpression of MAPKs in osteoarthritic rabbits’ joint cartilageObjectives To examines the expression of MAPKs in osteoarthritic rabbits’joint cartilage on different time after ACLT and partial excision of medial meniscus.Material and methods Thirty rabbits in experimental groups received unilateral ACLT and partial excision of medial meniscus,another 10 rabbits received unilateral arthrotomy as pseudo-operation control.Ten experimental rabbits were sacrificed at 4,8 and 12 weeks after surgery respectively,and 10 pseudo-operation rabbits were sacrificed at 12 weeks after surgery.Knees were harvested to observe the macro pathologic changes of joint cartilage.Cartilage near the lesion was obtained,and mRNA and protein of MAPK(including ERK1/2,P38MAPK and JNK) were detected by real-time PCR and western blot respectively.Results There are no significant changes in mRNA and total protein of MAPK on different time.Compared with that of control group, phospho-ERK1/2 is in high level expression on 4 weeks,8 weeks and 12 weeks postoperation,Phospho-P38 MAPK is in high level expression on 4 weeks postoperation and phospho-JNK is in high level expression on 4 weeks,8 weeks postoperation respectively.Conclusions Activation of MAPKs in osteoarthritic cartilage dependent on not total proteins expression,but proteins phosphorylated. There are phase differences between different MAPK.PartⅢInhibition of ERK-1/2 Pathway Reduce Joint Cartilage Destruction in Rabbit Experimental Osteoarthritis in vivoObjectives Observe the protective effect of PD98059,a selective inhibitor of ERK kinase1/2(MEK-1/2) for joint cartilage in vivo in experimental rabbit osteoarthritis;and to evaluate the contribution of ERK1/2 signaling pathway in the course of OA.Material and methods 30 rabbits receiving unilateral ACLT and excision of partial medial meniscus were randomly separated into 3 groups. From the 4th day post operation,PD98059 or placebo were injected into operated articular cavity,twice a week.The first and second group received 100μM and 40μM PD98059 respectively,and the third group received PBS as placebo.Another group contains 10 rabbits without any prior surgery received PBS as control group.Rabbits were killed at 8 weeks after surgery;knees were harvested to observe the macropathologic changes of joint cartilage.Cartilage near the lesion were obtained, protein of ERK1/2 and phospho-ERK1/2 were detected through Western blot; expression of MMP-1 and MMP-13 were detected through both real-time PCR and Western blot.Results Knees treated with PD98059 showed decreased in joint cartilage destruction compared with those treated with PBS. Phospho-ERK1/2 in joint cartilage treated with 100μM PD98059 depressed obviously.Both mRNA and protein expression of MMP-1 and MMP-13 in all those treated with PD98059 were lower than the placebo control.Results Knees treated with PD98059 showed decreased in joint cartilage destruction compared with those treated with PBS. Phospho-ERK1/2 in joint cartilage treated with 100μM PD98059 depressed obviously.Both mRNA and protein expression of MMP-1 and MMP-13 in all those treated with PD98059 were lower than the placebo control.Conclusions MAPK signaling pathways play active roles in the course of OA.PD98059,a selective inhibitor of MAPK kinase1/2(MEK-1/2) effectively reduces joint cartilage destruction in vivo experimental osteoarthritis of rabbits and the protective effect is positively associated with inhibition of ERK-1/2 activation.更多还原