重组人结缔组织生长因子对人成骨细胞膜型基质金属蛋白酶1及基质金属蛋白酶2表达的影响

使用重组人结缔组织生长因子(recombinant human connective tissue growth factor,rCTGF)干预体外培养的人成骨细胞,发现rCTGF可呈时间-剂量依赖性地促进人成骨细胞膜型基质金属蛋白酶1及基质金属蛋白酶2的表达,200 ng/ml CTGF作用24～48 h达最大效果.rCTGF可明显增强p38丝裂原活化的蛋白激酶(p38-MAPK)磷酸化,p38-MAPK阻断剂SB 23058可阻断rCTGF上调膜型基质金属蛋白酶1及基质金属蛋白酶2的效应.

Abstract：

Human osteoblast was treated with recombinant human connective tissue growth factor (rCTGF). This experiment showed that rCTGF increased membrane type-1 matrix metalloproteinase and matrix metalloproteinase-2 protein expression in a dose- and time-depentent manner in human osteoblasts. rCTGF induced activation of p38 MAPK in human osteoblasts. p38 MAPK inhibitor SB23058 abrogated the effect of rCTGF on the expressions of membrane type-1 matrix metalloproteinase and matrix metalloproteinase-2 in human osteoblasts.     

洛沙坦降低糖尿病大鼠肾组织膜3型基质金属蛋白酶mRNA的表达

目的　研究洛沙坦对糖尿病大鼠肾组织膜 3型基质金属蛋白酶 (MT3 MMP)mRNA表达的影响。方法　雄性Wistar大鼠分为 3组 ,A组 (11只 )为正常对照组 ,B组 (11只 )为糖尿病未干预组 ,C组 (9只 )为糖尿病大鼠洛沙坦 (血管紧张素Ⅱ 1型受体阻断剂 )干预组。以链脲佐菌素 (STZ)制备糖尿病大鼠模型。大鼠饲养 18周后取出肾脏检测MT3 MMPmRNA表达、电镜检测大鼠肾小球基底膜厚度及系膜基质密度 (系膜基质面积 /系膜面积 ) ;收集 2 4h尿测定尿白蛋白排泄量 (UAE)。mRNA表达采用RT PCR ,以 β actin作为内对照。UAE测定采用大鼠白蛋白特异的酶免疫分析试剂盒。结果　肾组织MT3 MMPmRNA表达在B组大鼠 (1.37± 0 .96 )显著高于A组 (0 .75± 0 .34,P <0 .0 5 )和C组 (0 .75± 0 .30 ,P <0 .0 5 ) ,而后两组比较差异无显著性。UAE、肾小球基底膜厚度及系膜基质密度在B组大鼠均显著高于A组和C组 (P <0 .0 5 )。结论　STZ糖尿病大鼠肾组织MT3 MMPmRNA表达明显增加 ,洛沙坦处理能延缓糖尿病肾病的发生 ,与此同时降低MT3 MMPmRNA表达。提示MT3 MMP与糖尿病肾病发病可能有一定关系。     

心肌组织基质金属蛋白酶-3及其抑制物-1的表达与心功能损害

目的 探讨心肌组织基质金属蛋白酶－3（MMP－3）及其抑制物－1（TIMP－1）相对含量与心力衰竭（心衰）的关系。方法 采用RT－PCR方法对31例风湿性心脏病（RHD）心衰患者及8例健康人心室肌组织中MMP－3及TIMP－1含量进行半定量分析;RHD患者术前心功能指标采用超声心动图检查。结果 瓣膜病所致心力衰竭（心衰）病人心肌组织呈心肌重型的病理改变;心衰病人心肌组织MMP－3 mRNA表达较正常人增高,且心功能越差,MMP－3相对含量越高;TIMP－1则相反。结论 MMP－3表达含量的增加及TIMP－1表达含量的降低参与心肌重构,是影响心衰患者心功能恢复的重要因素之一。     

大鼠肺纤维化模型间质成纤维细胞和肺泡巨噬细胞基质金属蛋白酶2及膜型基质金属蛋白酶mRNA的表达

目的探讨肺间质成纤维细胞和肺泡巨噬细胞基质金属蛋白酶2(MMP-2)和膜型基质金属蛋白酶(MT1-MMP)mRNA在肺纤维化中的表达及意义.方法清洁级SD大鼠50只,随机分为10组,实验组5组气管内注射盐酸博莱霉素A5,对照组5组代以等剂量生理盐水.于注射后1d、3d、7d、14d、28d分离和提取肺间质成纤维细胞及肺泡巨噬细胞,应用逆转录多聚酶链反应(RT-PCR)方法,观察其MMP-2及MT1-MMPmRNA的动态变化.结果(1)博莱霉素作用后1d成纤维细胞MMP-2基因转录就明显增强,达对照组的2.05倍(t=10.667,P＜0.01),且一直维持于高水平,直到用药28d才略下降.而此过程中肺泡巨噬细胞MMP-2mRNA的转录极微弱,仅于实验第1d较对照组增高(t=3.27,P＜0.05).(2)成纤维细胞和肺泡巨噬细胞MT1-MMPmRNA的转录均有增强,前者于实验第14d和第28d时分别为对照组的2.1倍(t=4.823,P＜0.01)和1.8倍(t=4.016,P＜0.01),后者于第28d时为对照组的2.4倍(t=5.851,P＜0.01).结论(1)成纤维细胞不单纯是肺纤维化效应细胞,其通过MMP-2基因转录的增强,参与了肺基膜结构的损伤,参与了肺间质纤维化的启动机制.(2)实验中后期成纤维细胞和肺泡巨噬细胞MT1-MMP表达增强,有助于MMP-2的持续活化,促进肺间质纤维化的进展.     

Expression of membrane type 1 matrix metalloproteinase, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 in human cartilaginous tumors with special emphasis on mesenchymal and dedifferentiated chondrosarcoma

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been identified as an activator of the proenzyme of matrix metalloproteinase 2 (MMP-2: gelatinase A), and has also been shown to play a crucial role in tumor invasion by activating proMMP2 in both lung and gastric carcinoma. The tissue inhibitor of metalloproteinase 2 (TIMP-2) plus the MT1-MMP complex also plays an important role in the activation of proMMP-2. In this study, the expressions of MT1-MMP, MMP-2 and TIMP-2 were evaluated in 10 enchondromas, 34 conventional chondrosarcomas, 5 clear-cell chondrosarcomas, 7 mesenchymal chondrosarcomas and 8 dedifferentiated chondrosarcomas. The expressions were immunohistochemically visualized on paraffin sections and the levels of expression were assessed semiquantitatively. The extent of staining was assessed by the extent score in order to determine the overall level of expression. The extent scores of MT1-MMP, MMP-2 and TIMP-2 in grade 2 chondrosarcoma were significantly higher than those in either enchondroma or grade 1 chondrosarcoma (P < 0.05). In conventional chondrosarcoma, significant correlations were found between the extent scores of MT1-MMP and MMP-2 (P < 0.001), MT1-MMP and TIMP-2 (P < 0.01), and MMP-2 and TIMP-2 (P < 0.01). The undifferentiated small round tumor cells of mesenchymal chondrosarcoma showed lower positive rates and extent scores for MT1-MMP (2/7, 0.7 ± 0.5) and MMP-2 (3/7, 0.7 ± 0.4) than for cartilaginous components of mesenchymal chondrosarcoma [MT1-MMP (4/7, 1.3 ± 0.5) and MMP-2 (7/7, 1.9 ± 0.3)] or conventional chondrosarcoma. In dedifferentiated chondrosarcoma, the extent scores of MT1-MMP, MMP-2 and TIMP-2 in low-grade cartilaginous components were not significantly different from those in conventional chondrosarcoma; however, the high-grade anaplastic components showed high extent scores for MT1-MMP, MMP-2 and TIMP-2, compared with the low-grade cartilaginous components of dedifferentiated chondrosarcoma or conventional chondrosarcoma. According to our results, the expression of MT1-MMP as well as that of MMP-2 or TIMP-2 demonstrated a significant correlation with the tumor grade in human cartilaginous tumors. Furthermore, the expressions of MT1-MMP, MMP-2 and TIMP-2 were also found to play a crucial role in invasion in the high-grade components of dedifferentiated chondrosarcoma. Received: 17 February 1999 / Accepted: 21 April 1999     

孕激素对人成骨样细胞膜型基质金属蛋白酶-1表达和明胶酶-A激活的影响

目的研究孕激素对人成骨样细胞MG-63膜型基质金属蛋白酶(MT1-MMP)表达和明胶酶-A激活的影响,探讨孕激素治疗绝经后骨质疏松症(OP)的作用机制.方法MG-63细胞用孕酮干预;半定量逆转录聚合酶链反应、Western杂交和激光共聚焦显微系统免疫荧光法分别检测MT1-MMPmRNA和蛋白质表达;明胶酶-A激活用明胶酶谱和酶联免疫吸附(ELISA)检测.结果观察到孕酮增强MG-63细胞MT1-MMPmRNA表达呈剂量依赖性,其中10-9mol/L孕酮组为对照组的(127±11)%(P＜0.01),10-8mol/L孕酮组为对照组的(171±19)%(P＜0.001).孕酮增强MG-63细胞MT1-MMP蛋白质表达呈剂量依赖关系,其中10-9mol/L孕酮组为对照组的(188±85)%(P＜0.001),10-8mol/L孕酮组为对照组的(233±25)%(P＜0.0001).激光共聚焦显微系统免疫荧光法证实孕酮促进MT1-MMP蛋白质表达增强,MT1-MMP蛋白质表达于细胞膜和细胞质中.孕酮对明胶酶-A激活无影响(P＞0.05),即孕酮诱导的MG-63细胞MT1-MMP表达增强不能增强明胶酶-A的激活.结论MT1-MMP在骨重建过程中起着维持骨形成的关键作用,孕激素可通过诱导人成骨样细胞MT1-MMP表达增强促进骨形成.     

Tubular complexes in cerulein- and oleic acid-induced pancreatitis in rats: glycoconjugate pattern, immunocytochemical, and ultrastructural findings

Ductlike tubular complexes in cerulein-induced pancreatitis and oleic acid-induced pancreatic insufficiency were studied to analyze further their origin and development. Immunocytochemistry for pancreatic enzymes, lectin-binding studies, and ultrastructural investigations were combined with autoradiographic quantitation of labeling indices of ductlike cells in tubular complexes. In one group of rats, pancreatitis was induced by infusion of cerulein (10 micrograms kg-1 h-1). In a second group, pancreatic insufficiency was induced by intraductal injection of oleic acid (50 microliters). The investigations were carried out at distinct intervals following induction of pancreatic injury. In both groups of animals, after 3 days, a significant widening of acinar lumina was paralleled by a decreasing height of acinar cells, which showed pronounced retrogressive changes. At this time, acinar cells bound all of the lectins used and retained their immunoreactivity for amylase, trypsinogen, chymotrypsinogen, and lipase. At further intervals, acinar structures formed typical ductlike complexes, with a progressive loss of immunoreactivity for pancreatic enzymes and a reduced lectin-binding for L-fucose and N-acetylgalactosamine. Autoradiographic quantitation demonstrated no significant labeling of acinar cells undergoing tubular dedifferentiation. In both models, tubular complexes were removed by macrophages. It is concluded that lining cells in tubular complexes represent degenerating acinar cells that have no regenerative potency and have lost their secretory and membrane characteristics.     

Exocrine pancreatic function in oleic acid-induced pancreatic insufficiency in rats

Pancreatic insufficiency was induced in rats by a single injection of 50 microliter oleic acid into the pancreatic duct over a period of 3 min. Exocrine tissue was destroyed within 3-6 days, and after 6 weeks the remaining pancreas equaled 2.7% of the original organ. The rats showed retardation of body weight in spite of normal food intake. After 7 weeks the fecal weight increased by 23%, and the fecal chymotrypsin activity decreased by 90% compared to controls. At this time plasma cholecystokinin (CCK) concentrations were significantly elevated. The amylase content in the remaining pancreas was reduced by 99%, and trypsin content was reduced by 93%. Unstimulated protein discharge from the remnant pancreas in vitro was threefold higher compared to secretion from control tissue. Thus a simple, reproducible model for inducing persistent pancreatic insufficiency was developed. To compensate for the loss of exocrine tissue, the remaining acinar cells adapt by a CCK-mediated increase in protein secretion.     

葛根素对兔动脉粥样硬化基质金属蛋白酶-9及其组织抑制物-1表达的影响

目的：观察葛根素对动脉粥样硬化兔髂动脉分泌和表达基质金属蛋白酶-9（MMP-9）及其组织抑制物-1（TIMP-1）的影响。方法：20只家兔分为正常对照组（正常饮食，6只）、病理对照组（球囊和高脂饮食，8只）和葛根素组（球囊、高脂饮食和葛根素，8只）。球囊损伤后4周处死，取一侧病变髂动脉做病理切片，应用免疫组化法测定MMP-9和TIMP-1的蛋白表达；取另一侧病变髂动脉抽提总RNA应用半定量逆转录多聚酶链式反应（RT—PCR）测定MMP-9和TIMP-1 mRNA的表达。结果：兔动脉粥样斑块MMP-9 mRNA（mRNA／GAP—DH mRNA）表达：正常对照组、病理对照组、葛根素组的分别为0．81±0．17，1．52±0．24，1.03±0．19，病理对照组、葛根素组的较正常对照组显著增加（P〈0．05），而葛根素组的较病理对照组显著下降（P〈0．05），上述三组的TIMP—1 mRNA的表达依次为1．44±0．14，2．63±0．16，2．67±0．12，病理对照组与葛根素组的较正常对照组显著增加（P〈0．05），但病理对照组与葛根素组间无显著差异（P〉0．05）。免疫组化检测显示葛根素抑制MMP-9蛋白质表达（P〈0．05），但对TIMP-1蛋白质的表达无影响。结论：葛根素可能是通过调节兔动脉粥样斑块分泌MMP-9途径发挥稳定动脉粥样硬化斑块的作用。     

实验性肝纤维化大鼠Ⅳ型胶原酶的表达

肝纤维化是以胶原为中心的细胞外基质(ECM)在肝脏异常的沉积。这种异常的沉积,更大程度是由于降解的绝对或相对减少而引起的。肝纤维化时,在ECM降解过程中起主导作用是基质金属蛋白酶(MMPs)。Ⅳ型胶原酶(MMP-2)是特异性Ⅳ型胶原降解酶,与肝纤维化的形成有关。现以四氯化碳(CCl4)大鼠肝纤维化模型,采用northern印迹杂交,明胶酶谱     

环氧合酶-2和基质金属蛋白酶-2在大鼠非酒精性脂肪肝炎中的作用

目的:探讨环氧合酶-2(cyclooxygenase,COX-2)和基质金属蛋白酶-2(matrix metalloproteinase,MMP-2)在非酒精性脂肪性肝炎(nonalcoholic steamhepatitis,NASH)的发生、发展过程的作用.方法:取30只Wistar大鼠随机分成2组:模型组24只饲以高脂饲料(100 g/L猪油、20 g/L胆固醇、5 g/L胆酸钠、875 g/L基础饲料)和正常对照组6只以基础饲料饲养.分别于实验第12,16,24周随机处死模型组大鼠8只和正常对照组2只,取肝组织用于HE染色和RNA提取,应用逆转录聚合酶链反应(RT-PCR)检测肝组织COX-2 mRNA和MMP-2 mRNA的相对含量.结果:正常对照组肝组织COX-2 mRNA无表达;模型组均表达,并且随着造模时间延长COX-2 mRNA表达增强,造模第24周肝组织COX-2 mRNA表达明显高于第12,16周(1.035±0.040 vs 0.699±0.062,0.533±0.059,P<0.05);MMP-2 mRNA相对表达量在造模第12周与正常组相比无明显升高,而造模第16,24周,显著高于第12周(0.952±0.124,0.726±0.064 vs 0.454±0.06l,P<0.05),造模第24周和第16周之间亦有差别(P<0.05);NASH大鼠肝组织COX-2 mRNA和MMP-2 mRNA表达有相关性(r=0.794,P<0.05).结论:NASH大鼠肝组织中高表达COX-2 mRNA和MMP-2 mRNA,他们可能共同参与了NASH的形成、发展过程.     

基质金属蛋白酶抑制剂BB-94对急性胰腺炎及其相关性肺损伤的影响

目的探讨基质金属蛋白酶抑制剂对急性胰腺炎(AP)及其相关性肺损伤(APALI)的影响及其作用.方法腹腔注射L-精氨酸制备大鼠急性坏死型胰腺炎(ANP)模型.44只大鼠随机分为BB-94(40 mg/kg/24 h,诱导AP前腹腔注射BB-94 3次)治疗组和生理盐水对照组(20 ml/kg/24 h,诱导AP前腹腔注射生理盐水3次),每组各10只大鼠观察生存率,其余测定血清淀粉酶,胰腺和肺组织病理评分,明胶酶谱法检测MMP9和MMP2活性,检测肺脏髓过氧化酶(MPO)活性和肺血管Evans blue渗透性.结果 BB-94组生存率较对照组提高(100% vs 75%),血清淀粉酶明显降低(P ＜ 0.01),肺的MMP9和MMP2活性明显降低(P ＜ 0.01)、粒细胞浸润和蛋白渗漏明显降低(P ＜ 0.05).结论 BB-94可减轻AP时胰腺和肺脏的病理变化,降低AP所引起的肺组织MMP9和MMP2活性和蛋白渗漏.     

Eotaxin-induced expression of membrane type 1 matrix metalloproteinase mRNA in human eosinophils

Eosinophil penetration across the basement membrane (BM) is thought to be dependent on the degradation of membrane components. In this process, matrix metalloproteinases (MMP) appear to be primarily responsible for degradation of the BM. Matrix metalloproteinases -2 and MMP-9 degrade type IV collagen, which is a major component of the BM. In the present study, the effects of eotaxin, a selective chemoattractant for eosinophils, on the expression of MMP mRNA were examined. Incubation with chemotactically active concentrations of eotaxin for 24 h enhanced the expression of mRNA for membrane-type 1 MMP (MT1-MMP), but not of mRNA for MMP-2 and MMP-9. An increase in the protein level of MT1-MMP was also detected in the cell lysate of eotaxin-treated eosinophils. These results suggest that up-regulation of MT1-MMP expression may be involved in the eotaxin-induced penetration of eosinophils.     

环孢素A对链脲佐菌素诱导的糖尿病大鼠肾脏基质金属蛋白酶-2和基质金属蛋白酶-9表达的影响

目的 观察免疫抑制剂环孢素A(CsA)对糖尿病大鼠肾脏基质金属蛋白酶(MMP)表达的影响.方法 健康雄性SD大鼠74只,平均体重250 g,采用数字表法随机分为正常对照组(n=10)、单纯糖尿病组(n=8)、胰岛素治疗组(n=9)、造模前低剂量CsA治疗组(n=9;造模前1周皮下注射1μg·g-1·d-1 CsA)、造模前中剂量CsA治疗组(n=8;造模前1周皮下注射4μg·g-1·d-1CsA)、造模前高剂量CsA治疗组(n=8;造模前1周皮下注射8μg·g-1·d-1CsA)、造模后低剂量CsA治疗组(n=8;造模后1周皮下注射1μg·g-1·d-1CsA)、造模后中剂量CsA治疗组(n=7;造模后1周皮下注射4μg·g-1·d-1 CsA)和造模后高剂量CsA治疗组(n=7;造模后1周皮下注射8μg·g-1·d-1 CsA).8周后处死动物,采用免疫组织化学法、逆转录-聚合酶链反应和Western blot检测肾脏MMP-2、MMP-9 mRNA和蛋白表达水平.采用单因素方差分析和直线相关分析进行统计学分析.结果 8周时,单纯糖尿病组24 h尿微量蛋白显著高于正常对照组[分别为(5.80±3.23)、(1.24±0.21)mg/24 h,F=4.229,P＜0.01];各CsA治疗组24 h尿微量蛋白不同程度降低,与正常对照组比较差异无统计学意义.单纯糖尿病组肾小管上皮细胞和肾小球系膜基质MMP-2、MMP-9mRNA(分别为2.22±0.08、2.55±1.10)和蛋白表达(分别为3.1±1.5、2.8±1.0)显著高于正常对照组(MMP-2 mRNA:0.70±0.26,F=6.031;MMP-9 mRNA:0.37±0.24,F=5.193;MMP-2蛋白:1.0±0.0,F=7.532;MMP-9蛋白:1.0±0.0,F=6.100;均P＜0.01).胰岛素治疗对其表达无影响,但CsA干预可下调MMP-2、MMP-9 mRNA和蛋白的异常表达.MMP-2 mRNA与蛋白表达呈显著正相关(r=0.618,P＜0.01),而MMP-9 mRNA与蛋白表达未见相关性(r=0.420,P＞0.05).结论 CsA可减少糖尿病大鼠肾脏MMP-2和MMP-9基因转录和蛋白表达,改善细胞外基质代谢紊乱,可能具有延缓糖尿病肾病发生的作用.     

基质金属蛋白酶抑制剂BB-94对急性胰腺炎及其相关性肺损伤的影响

目的探讨基质金属蛋白酶抑制剂对急性胰腺炎(AP)及其相关性肺损伤(APALI)的影响及其作用。方法腹腔注射L-精氮酸制备大鼠急性坏死型胰腺炎(ANP)模型。44只大鼠随机分为BB- 94(40 mg/kg/24 h,诱导AP前腹腔注射BB-94 3次)治疗组和生理盐水对照组(20 ml/kg/24 h,诱导AP 前腹腔注射生理盐水3次),每组各10只大鼠观察生存率,其余测定血清淀粉酶,胰腺和肺组织病理评分,明胶酶谱法检测MMP9和MMP2活性,检测肺脏髓过氧化酶(MPO)活性和肺血管Evans blue渗透性。结果BB-94组生存率较对照组提高(100% vs 75%),血清淀粉酶明显降低(P<0.01),肺的MMP9 和MMP2活性明显降低(P<0.01)、粒细胞浸润和蛋白渗漏明显降低(P<0.05)。结论BB-94可减轻AP时胰腺和肺脏的病理变化,降低AP所引起的肺组织MMP9和MMP2活性和蛋白渗漏。     

膜型基质金属蛋白酶-1与肿瘤的侵袭和转移的研究进展

基质金属蛋白酶在肿瘤侵袭和转移中起着非常重要作用,而膜型基质金属蛋白酶是一类基质金属蛋白酶家族的新成员,其中膜型基质金属蛋白酶-1是研究最深、意义极为重要的一种,它直接或间接降解细胞外基质中的多种成分,通过调节多种细胞效应分子,影响肿瘤细胞的浸润、转移以及血管生成等过程,在肿瘤的浸润和转移的过程中有重要作用.     

MMP-2和MMP-9蛋白在结肠癌中的表达

目的:分析基质金属蛋白酶-2(MMP-2)和MMP-9蛋白与结肠癌病理因素的关系,探讨MMP蛋白在结肠癌发生中的临床意义.方法:用SP免疫组化法检测31例结肠癌中MMP-2和MMP-9蛋白表达情况,并用SPSS10.0forWindows软件统计分析MMP蛋白与临床病理因素的关系.以20例溃疡性结肠炎、21例结肠腺瘤和10例正常结肠黏膜作为对照组.结果:MMP-2除结肠癌组和正常结肠黏膜组间相比具有显著性差异(10.0%vs54.8%,P<0.05)外,其余各组间比较差异无统计学意义,MMP-9各组间比较差异无统计学意义,从溃疡性结肠炎、结肠腺瘤到结肠癌中MMP-2和MMP-9蛋白表达阳性率不同且具有递增趋势.MMP-2和MMP-9蛋白表达与结肠癌的Duke’s分期及有无淋巴结转移显著相关(A B期:38.9%和27.8%;C D期:76.9%和84.6%;无淋巴结转移:38.9%和27.8%;有淋巴结转移:76.9%和84.6%,P<0.05).结论:MMP-2和MMP-9蛋白过度表达对结肠癌的诊断、Duke's分期及有无淋巴结转移判断具有重要的临床意义.