Characterization of opticin digestion by proteases involved in osteoarthritis development

ObjectiveOpticin is a class III member of the small leucine-rich repeat proteoglycan (SLRP) family, produced in articular joint tissues. In normal and osteoarthritic (OA) cartilage, opticin is degraded. This study aimed to assess whether human cartilage opticin is degraded by the main proteases involved in OA pathophysiology, and to determine the protease cleavage sites of this SLRP.MethodsWe analyzed the proteolytic activity of matrix metalloproteinases (MMPs)-1, -2, -3, -7, -8 and -9, and ADAMTS-4 and -5 on proteoglycan extracts from normal and moderately fibrillated OA human cartilage, and on recombinant human opticin. Opticin degradation was analyzed by Western blotting and cleavage sites were determined by sequence analysis.ResultsAll eight proteases digested opticin from proteoglycan extracts from both normal and OA samples, as well as recombinant human opticin, MMP-2 and MMP-7 are the proteases that degrade recombinant human opticin most efficiently. The opticin cleavage site determined for these MMPs was between the glycosylation and leucine-rich repeat domains. MMP-7 had two additional digestion sites near the N-terminal end of opticin.ConclusionOpticin is a substrate for several MMPs and aggrecanases involved during OA cartilage degradation, and seems to be a preferential substrate for MMP-7. The role of opticin in cartilage degeneration could be related to decreased levels of intact opticin, followed by its proteolytic degradation, which in turn may stimulate some of the modifications observed in the OA cartilage, such as neovascularisation and changes in the extracellular matrix.     

血清ADAM 12-S在孕早期孕妇异位妊娠中的诊断价值

[目的]探讨血清解整合素-金属蛋白酶(ADAM12-S)在孕早期孕妇异位妊娠中的诊断价值.[方法]选取异位妊娠孕妇92例(异位妊娠组)和106例正常单胎妊娠孕妇(正常妊娠组),采用时间分辨荧光免疫分析法检测血清ADAM 12-S水平,分析异位妊娠的工作曲线(ROC).[结果]在不同孕周,正常妊娠组孕妇ADAM 12-S水平均显著高于异位妊娠组(P<0.05);正常妊娠组孕妇ADAM 12-S水平随孕周呈二次函数式上升趋势(P<0.05);在ROC曲线中当ADAM 12-S水平为1.565μg/L时,其诊断异位妊娠的灵敏度为99.06%,特异度为100.00%.[结论]ADAM 12-S可作为诊断异位妊娠的生物标志物,具有较高的灵敏度和特异度,值得在临床上推广应用.     

Proteomic profiling of snake venom metalloproteinases (SVMPs): Insights into venom induced pathology

Bothrops sp. snakebites account for the majority of envenomations in South and Central America. Bothrops jararaca accidents are characterized by edema, hemorrhage and necrosis, mainly attributed to the action of hemorrhagic snake venom metalloproteinases (SVMPs). Interestingly, accidents involving Bothrops lanceolatus (Fer-de-Lance) have a prothrombotic profile with necrosis and hemorrhage rarely reported. Here we describe biochemical and proteomic approaches to compare the venom composition of these snakes, focusing on the presence and activity of SVMPs. The total relative amount of SVMPs was found to be approximately the same in the venom of both species, the difference being in the distribution of SVMPs subgroups. Fer-de-Lance venom has relatively more PI SVMPs peptides identified (23–16%) while Jararaca venom has a higher amount of PIII SVMPs (54–43%). Gelatinolytic activity in the PIII mass range is also higher in Jararaca venom. Interestingly, the homologous band region in the Fer-de-Lance zymogram was only very weakly gelatinolytic. According to these findings it is feasible that the different distribution of SVMPs subgroups and their particular biochemical and pharmacological characteristics are two of the main factors contributing to these two radically different venom induced pathologies.     

大鼠性别对腹主动脉瘤模型建立的影响

目的通过对雌、雄性大鼠腹主动脉瘤(abdominal aortic aneurysm,AAA)动物模型组织中基质金属蛋白酶-2、9(matrix metalloproteinases-2、9,MMP-2、9)mRNA及蛋白表达的对比,探讨性别差异对腹主动脉瘤模型形成的影响。方法雌、雄Wistar大鼠各15只,分别用弹性蛋白酶、生理盐水灌注肾下腹主动脉制作AAA模型。14 d后取出腹主动脉,用免疫组化、适时RT-PCR测定动脉瘤组织MMP-2、9的mRNA及蛋白的表达。结果雌性大鼠实验组与雄性大鼠实验组比较,动脉瘤扩张率分别为(104±6)％和(179±11)％(P<0．01)、MMP-2 mRNA的相对表达量分别为0．02±0．01和0．46±0．25(P<0．01)、MMP-9 mRNA的相对表达量分别为0．42±0．24和4．30±2．87 (P<0．01)、MMP-2的表达率分别为(22±5)％和(46±9)％(P<0．01)、MMP-9的表达率分别为(26±2)％和(43±3)％(P<0．01)。结论在腹主动脉瘤形成的过程中雌性大鼠的MMP-2、9的mRNA和蛋白的表达以及炎性细胞的浸润均明显少于雄性大鼠,导致动脉瘤扩张率出现明显差异。     

Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶在老年大鼠心肌缺血预适应中的作用

目的 观察缺血预适应(ischemic preconditioning,IPC)对老年大鼠心肌Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(a disintesrin and metalloprotease with thrombospondin type 1motifs,ADAMTS-1)表达及ADAMTS-1特异小于扰核糖核酸(small interfering ribonucleic acid,siRNA)干预对老年IPC保护作用的影响. 方法成年(4月龄)和老年(24月龄)SD大鼠各32只,按抽签法分别随机分入IPC组20只和假手术组12只,IPC后留取缺血再灌注部位心肌行免疫组织化学和免疫蛋白印迹检测ADAMTS-1表达.另老年SD大鼠110只,随机分人ADAMTS-1 siRNA组55只和对照组55只,观察ADAMTS-1 siRNA转染对IPC后ADAMTS-1蛋白表达的影响,同时观察ADAMTS-1 siRNA转染对IPC后心肌梗死存活率、心功能和心肌梗死面积的影响. 结果成年和老年大鼠IPC后24 h时缺血再灌注部位心肌ADAMTS-1蛋白表达明显升高(P＜0.05),同时老年大鼠心肌的ADAMTS-1蛋白表达高于成年大鼠(P＜0.05).成年大鼠IPC后0 h和24 h时ADAMTS-1蛋白表达的免疫组织化学检测吸光度分别为0.05±0.01和0.12±0.03,免疫蛋白印迹检测吸光度为0.68±0.16和1.17±0.21.老年大鼠IPC后0 h和24 h时ADAMTS-1蛋白表达的免疫组织化学检测吸光度分别为0.07±0.03和0.21±0.04,免疫蛋白印迹检测吸光度为0.76±0.21和1.48±0.17.ADAMTS-1 siRNA干预抑制老年大鼠IPC后的ADAMTS-1蛋白表达(IPC后0 h和24 h时的ADAMTS-1免疫蛋白印迹检测吸光度分别为0.66±0.19和0.78±0.21,P＞0.05),但不影响老年IPC大鼠心肌梗死存活率[ADAMTS-1 siRNA组和对照组老年大鼠分别为14.3%(5/35)和17.1%(6/35),P＞0.05]、心肌梗死面积[分别为(39.0±4.1)%和(38.0±5.3)%,P＞0.05]和左心室短轴缩短率[分别为(14.0±3.2)%和(13.0±2.9)%,P＞0.05]. 结论延迟IPC促使老年心肌ADAMTS-1表达增加,ADAMTS-1 siRNA转染能够抑制ADAMTS-1的表达但不能恢复老年心肌的IPC保护作用.

Abstract：

Objective To investigate the effect of ischemic preconditioning (IPC) on the expression of a disintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), and to study whether the application of small interfering (si)RNA specifically targeting ADAMTS-1 would help to recover IPC protection in the aged heart. Methods The 32 young (4 months) and 32 aged(24 months) male Sprague-Dawley (SD) rats were assigned randomly to IPC group (n=20) and sham operated group (n= 12) respectively. Myocardial samples from the ischemic-reperfused region were harvested for detecting the ADAMTS-1 expression. In addition, the 110 aged SD rats were assignedrandomly to ADAMTS-1 siRNA group and control group (n=55, each). The effects of ADAMTS-1siRNA transfcction on the expression of ADAMTS-1 protein, myocardial infarction survival rate,heart function and myocardial infarction size after IPC were observed.Results Twenty-four hours after IPC, the ADAMTS-1 protein expression increased significantly in iscbemic-reperfused region both in young and aged rats (P＜0. 05), and the protein expression was higher in aged rats than in young rats (P＜0.05). In young-IPC group, the absorbency showed ADAMTS-1 protein expression at 0 hrs and 24 hrs after IPC were 0. 05±0.01 and 0.12±0.03 by immunohistochemical staining, and were 0.68±0. 16 and 1. 17±0.21 by Western blots respectively. In aged-IPC group, the absorbency showed ADAMTS-1 protein expression at 0 hrs and 24 hrs after IPC were 0.07±0. 03 and 0.21 ±0.04 by immunohistochemical staining, and were 0. 76±0. 21 and 1. 48±0. 17 by Western blots. In the aged rats, ADAMTS-1 siRNA transfection inhibited ADAMTS-1 protein expression (0. 66±0. 19and 0.78±0.21, by Western blots at 0 hrs and 24 hrs after IPC, P＞0.05), but didn't improve myocardial infarction survival rates [ADAMTS-1 siRNA group and sham operated group: 14.3% (5/35) vs. 17.1 %(6/35), P＞0.05], left ventricular fractional shortening [(14.0±3.2)% vs. (13.0±2.9)%, P＞0.05] and myocardial infarction size[(39.0±4.1)% vs. (38.0±5.3)%, P＞0.05].Conclusions ADAMTS-1 expression induced by IPC increases significantly in aged versus in young rats. ADAMTS-1 knockdown by siRNA inhibits ADAMTS-1 protein expression but cannot recover the age-associated loss of IPC protection.     

解整合素-金属蛋白酶12在染色体非整倍体筛查中的应用

解整合素-金属蛋白酶12（A disintegrin and metalloprotease 12,ADAM12）是细胞膜表面具备多功能的糖蛋白家族成员之一,具有金属蛋白酶的特性,可以水解类胰岛素样生长因子结合蛋白（insulin-like growth factor binding proteins,IGFBP）。妊娠妇女血清中有分泌性/可溶性形式的ADAM12,即ADAM12-S,非妊娠妇女血清中则不存在。妊娠正常胎儿时,ADAM12-S的浓度随妊娠周的增加而升高。ADAM12-S在妊娠妇女血清中浓度的高低有种族差异。ADAM12浓度的高低与妊娠妇女的体质量有关,但与年龄无关。吸烟的妊娠妇女血清中ADAM12浓度低于非吸烟者。在妊娠早期尤其在10周前,妊娠唐氏综合征（DS）患儿妇女的ADAM12浓度明显低于妊娠正常胎儿者,在妊娠中期则高于妊娠正常胎儿者。在早期妊娠18-三体综合征、13-三体综合征、45,X和三倍体胎儿的妇女血清中,ADAM12浓度明显低于妊娠正常胎儿者。ADAM12作为一个新的血清标记物还需进行深入研究。     

CD68和MMP-9在小儿先天性胆总管囊肿中的表达及意义

目的通过观察先天性胆总管囊肿(congenital choledochocgst,CCC)囊壁CD68和MMP-9的表达,探讨其与CCC发病机制的关系。方法收集48例先天性胆总管囊肿患儿的临床资料以及组织标本,为观察组,取正常胆囊组织11例作为对照组。采用免疫组化方法检测CD68和MMP-9。结果观察组CD68和MMP-9均有较高阳性表达,与对照组比较,差异有统计学意义(P≤0.05),其中MMP-9在囊性型囊肿和梭状型囊肿囊壁组织中的表达差异有统计学意义(P≤0.05)。结论 CD68和MMP-9的高表达,提示炎性因素可能参与了胆总管囊肿的形成过程。