【作者】 张汉庆；

【导师】 熊昌源；

【作者基本信息】 湖北中医学院 ， 中医骨伤科学， 2009， 博士

【摘要】 目的膝关节骨性关节炎是临床常见病、多发病,随着社会人口的老龄化,该病越来越成为社会和家庭沉重的负担。该病以关节软骨变性、破坏、软骨下骨硬化为主要病理特点,其中关节软骨的退行性改变是骨性关节炎发病的病理基础和核心。本研究从对关节软骨中基质金属蛋白酶及其抑制剂着手,探讨葛根素对骨性关节炎关节软骨的影响,以及其治疗膝关节骨性关节炎的作用机制,初步阐明葛根素治疗骨性关节炎的作用机理。方法本实验设5组,将50只体重2kg(±200g)健康成年大白兔(雌雄各半)将动物随机分为正常组、模型组、生理盐水组、透明质酸组、葛根素组。随机抽出的10只作为正常组,其余40只采用Hulth方法进行造模,即通过切断内侧副韧带、内侧半月板前角、前交叉韧带的方法造模,造模手术1周后行驱赶4周,即得明显的KOA模型。待骨性关节炎模型建立后,正常组不做任何处理;模型组仅按Hulth法造模,不给药治疗;生理盐水组、透明质酸组、葛根素组分别于动物模型制备后的第1周开始在严格无菌操作下,膝关节内注射0.9%生理盐水、透明质酸、葛根素各0.3ml,其后每周注射2次,共10次,即用药5周。治疗结束1周后,分别空气栓塞处死,显露膝关节腔。①肉眼观察关节软骨、照相,观察形态学改变;并切取制备关节软骨标本,在光镜下观察组织学变化。②取股骨内髁退变软骨制成软骨组织切片,用免疫组化的方法检测各组软骨中基质金属蛋白酶-1(MMP-1)及及其抑制剂(TIMP-1)的表达。③用反转录-多聚酶链反应(RT-PCR)的方法检测各组软骨中MMP-1及TIMP-1mRNA表达。结果①肉眼见正常组关节面光滑有光泽,软骨透明无明显缺损和新生物物,滑膜无充血。造模组关节面不光滑,无光泽,软骨不透明,有明显缺损和新生物,滑膜色暗充血肿胀。葛根素组关节腔未见明显新生物,色泽较暗,软骨较光滑但透明差。生理盐水组表现同模型组;透明质酸组与葛根素组类似。光镜观察见葛根素组软骨细胞较平整,表层软骨细胞坏死变性较模型组和生理盐水组轻,炎性细胞渗出物少见,可见新生血管和成纤维细胞增生。②正常组有少许MMP-1及TIMP-1的阳性表达,模型组MMP-1的阳性细胞计数较正常组明显增多,表达量增加,而TIMP-1表达呈阴性,生理盐水组与模型组相似;葛根素组和透明质酸组MMP-1的阳性细胞数较模型组减少,表达量较模型组降低,葛根素组与模型组和生理盐水组比较减少明显有差异(P<0.05)。TIMP-1在葛根素组和透明质酸组中的表达均较低,且无显著的差异。③MMP-1和TIMP-1的mRNA阳性表达率:正常组MMP-1及TIMP-1在软骨组织mRNA表达较低,模型组MMP-1软骨组织mRNA表达与正常组比较显著增高(P<0.05),生理盐水组与模型组类似。透明质酸组和葛根素组MMP-1 mRNA表达高于正常组,较模型组低。TIMP-1在模型组呈阴性表达,生理盐水组与模型组类似;透明质酸组和葛根素组关节软骨中TIMP-1mRNA表达均较低,与模型组和生理盐水组比较有显著差异(P<0.05)。结论基质金属蛋白酶较能反映骨关节软骨代谢变化,它对关节软骨细胞外基质具有降解作用,对加重软骨退变破坏有着十分重要的关系。通过实验研究发现兔膝关节软骨组织的MMP-1表达明显升高,而TIMP-1表达极少,且软骨组织细胞外基质Ⅱ型胶原含量明显下降:而经过葛根素治疗的动物软骨中MMP-1的表达下降,TIMP-1的表达升高,说明葛根素能使关节软骨中MMP-1的含量降低,使得细胞外基质的胶原含量升高,说明葛根素具有一定的抑制细胞外基质中胶原的降解作用,从而有可能减缓骨性关节炎的病程进展,为葛根素治疗膝关节骨性关节炎提供了实验研究的依据。更多还原

【Abstract】 Objective:Knee osteoarthritis(KOA) is one of the most clinical common,frequently-occurring disease,with the aging population,the disease is increasingly becoming social and family burden.Articular cartilage degeneration in the disease, destruction,subchondral bone sclerosis as the main pathological features of the degenerative articular cartilage changes in the incidence of osteoarthritis(OA) and the core of the pathological basis.This study on the articular cartilage matrix metalloproteinases and their inhibitors tries to explore the effect of Puerarin(Pue) on OA articular cartilage, as well as its mechanism on treatment of KOA.Method:5 for the experimental group 50 weight 2kg(±200g) healthy adult rabbits(half male and female) animals were randomly divided into normal group,model group,Sodium Chloride group,Hyaluronic Acid group,Puerarin group by 50 healthy adult rabbits(half male and female),each one weights 2kg(±200g).Of 10 randomly selected as the normal group,and the remaining 40 were duplicated by Hulth technique,or cutting the medial collateral ligament,anterior horn of medial meniscus,anterior cruciate ligament model approach.After model surgery 1 week all rabbits were drived for 4 weeks,that is a model of KOA.Osteoarthritis model to be established,did not do anything to deal with the normal group;the model group only by Hulth model and non-drug treatment;Sodium Chloride group,Hyaluronic Acid(HA) group,Pue group in the first 1 week after preparation of animal models were put into strict aseptic operation,were given the knee joint injection of 0.9%Sodium Chloride,HA,Pue each 0.3ml,2 times per week,that is,5 weeks continuously.In 1 week after end of treatment,all were sacrificed by air embolism,and the knee joint cavity was exposed.①To observate,photograph and survey the morphological and histological changes of rabbits knees articular cartilage cartilage with eyes and microscope.②To detect the expression of MMP-1 and TIMP-1 by immunohistochemistry.③To survey the mRNA expression of MMP-1 and TIMP-1 by RT-PCR.Results:①Smooth articular surface shiny,transparent and no obvious defect in the cartilage and the new biological material,without synovial hyperemia were found by naked eye in normal group.Articular surface of the model is not smooth, matte,opaque cartilage there is an obvious defect and the new biology,synovial swelling congestive dark color.Articular cavity Pue group was no significant new biological,dark color, cartilage worse than the smooth but transparent.Sodium Chloride group was similar to the model group;HA group was similar to groups.Pue group is more formation of cartilage cells,cartilage cells,necrotic degeneration of the surface than the model group and the Sodium Chloride group with light rare exudation of inflammatory cells by microscope,we could see new blood vessels and fibroblasts.②2The expression rates of MMP-1 and TIMP-1:there was a little MMP-1 and TIMP-1 positive expression in normal group.In model group,MMP-1 positive cell count was significantly increased than the normal group,and its expression of TIMP-1 was negative.Sodium Chloride group was similar to model group.MMP-1 positive cells of Pue group were lower than the model group,decreased expression than the model group,HA group is similar to Pue group.TIMP-1 in the puerarin group and the expression of hyaluronic acid group were lower,and no significant differences.③The Positive expression rates of MMP-1 and TIMP-1 mRNA:the mRNA expression rates of MMP-1 and TIMP-1 in knee cartilage of nomal groups were very low.The mRNA expression rates of MMP-1 and TIMP-1 in knee cartilage of model group and significantly increased than nomal group(P<0.05).The expression of TIMP-1 was negative in model group.The mRNA expression rates of MMP-1 of Pue and HA group were higher than nomal group and lower than model group.The mRNA expression rate Of TIMP-1 Of the group of the Pue was significantly different than model the and Sodium Chloride group(P<0.05).The mRNA expresion rates of TIMP-1 in Pue group was very low and no significant difference(P>0.05).Conclusions:Matrix metalloproteinase is able to reflect changes in bone metabolism of articular cartilage,its articular cartilage extracellular matrix degradation have to increase the damage to cartilage degeneration,that has a very important relationship.The study found,through experimental animal model of cartilage tissue of MMP-1 expression was significantly increased,and TIMP-1 expression in very few,and the cartilage extracellular matrix of collagen typeⅡcontent decreased.After the treatment of animals cartilage MMP-1 expression decreased in Pue group,while TIMP-1 expression increased on articular cartilage,the extracellular matrix increases collagen content.That is,Puerarin could put off extracellular matrix collagen degradation with a certain degree, and may slow down the course of osteoarthritis progress.The study supply theory foundation for Puerarin on OA treatment.更多还原