关节内注射尿激酶型纤溶酶原激活物对关节软骨的实验研究

探讨了尿激酶型纤溶酶原激活物（urokinase-type plasminogen activator,uPA）对关节软骨的影响.选择健康新西兰大白兔42只,随机分为实验组和对照组,实验组24只,对照组18只;实验组在右膝关节内注射uPA 0.4μg/0.2mL,对照组注射等容量的生理盐水,在注射后4周、8周、12周分批取兔软骨进行组织学观察及电镜检查.结果显示：实验组关节软骨表面不规则、缺损及结构破坏,软骨细胞数目减少、纤维增生及异染性降低,潮线的完整性破坏.结果表明：尿激酶型纤溶酶原激活物可能是导致关节软骨破坏的相关因素之一.     

用尿激酶型纤溶酶原激活物建立兔骨关节炎模型的研究

为探讨在兔膝关节腔注射尿激酶型纤溶酶原激活物（urokinase-type plasminogen activator, uPA）,建立兔膝关节骨关节炎动物模型的可行性.选择健康新西兰大白兔42只并随机分为实验组和对照组,实验组24只,对照组18只;实验组在右膝关节内注射uPA0.4μg/0.2mL,对照组注射等容量的生理盐水,注射完成后在4周、8周、12周分批取兔滑膜进行组织学观察、取软骨行组织学观察及电镜检查.结果显示：实验组兔膝关节滑膜均存在不同程度增生、肥厚、水肿,光镜及电镜下见实验组关节软骨呈明显退行性变.Mankin＇s评分实验组得分显著高于对照组（P〈0.05）.结论：兔膝关节注射uPA可使滑膜发生炎症、关节软骨发生退变,而且随着时间退变的程度逐渐加重,这一模型可为研究膝关节OA的病因、发病机理及治疗方法提供实验工具.     

单关节类风湿性关节炎滑膜组织中uPA、uPAR、PAI-1蛋白表达及意义

为探讨尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,uPA)及其受体(urokinase-type plas-minogen activator receptor,uPAR)、纤溶酶原激活物抑制剂(plasminogen activator inhibitor,PAI-1)在单关节发病类风湿关节炎(rheumatoid arthritis,RA)的表达。采用免疫组化方法检测13例单关节发病RA、19例典型RA和20例正常滑膜组织中uPAu、PAR、PAI-1蛋白表达情况。结果显示:13例单关节RA滑膜组织中uPA、u PAR、PAI-1阳性表达主要分布在滑膜衬里细胞、滑膜下层单核细胞及血管内皮细胞,阳性表达部位与典型RA相同,但表达强度明显低于典型RA(P<0.05)。与正常滑膜组织相比,单关节RA滑膜中uPA、uPAR、PAI-1蛋白表达明显增高(P<0.05)。由此可知:单关节RA滑膜组织中uPA、uPAR、PAI-1表达明显低于典型RA滑膜组织,可能与单关节RA为典型RA病程早期阶段,滑膜中uPA、uPAR、PAI-1蛋白尚处于低水平表达阶段有关。     

骨关节炎滑膜uPA、uPAR、PAI-1表达及意义

探讨尿激酶型纤溶酶原激活物(uPA)及其受体(uPAR)和其抑制剂(PAI-1)在OA的发生发展中的意义。方法:采用免疫组化方法检测OA滑膜组织中uPA,uPAR,PAI-1的表达。结果显示:36例OA滑膜中阳性表达uPA 29例(80.56%)、uPAR 25例(69.44%)P、AI-1 27例(75.00%)。21例对照滑膜中阳性表达uPA 6例(28.57%)、uPAR 6例(28.57%)、PAI-1 3例(14.29%)。实验组与对照组间3种蛋白表达差异均有统计学意义(P<0.05);再将实验组按年龄及软骨破坏程度分组,按年龄分组3种蛋白表达差异均无统计学意义(P>0.05);按软骨破坏程度分组3种蛋白表达差异均有统计学意义(P<0.05)。阳性表达主要分布在滑膜衬里层细胞。结论:uPA系统可能参与介导软骨降解、促进OA的发生发展;而PAI-1则通过抑制uPA活性延缓OA的发生发展。     

甘糖酯对大鼠尿激酶型纤溶酶原激活物活性的诱导作用

为了研究甘糖酯对大鼠体内尿激酶型纤溶酶原激活物（urokinase Plasminogen Activator,uPA)活性的影响,采用溶解圈中和抑制法和发色底物法检测了大鼠口服甘糖酯后体内uPA活性的变化情况。结果表明：甘糖酯可提高大鼠体内血液的纤溶活性,主要表现为uPA活性升高。以不同剂量的甘糖酯连续喂药10d后,发现喂药组大鼠uPA活性明显高于对照组,喂药组大鼠血浆uPA活性升高同喂药剂量正相关,在0－100mg/kd的剂量范围内,随喂药剂量增加而升高。提示在一定的剂量范围内甘糖酯可提高大鼠体内uPA活性,激活纤溶系统,提高血液纤溶活性,表现出良好的抗栓作用。     

Kunitz抑制剂家族成员抑肽酶对纤溶酶原活化的抑制作用

抑肽酶属Kunitz抑制剂家族成员，能够抑制激肽释放酶、纤溶酶及胰蛋白酶的蛋白水解活性．研究表明，抑肽酶能够抑制尿激酶型-纤溶酶原激活刹（u—PA）和组织型纤溶酶原激活剂（t—PA）对纤溶酶原的激活，但不影响u—PA和t—PA对小分子底物的酰胺水解活性．用u—PA研究了上述作用的机制，发现抑肽酶与u—PA的丝氨酸蛋白酶功能区特异性结合，而与纤溶酶原没有相互作用．抑肽酶与uPA的结合并不阻断u—PA的活性位点，因为u—PA对小分子底物的水解活性仍然保持．上述发现提示抑肽酶可能存在另一种抑制作用模式，该模式不同于以前报道的关于Kunitz抑制剂或纤溶酶原激活酶抑制剂的作用．由于人体内的Kunitz抑制剂与抑肽酶在结构上非常相似，根据研究结果，推测体内纤溶酶原的激活作用并非仅受丝氨酸蛋白酶抑制剂的控制。     

甘糖酯对大鼠尿激酶型纤溶酶原激活物（uPA）mRNA水平的影响

从分子水平上探讨海洋新药甘糖酯的抗栓作用机理。实验以大鼠为材料，采用定量ＲＴ－ＰＣＲ方法定量检测口服甘糖酯后大鼠尿激酶型纤溶酶原激活物（ｕＰＡ）基因ｍＲＮＡ水平变化。研究表明大鼠口服甘糖酯后ｕＰＡ基因ｍＲＮＡ水平高于对照组，差异显著。结论认为甘糖酯可提高体内ｕＰＡ基因的ｍＲＮＡ水平，从而提高ｕＰＡ蛋白的活性，激活机体的纤溶系统，达到抗栓作用。     

尿激酶原激活是其催化纤溶酶原激活的限速步骤

尿激酶原催化纤溶酶原的激活包括3个反应：（1）尿激酶原内在酶活性催化纤溶酶原转化为纤溶酶；（2）纤溶酶激活尿激酶原变成尿激酶；（3）尿激酶激活纤溶酶原，所以一直以来人们对尿激酶原激活纤溶酶原的过程知之甚少．研究发现EACA可以抑制尿激酶原催化的纤溶酶原激活。通过EACA对上述3个反应的影响进行逐个分析，观察到在EACA浓度为2．0mmol／L时，尿激酶原内在酶活性催化纤溶酶原激活的反应速度提高了4．5倍，尿激酶激活纤溶酶原的反应速度提高了6倍．相反地，纤溶酶激活尿激酶原变成尿激酶的反直被抑制了50％，此反应决定了EACA对尿激酶原催化纤溶酶原激活反应的抑制作用．该结果提示尿激酶原催化纤溶酶原激活反应的限速步骤是纤溶酶激活尿激酶原变成尿激酶，缘于纤溶酶和尿激酶原的赖氨酸结合位点的相互作用．     

尿激酶型纤溶酶原激活剂(uPA)系统在子宫内膜癌中的研究进展

尿激酶型纤溶酶原激活系统是纤溶系统的重要组成部分，它通过水解细胞外间质，参与组织改造和细胞迁移，在肿瘤细胞的侵袭和转移中发挥作用；讨论了uPA系统的结构、作用机理及与子宫内膜癌的关系，旨在为子宫内膜癌的诊断和治疗提供新方法。     

细胞外基质对体外培养兔关节软骨细胞增殖及基质代谢的影响

目的：观察软骨组织的主要细胞外基质成分：Ⅱ型胶原、蛋白多糖、透明质酸对体外单层培养的兔关节软骨细胞的增殖及基质合成的影响。方法：原代分离、培养兔关节软骨细胞，分别向其中添加Ⅱ型胶原、蛋白多糖和透明质酸。采用改良MTT法检测细胞的增殖情况，Aleian Blue法检测基质蛋白多糖含量及羟脯氨酸法测定软骨细胞胶原产量。结果：单独添加Ⅱ型胶原（50μg／mL）组、蛋白多糖（50μg／mL）组和透明质酸（100μg／mL）组及三者联合添加实验组与对照组相比，对关节软骨细胞的增殖及胶原、蛋白多糖的合成未见明显统计学差异（P〉0．05）。结论：正常软骨组织的主要基质成分对体外培养的兔关节软骨细胞增殖、蛋白多糖和胶原的合成无明显影响。     

射频消融处理时间对软骨基质影响的初步观察

建立膝关节软骨退变模型,模拟膝关节镜环境,观察射频消融能量在不同处理时间对关节软骨的即时损伤。射频消融气化仪在同一能量设置条件下处理牛膝关节退变软骨,按处理时间分为对照组、10s组、30s组、60s组,共4组,每组6份标本,随后行软骨组织块培养,阿利新蓝法测定组织块每日GAG释放率以确定基质的损伤程度,SEM观察软骨网状胶原纤维的超微结构改变。随着射频消融处理时间的增加,GAG释放率明显减少,培养后第3天,各组释放率较第1天都明显减少,30s组、60s组GAG释放率几乎为0。SEM超微结构提示,网状胶原纤维出现排列紊乱,正常网状结构消失,结构致密,胶原纤维断裂。随着射频消融处理时间的延长,软骨基质损伤明显加重,射频消融处理时间与关节软骨基质的损伤呈正相关。     

兔膝骨关节炎模型的建立及评价

目的 通过兔软骨缺损制备兔膝骨关节炎模型并进行评价。方法 将动物按体质量和性别随机分成3组：假手术组,模型组和阳性对照氨基葡萄糖组,每组12只。造模4周后开始灌胃给药,每天1次,连续给药4周。末次给药后,将动物处死,ELISA试剂盒检测血清及关节液中IL-1β和TNF-α水平,对关节软骨和滑膜进行HE染色,并对关节软骨进行Mankin’s评分以评价其损伤情况。结果 与假手术组相比,模型组血清、关节液中IL-1β、TNF-α水平明显升高,而给予氨基葡萄糖可降低血清、关节液中IL-1β、TNF-α水平。HE染色见模型组软骨结构改变、缺损,滑膜有炎细胞浸润;软骨评分明显高于假手术组。给予氨基葡萄糖可以减轻软骨缺损,减少滑膜炎细胞浸润,降低软骨评分。结论 关节软骨钻孔可建立兔膝骨关节炎模型,反映其主要特征,并能通过给予阳性对照药减轻损伤和炎症反应。该模型可用于治疗关节炎药物的评价和筛选。     

Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis

Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.     

聚乙烯醇水凝胶与人关节软骨摩擦特性比较

利用自行研制的振子式摩擦仪测定了聚乙烯醇水凝胶自摩擦副、人关节软骨/聚乙烯醇水凝胶摩擦副的摩擦因数,并根据其Stribeck曲线对其摩擦润滑机理进行初步探讨.实验结果表明,在透明质酸润滑条件下,聚乙烯醇水凝胶与人关节软骨具有相似的摩擦学特性,且聚乙烯醇水凝胶自摩擦副的摩擦因数略小于聚乙烯醇水凝胶/人软骨的摩擦因数,由其Stribeck曲线初步判定其润滑机制为液膜润滑.     

BMSCs过表达BMP-4复合同种异体骨修复软骨缺损

目的 针对关节软骨损伤后修复困难,采用腺病毒(Ad)载体介导骨形态发生蛋白(BMP-4)修饰骨髓间充质干细胞(BMSCs),并复合同种异体骨软骨柱支架,以期实现关节软骨有效再生.方法 制备同种异体骨软骨柱,BMSCs过表达BMP-4复合同种异体骨修复关节软骨损伤,膝关节标本取材大体观察及分析,HE、番红固绿、Masso...     

Difference in apoptosis-associated genes expression profiling and immunohistology analysis between Kashin-Beck disease and primary osteoarthritis

Kashin-Beck disease(KBD)is a chronic and deformed endemic osteoarthritis,without fully known etiology.As compared to primary osteoarthritis(POA),its pathogenesis still exists controversial.We performed this study to discriminate the difference in genes expression involved in apoptosis from KBD cartilages and POA cartilages.Microarray analysis and Ingenuity Pathway Analysis(IPA)were used to identify the molecular mechanisms/canonical pathways implicated in KBD arthritis.Immunohistochemistry staining was carried out to detect tissue distribution of PDCD5 and EGR1 in the knee cartilages from KBD and POA.The 23 up-regulated apoptosis-related genes with[2-fold change were identified.‘‘Role of Macrophages,Fibroblasts,and Endothelial Cells in Rheumatoid Arthritis’’signaling was screened as the most relevant pathway through IPA.The 8 differentially regulated genes were verified by qRT-PCR analysis.Programmed cell death 5 in middle and deeper zones and early growth response 1 in superficial and middle zones showed protein overexpressed in KBD cartilage samples comparing to those in POA cartilage samples.Data indicates a higher expression level of apoptosis-related functional genes in KBD articular cartilage compared with POA articular cartilage.     

关节腔内注射碘乙酸快速建立小鼠骨关节炎模型

摘要:目的 观察分析不同剂量碘乙酸(M IA)对于小鼠软骨和骨组织的损害,找出适合不同程度的骨关节炎小鼠模型的浓度。方法 C57BL/6小鼠随机分为4组,每组左右腿互为对照注射不同剂量的碘乙酸和对照用PBS,1,2,4周后测量膝关节宽度,进行相关评分,分析病变情况。结果 注射后出现不同程度病L变化。实验组关节评分明显大于对照组(P<0.05),且实验组中表现出较大剂量组(≥0.1 mg)与较小剂量组(<0.1 mg)两对组间差异。结论 推荐0.1 mg为较大剂量组代表剂量,产生明显软骨组织破坏和炎症反应,0.05 mg为较小剂量组代表剂量,产生轻度软骨组织的破坏和可逆炎症反应。     

Role of plasminogen activator and plasminogen activator inhibitor type-1 in luteolysis—a minireview

This minireview summarized our recent studies on the role of plasminogen activator (PA) and inhibitor type-1 (PAI-1) in luteolysis. We have demonstrated that (1) both tissue type and urokinase type plasminogen activators (tPA and uPA) and a plasminogen activator inhibitor type-1 (PAI-1) were present in the corpus luteum of rat and rhesus monkey; (2) decrease in progesterone production in corpus luteum was well correlated with a sharp increase in tPA (but not uPA) and PAI-1 secretion; (3) exogenous tPA decreased luteal progesterone synthesis while monoclonal antibodies increased progesterone production; (4) interferony inhibited luteal progesterone synthesis and stimulated tPA production while LH plus prolactin increased progesterone production and decreased tPA (hut not uPA) activity in cultured luteal cells; (5) increase in proteolysis in the corpus luteum was also correlated with decrease in progesterone production in mouse. These data suggest that local degradation of extracellular matrix controlled by plasminogen activator and inhibitor is involved in the processes of luteolysis.     

Role of plasminogen activator and plasminogen activator inhibitor type-1 in luteolysis——a minireview

This minireview summarized our recent studies on the role of plasminogen activator (PA) and inhibitor type-1 (PAI-1) in luteolysis. We have demonstrated that (1) both tissue type and urokinase type plasminogen activators (tPA and uPA) and a plasminogen activator inhibitor type-1 (PAI-1) were present in the corpus luteum of rat and rhesus monkey; (2) decrease in progesterone production in corpus luteum was well correlated with a sharp increase in tPA (but not uPA) and PAI-1 secretion; (3) exogenous tPA decreased luteal progesterone synthesis while monoclonal antibodies increased progesterone production; (4) interferon y inhibited luteal progesterone synthesis and stimulated tPA production while LH plus pro-lactin increased progesterone production and decreased tPA (but not uPA) activity in cultured luteal cells; (5) increase in proteolysis in the corpus luteum was also correlated with decrease in progesterone production in mouse. These data suggest that local degradation of extracellular matrix controlled by plasminogen activator and inhibitor is involved in the processes of luteolysis.