兔前交叉韧带切断骨关节炎模型软骨MMP-1 MMP-3及诱导型一氧化氮合酶表达的研究

目的观察兔前交叉韧带切断（ACLT）骨关节炎（OA）模型软骨中不同造模时期基质金属蛋白酶（MMP）-1、3及诱导型一氧化氮合酶（iNOS）的表达，为该模型用于OA防治提供理论依据。方法36只大白兔，随机选择27只，每只均行右侧膝关节腔切开前交叉韧带切断术（ACLT），另9只单纯行右侧膝关节腔切开术作为假手术对照组。术后4、8及12周随机处死实验组大白兔各9只及假手术对照组大白兔3只。对比各组大白兔股骨髁关节软骨的大体改变，用反转录-聚合酶链反应及免疫组织化学的方法检测MMP-1、3及iNOS在软骨中的mRNA和蛋白的表达结果。结果ACLT造模术后4周即开始出现软骨早期退变表现，造模8周软骨退变加重，造模12周出现OA晚期软骨退变表现，不同造模时期的大体评分差异有统计学意义，造模4周MMP-1、3及iNOS表达量明显高于对照组，随着造模时间延长MMP-1、3及iNOS表达量进一步升高，MMP-1、3及iNOS在退变软骨的不同阶段表达分布有各自的特点。结论ACLT模型能够表现OA软骨降解退变的全过程，适宜用于OA防治研究，MMP-1、3及iNOS的高表达在OA软骨退变的病理过程中起着重要的作用，随着造模时间延长、软骨退变的加重，表达量持续升高，适合作为OA防治研究中的评价指标。     

兔前交叉韧带切断骨关节炎模型中MMP-1 MMP-13及TIMP-1的mRNA表达研究

目的；研究兔膝关节前交叉韧带切断（ACLT）骨关节炎模型关节软骨及滑膜中基质金属蛋白酶（MMP）－1，MMP－13及组织源性基质金属蛋白酶抑制剂（TIMP）－1在不同造模时期 的表达情况，探讨上述因子在骨关节炎（OA）发病过程中的作用。为该模型作OA防治研究提供理论依据。方法：实验组ACLT模型兔20史，分别于造模4周各处死10只，假手术对照组大白兔10只于术后8周处死。解剖显微镜下行股骨髁关节软骨退变的大体评分，取股骨内髁内侧退变软骨及邻近滑膜，用反转录－多聚酶链反应（RT－PCR）的方法检测MP－1，MMP－13及TIMP－1的mRNA表达。结果：ACLT术后4周即可见软骨退变，8周时退变进一步加重（P＜0．02），对照组无明显软骨退变。对照组软骨及滑膜中MMP－1及TIMP－1的检出率较低，造模4周时检出率明显增高（P＜0．05），部分阳性标本表达量有一定增高，造模8周时MMP－1仍有很高的检出率，表达量持续增高，而TIMP－1检出率较4周时下降；MMP－13在对照组滑膜中没有检测出，造模4周时有一定的检出率，8周时检出率明显增加（P＜0．002），软骨 中对照组MPP－13的表达及表达量都很低，造模4周时表达率明显增高（P＜0．05），8周时表达率却明显下降。结论：MMP－1、MMP－13及TIMP－1在骨关节炎软骨退变过程中起重要作用。其中MMP－1在造模4周和8周都有很高的检出率，且从阳性标本的电泳情况看，其表达量逐渐增高，适合作为OA防治研究中的评价指标。     

兔膝骨关节炎负重模型的建立

目的 建立兔膝骨关节炎（osteoarthfitis，OA）负重模型，为骨关节炎实验研究奠定基础。方法 兔右侧膝关节前交叉韧带切断建成ACLT模型后，以石膏将左腿固定于腹部，右腿负重锻炼建成重模型，通过大体形态观察、关节液白介素-1（IL-1）、一氧化氮（NO）及软骨基质金属蛋白酶-1（MMP-I）含量的检测反映关节损害程度，比较负重模型与ACLT模型在不同造模时间关节损害的差别。结果 负重模型组较同期ACLT模型组关节退变更为明显；与ACLT模型组相比，负重模型能显著增加OA关节液IL-1、NO的产生和软骨MMP-1的表达（P〈0．05；P〈0．01），在造模4w升高6．5％、22．8％和26．2％，在8w升高3．0％、5．0％和3．6％。结论 兔膝骨关节炎负重模型可加快关节退行性变，缩短造模时间，是一种自然有效且造模时间较短的OA模型。     

羧甲基化几丁质降低兔实验性骨关节炎软骨基质金属蛋白酶-1表达的实验研究

目的 观察羧甲基化几丁质(CMC)经关节内注射后对兔前交叉韧带切断(ACLT)骨关节炎模型中软骨退变及软骨基质金属蛋白酶-1(MMP-1)表达及分布的影响。方法 20只大白兔行单侧ACLT，随机选取10只作实验组，于术后第1、3、5周分别给予0.3ml 2% CMC关节内注射，另10只于同一时间点给予0．3m1生理盐水关节内注射作对照组。术后第6周处死大白兔，对比两组大白兔股骨髁关节软骨的大体改变和光镜下的病理改变，并用反转录-聚合酶链反应（RT-PCR)及免疫组织化学的方法检测MMP—1在软骨中的mRNA和蛋白表达。结果 实验组软骨退变的大体评分和光镜下Mankin‘s评分都显著低于对照组，RT—PCR亦显示MMP-1在实验组中的表达明显低于其在对照组中的表达。免疫组织化学显示MMP-1主要表达于软骨表层及中上层，实验组MMP-1表达量亦明显低于对照组．结论 CMC能明显降低骨关节炎(OA)软骨中MMP-1的mRNA和生白表达水平，并明显降低软骨退变的程度，有可能成为防治OA的良好药物。     

盘状结构域受体2与软骨破坏程度的相关研究

目的 观察盘状结构域受体(DDR)2和基质金属蛋白酶(MMP)-13在膝骨关节炎(OA)大鼠不同时期软骨及滑膜中的表达,探讨DDR2与关节软骨破坏之间的关系.方法 采用改良膝关节腔内注射木瓜蛋白酶法制作OA大鼠模型,从蛋白水平检测造模后不同病理阶段关节软骨及滑膜中DDR2和MMP-13的表达规律和分布特点.结果 DDR2在各模型组关节软骨及滑膜中的表达较健康组增高(P＜0.01),并且各组中软骨表达高于相应滑膜,MMP-13表达呈现与DDR2相同的特点,二者相关系数r=0.93(P＜0.01).结论 初步证明"DDR2-MMP-13-软骨破坏"途径在OA病理过程中起重要作用.软骨和滑膜DDR2的表达升高共同促进软骨退变.     

关节软骨中整合素αvβ3表达与老年膝骨关节炎退变程度的相关性

目的研究关节软骨中整合素(ITG)αvβ3表达水平与老年膝骨关节炎退变程度的相关性。方法回顾性分析20例老年膝骨关节炎患者行全膝关节置换术得到的不同退变程度的71个关节软骨标本,根据Bobinac分期和苏木素-伊红(HE)染色后Mankin评分将标本分为正常组(n=5)、轻度退变组(n=23)、中度退变组(n=24)、重度退变组(n=19),利用qRT-PCR、Western印迹检测不同组标本中ITGαvβ3、基质金属蛋白酶(MMP)-9、MMP-13表达水平。结果 qRT-PCR、Western印迹检测方式结果均显示ITGαvβ3、MMP-9、MMP-13的表达水平在膝关节炎关节软骨中存在差异性表达,并且随着关节软骨退变程度增加其表达水平明显升高,组间相比有统计学意义(P0.05);相关性分析发现,ITGαvβ3蛋白表达水平与MMP-9蛋白表达水平呈显著正相关(r=0.781 8,P0.001),ITGαvβ3蛋白表达水平与MMP-13蛋白表达水平呈显著正相关(r=0.719 1,P0.001);MMP-9蛋白表达水平与MMP-13蛋白表达水平之间呈正相关(r=0.847 5,P0.001),并且三者表达水平均与退变程度呈显著正相关(r=0.848 2,P0.001;r=0.931 5,P0.001;r=0.909 2,P0.001)。结论老年膝骨关节炎退变程度越高,其关节软骨中ITGαvβ3、MMP-9、MMP-13的表达水平越高。     

壳聚糖膝关节腔内注射疗法对兔骨关节炎关节软骨的影响

目的观察关节内注射羧甲基壳聚糖(CMCTS)对兔骨关节炎(OA)关节软骨退变及软骨基质金属蛋白酶(MMP)-1,-3mRNA表达的影响。方法24只大耳白兔行单侧膝关节前交叉韧带切断术,术后5周将动物随机分为3组,A组关节内注射2%CMCTS0.3ml,每2周1次;B组关节内注射1%透明质酸钠(HA)0.3ml,每周1次;C组关节内不注射药物。术后11周处死动物,观察各组股骨内髁关节软骨的大体变化,采用反转录-聚合酶链反应(RT-PCR)方法检测股骨内髁退变软骨中MMP-1和MMP-3的mRNA表达。结果CMCTS和HA注射组软骨退变程度较不用药组明显减轻,CMCTS注射组软骨MMP-1、MMP-3的mRNA表达明显低于HA注射组和不用药组。HA注射组软骨MMP-1和MMP-3的mRNA表达与不用药组比较,差异没有显著性意义。结论CMCTS能够明显抑制OA软骨MMP-1和MMP-3的mRNA表达,明显减轻软骨退变的程度,CMCTS对早期OA软骨有修复保护作用。     

高脱乙酰度羧甲基壳聚糖对兔膝骨关节炎软骨诱导型一氧化氮合酶表达的影响

目的观察高脱乙酰度羧甲基壳聚糖(HD-CMC)经关节腔注射后对兔膝骨关节炎(OA)软骨诱导型一氧化氮合酶(iNOS)的mRNA和蛋白表达的影响,探讨其用于OA防治的机制及可能性。方法36只日本大耳白兔,行右侧膝关节前交叉韧带切断术(ACLT),术后随机分为2组,实验组于术后当天开始关节腔内注射2%HD-CMC 0．15mg/kg,每2周给药1次,对照组同一时间点关节腔内注射0．9%生理盐水0．15mg/kg。实验组与对照组于第6周分别随机处死大白兔各9只,剩余大白兔于第12周处死,对比两组大白兔股骨髁关节软骨的大体改变,用反转录聚合酶链反应(RT-PCR)及免疫组织化学的方法检测iNOS在软骨中的mRNA和蛋白的表达。结果实验组退变软骨的大体评分轻于对照组,NOS的mRNA和蛋白表达水平均低于对照组。结论HD-CMC能够降低OA退变软骨iNOS的表达,并延缓软骨的退行性变,对OA退变软骨具有修复保护作用。     

透明质酸钠对兔骨关节炎软骨和滑膜中MMP-1-3及其组织抑制物mRNA表达的影响

目的观察透明质酸钠(HA)关节腔注射对骨关节炎(OA)模型关节软骨及滑膜中的基质金属蛋白酶(MMP)-1、MMP-3及其组织抑制物(TIMP)-1mRNA表达水平的影响。方法16只大耳白兔行单侧前交叉韧带切断术,术后5周将动物随机分为实验组和对照组,实验组关节腔注射1%HA0.3ml,每周1次,连续5周,对照组则注射等量生理盐水。术后10周观察两组动物股骨内髁关节软骨光镜下的病理改变,采用反转录-聚合酶链反应(RT-PCR)方法检测关节软骨及滑膜中MMP-1、MMP-3及TIMP-1mRNA的表达。结果实验组软骨退变程度较对照组明显减轻,实验组滑膜中MMP-3的mRNA表达水平显著低于对照组(0.40±0.10vs0.62±0.13),而软骨中的MMP-3的表达较对照组差异无显著性,MMP-1和TIMP-1在实验组和对照组软骨及滑膜中的mRNA表达差异无显著性。结论HA能有效地减轻早期OA关节软骨的退变,其对早期OA的治疗作用的机制之一可能是抑制滑膜MMP-3的表达。     

复元胶囊对实验性骨关节炎软骨中MMP13、TIMP1及TIMP2的影响

目的观察复元胶囊对实验性骨关节炎软骨中基质金属蛋白酶13、组织型金属蛋白酶抑制剂1,2及病理形态方面的影响。方法选用新西兰兔66只,随机分为:正常对照组6只,不给予任何处理;模型对照组12只,造模后生理盐水灌胃;四环素组、复元胶囊低、中、高剂量组各12只,造模后分别用四环素、复元胶囊灌胃。连续灌胃4~12w后,比较各组关节软骨病理变化;免疫组化法检测兔膝关节软骨中MMP13、TIMP2蛋白质水平的变化;RT-PCR法检测关节软骨中MMP13、TIMP1及TIMP2mRNA表达。结果实验组软骨Mankin′s评分都显著低于模型对照组,实验组MMP13的mRNA表达水平明显低于模型对照组,而其TIMP1、TIMP2的mRNA的水平较模型对照组有所增加。MMP13主要表达于软骨表层及中上层,实验组MMP13表达量低于模型对照组(P<0.05)。结论复元胶囊能明显抑制骨关节炎软骨中MMP13的蛋白和mRNA的水平,能提高OA软骨中TIMP1,2的蛋白和mRNA的水平,稳定二者水平,对软骨退变有一定的防治作用。     

Twist1,MMP-2和MMP-9在结直肠癌组织中的表达及意义

目的:研究Twist1、MMP-2和MMP-9蛋白在结直肠癌组织中的表达及其相互关系.方法:建立组织微阵列平台,应用免疫组织化学方法检测92例结直肠癌组织Twist1、MMP-2和MMP-9蛋白的表达情况.结果:结直肠癌中Twist1的表达率为64.1%,MMP-2和MMP-9阳性率分别为66.3%和67.4%;Twi...     

成纤维细胞激活蛋白和基质金属蛋白酶-1在小鼠肝纤维化组织中的表达及其相关性

目的探讨成纤维细胞激活蛋白(FAP)和基质金属蛋白酶-1(MMP-1)在小鼠肝纤维化组织中的表达和两者之间的关系,及其在肝纤维化形成过程中的作用机制。方法建立四氯化碳诱导的小鼠实验性肝纤维化模型,HE染色和Masson染色法判定肝纤维化程度,采用免疫组织化学染色法测定FAP、MMP-1在肝纤维化过程中的时序动态表达。结果模型组中FAP和MMP-1的表达部位和范围都一致,阳性表达主要分布于门管区内血管壁周围及纤维间隔内的成纤维细胞的胞浆中。线性相关分析显示FAP和MMP-1与小鼠肝纤维化程度呈正相关(r分别为0.672,0.684;P0.0001);且FAP与MMP-1的表达也呈正相关(r为0.828;P0.0001)。结论 FAP和MMP-1可能协同作用参与降解细胞外基质和促进肝星形细胞的活化,从而在肝纤维化的发生发展中起作用。     

Collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) are expressed by migrating enterocytes during intestinal wound healing

BACKGROUND: Matrix metalloproteinases (MMPs) play a crucial role in wound healing of the skin, airways, and cornea, but data on MMPs in normal intestinal wound healing is limited. The aim of this study was to clarify the role of collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) in intestinal wound repair and to determine the effect of cytokines on the expression of these MMPs in intestinal epithelial cell lines. METHODS: Surgical specimens from patients with ischemic colitis (n = 5) were used as an in vivo model of intestinal re-epithelialization. Fetal ileal explants were used as an ex vivo model. In situ hybridization for MMPs -1, -3, -7, and -10 was performed and immunohistochemical stainings were used to localize MMP-7 and -9 expressing cells. Stainings for cytokeratin and laminin-5 were performed to identify epithelial cells and migrating enterocytes, respectively. Caco-2, HT-29, and WiDr cell lines were treated for 6-48 h with different cytokines (e.g. EGF, KGF, IL-1 beta, TGF-alpha, TNF-alpha, and TGF-beta1) and Taqman real-time quantitative RT-PCR was used to investigate their effect on the expression of MMPs-1, -7, and -10. RESULTS: MMP-7, MMP-10, and MMP-1 were expressed by migrating enterocytes bordering intestinal ulcers in 5/5, 3/5, and 3/5 samples, respectively. In the fetal gut model, MMP-1 and MMP-10 were expressed by migrating enterocytes, but matrilysin-1 expression was not detected. Matrilysin-1 was up-regulated by TNF-alpha and IL-1 beta, and stromelysin-2 by TNF-alpha and EGF in Caco-2 and WiDr cell cultures. MMP-1 was up-regulated in Caco-2 cells by TGF-beta, EGF, and IL-1 beta, but only by EGF in WiDR cells. CONCLUSIONS: It is concluded that collagenase-1, stromelysin-2, and matrilysin-1 are involved in intestinal re-epithelialization in vivo and that they are up-regulated by cytokines relevant in wound repair.     

Collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) are expressed by migrating enterocytes during intestinal wound healing

Background: Matrix metalloproteinases (MMPs) play a crucial role in wound healing of the skin, airways, and cornea, but data on MMPs in normal intestinal wound healing is limited. The aim of this study was to clarify the role of collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) in intestinal wound repair and to determine the effect of cytokines on the expression of these MMPs in intestinal epithelial cell lines. Methods: Surgical specimens from patients with ischemic colitis (n?=?5) were used as an in vivo model of intestinal re-epithelialization. Fetal ileal explants were used as an ex vivo model. In situ hybridization for MMPs -1, -3, -7, and -10 was performed and immunohistochemical stainings were used to localize MMP-7 and -9 expressing cells. Stainings for cytokeratin and laminin-5 were performed to identify epithelial cells and migrating enterocytes, respectively. Caco-2, HT-29, and WiDr cell lines were treated for 6-48?h with different cytokines (e.g. EGF, KGF, IL-1β, TGF-α, TNF-α, and TGF-β1) and Taqman real-time quantitative RT-PCR was used to investigate their effect on the expression of MMPs-1, -7, and -10. Results: MMP-7, MMP-10, and MMP-1 were expressed by migrating enterocytes bordering intestinal ulcers in 5/5, 3/5, and 3/5 samples, respectively. In the fetal gut model, MMP-1 and MMP-10 were expressed by migrating enterocytes, but matrilysin-1 expression was not detected. Matrilysin-1 was up-regulated by TNF-α and IL-1β, and stromelysin-2 by TNF-α and EGF in Caco-2 and WiDr cell cultures. MMP-1 was up-regulated in Caco-2 cells by TGF-beta, EGF, and IL-1β, but only by EGF in WiDR cells. Conclusions: It is concluded that collagenase-1, stromelysin-2, and matrilysin-1 are involved in intestinal re-epithelialization in vivo and that they are up-regulated by cytokines relevant in wound repair.     

Expression levels and association of gelatinases MMP-2 and MMP-9 and collagenases MMP-1 and MMP-13 with VEGF in synovial fluid of patients with arthritis

This study was performed to provide evidence, albeit indirectly, as to which matrix metalloproteinases (MMPs), among the gelatinases MMP-2 and MMP-9 and the collagenases MMP-1 and MMP-13, play a more proactive role in the angiogenic process in arthritic joint. Joint fluid was collected from 33 patients with rhuematoid arthritis (RA) and osteoarthritis (OA), and protein (MMPs and vascular endothelial growth factor (VEGF)) levels were measured by ELISA, and the association of MMPs with VEGF was evaluated in joint fluid of patients with RA or OA. The levels of collagenases (total MMP-1 and total MMP-13) and gelatinases (total MMP-2 and total MMP-9) in RA joint fluid were significantly higher than those in OA fluid. Total MMP-9 levels were significantly associated with VEGF levels in RA fluids, but not in OA fluid, while total MMP-13 levels were strongly associated with VEGF levels in both RA and OA fluid. However, total MMP-2 and total MMP-1 levels were not associated with VEGF levels in either RA or OA joint fluid. Our results indirectly suggest that in RA and OA, MMP-9 and MMP-13 may play a more important role in angiogenesis than MMP-2 and MMP-1.     

Enhanced production of MMP-1, MMP-3, MMP-13, and RANTES by interaction of chondrocytes with autologous T cells

It has been reported that T cells and chondrocytes interact through cell surface molecules such as MHC, CD4 or CD8 in osteoarthritis (OA) and T cells are activated. The objective of this study is to investigate the responses of chondrocyte–T cell interaction in terms of metalloprotease (MMP) and chemokine production. Articular cartilage and autologous blood were obtained from patients with OA and fracture who under went prosthetic surgery. Synovial fluid (SF) was collected from OA patients. Isolated chondrocytes were co-cultured with autologous T cells. SF cells were analyzed by immunostaining or Alcian blue staining. The production of MMP-1, MMP-3, MMP-13, and regulated on activation, normal T expressed and secreted (RANTES) was enhanced by direct co-culture compared to indirect co-culture using Transwell. Production ratio of RANTES in OA was significantly higher than non-arthritic samples. CD3 positive mononuclear cells and chondrocyte-like cells were found in SF. Chondrocyte–T cell contact was more adhesive in OA samples. These results showed the production of MMPs and RANTES was enhanced by the interaction and that chondrocyte–T cell contact was possible in vivo.