尿激酶型纤维蛋白酶原激活物受体的反义基因片断抑制胶质瘤u251细胞的体外侵袭

目的:研究尿激酶型纤维蛋白酶原激活物受体(uPAR)的反义基因片断对胶质瘤细胞株u251体外侵袭能力的影响。方法:脂质体介导寡核苷酸转染培养的u251胶质瘤细胞,用RT-PCR、原位杂交和免疫组化方法分别检测相关基因和蛋白的表达,Boyden小室模型研究对肿瘤细胞侵袭能力的影响。结果:uPAR反义寡核苷酸作用u251胶质瘤细胞后相关基因的mRNA及蛋白的表达明显减弱,细胞的体外侵袭能力被明显抑制。结论:uPAR的反义寡核苷酸片断能有效地抑制胶质瘤细胞u251相关基因的表达,并能抑制胶质瘤细胞的体外侵袭能力。     

反义基因片段抑制人胶质瘤u251增殖和侵袭的体外研究

目的:研究反义PCNA及uPAR基因片段对胶质瘤细胞株u2 5 1体外增殖和侵袭能力的影响。方法:用脂质体介导寡核苷酸转染培养的u2 5 1胶质瘤细胞,RT PCR、原位杂交和免疫组化等方法分别检测相关基因和蛋白的表达,MTT比色法研究对胶质瘤细胞增殖能力的影响,Boyden小室模型研究对细胞侵袭能力的影响。结果:PCNA及uPAR反义寡核苷酸作用u2 5 1胶质瘤细胞后相关基因的mRNA及蛋白的表达明显减弱,细胞的体外增殖和侵袭能力明显被抑制。结论:反义PCNA及uPAR寡核苷酸片段能有效地抑制胶质瘤细胞的u2 5 1相关基因的表达,并能抑制胶质瘤细胞的体外增殖和侵袭能力。     

尿激酶型纤溶酶原激活物受体在人脑胶质瘤中的表达及意义

一、资料与方法1.尿激酶型纤溶酶原激活物受体(uPAR)在胶质瘤组织中的表达:星形胶质细胞瘤患者共58例,Ⅰ级14例,Ⅱ级15例,Ⅲ级15例,Ⅳ级14例。男30例,女28例,平均年龄(43.2±18.1)岁。病理切片行免疫组化染色(SABC法),计算阳性细胞率(PI)。     

脑出血患者尿激酶型纤溶酶原激活物及其受体的变化

目的探讨脑出血患者血浆尿激酶型纤溶酶原激活物(uPA)及其受体(uPAR)的变化及临床意义。方法对64例脑出血患者和30例健康对照组应用酶联免疫吸附双抗体夹心法(ELISA法)定量测定血浆uPA和u-PAR的水平。结果脑出血组uPA含量(2866±788ng/L)和uPAR含量(3645±322ng/L)明显高于正常对照组的uPA含量(1075±244ng/L)和uPAR含量(877±216nP<0.01)。随脑出血患者病情严重程度的加重,血浆uPA和u-PAR的含量增高越明显。结论血浆uPA和uPAR的增高参与了脑出血时继发性损害,与脑出血患者的病情密切相关。     

急性脑梗死患者尿激酶型纤溶酶原激活物及受体的变化

目的探讨急性脑梗死患者血浆尿激酶型纤溶酶原激活物(uPA)及受体(uPAR)的变化和临床意义。方法对66例急性脑梗死患者和30例健康者应用酶联免疫吸附双抗体夹心法(ELISA法)定量测定血浆uPA和uPAR的水平。结果与正常对照组相比,急性脑梗死组uPA含量(4756±632)ng/L,uPAR含量(3102±256)ng/L,明显高于正常对照组的uPA(1075±244)ng/L和uPAR(877±216)ng/L,差异有显著性(P<0.01)。随急性脑梗死患者病情严重程度的加重,血浆uPA和uPAR含量增高越明显。结论急性脑梗死患者血浆uPA和uPAR的水平明显升高,且与急性脑梗死患者的病情密切相关。     

尿激酶型纤溶酶原激活因子及其受体在人脑胶质瘤中的表达和意义

目的探讨尿激酶型纤溶酶原激活剂(urokinase-type plasminogen activator，uPA)及其受体(uPAR)的表达与人脑胶质瘤恶性程度的关系。方法采用免疫组化法，对58例人脑胶质瘤手术切除标本，10例内减压术中切除的正常脑组织标本的uPA、uPAR蛋白表达水平进行检测。结果随着胶质瘤恶性度的升高，其uPA、uPAR蛋白表达率和表达水平逐渐增高，而在正常组织中均无uPA和uPAR表达。结论uPA／uPAR蛋白高表达反映了胶质瘤的恶性生物学行为。     

肝细胞生长因子受体反义寡核苷酸对胶质瘤细胞的作用

近期的研究认为肝细胞生长因子(hepatocyte growth factor,HGF)及其受体c-Met信号通路的激活对肿瘤发生、生长和侵袭起着关键的作用,而选择性地阻滞肿瘤细胞的信号通路,控制肿瘤的增殖和侵袭是当前的研究热点,这也是研究肿瘤信号通     

Expression of plasminogen activator inhibitors type 1 and type 3 and urokinase plasminogen activator protein and mRNA in breast cancer

BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.     

Urokinase‐type plasminogen activator modulates mammalian circadian clock phase regulation in tissue‐type plasminogen activator knockout mice

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue‐type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA^?/?; B6.129S2‐Plat^tm1Mlg/J) mice, and identify urokinase‐type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA^?/? mice entrain to a light‐dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA^?/? mice take significantly longer to entrain than C57BL/6J wild‐type (WT) mice. SCN brain slices from tPA^?/? mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose‐dependency. Pre‐treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor‐1 (PAI‐1), inhibits glutamate‐induced phase delays in tPA^?/? slices. Selective inhibition of uPA with UK122 prevents glutamate‐induced phase resetting in tPA^?/? but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA^?/? slices. Casein‐plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA^?/? SCN. Finally, tPA^?/? SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.     

The plasminogen activator and esterase activities of the two forms of urokinase

High and low mol. wt. urokinase (UK) purified to homogeneity by affinity chromatography, and partially purified commercial preparations with varying ratios of high to low mol. wt. forms were assayed for plasminogen activator activity by: physiologic clot lysis time using varying amounts of native and degraded plasminogen; plasminogen activator activity by protamine assay; and esterase activity on the synthetic peptide ester N-Ac-Gly-Lys-O-Me (AGLMe); as well as by active-site titration using p-nitrophenyl-p′-guanidino-benzoate (NPGB). The molar activity of both high and low mol. wt. UK by the three rate assays was about 1 × 10¹³ IU/mole active site and was not affected by molecular size or low mol. wt. impurities. Depending on the particular UK preparation and the type of plasminogen, one or more impurities may affect the fibrinolytic assay at limiting substrate concentrations. Gel filtration or affinity chromatography abolishes the effect. NPGB titration provides another rapid and simple means of estimating UK activity and relating all three rate assays to molar activities. Correlation of the results of protamine and AGLMe assays with those of the fibrinolytic assays was generally good, except for the tissue culture UK which showed at least 25% higher values by both the protamine and AGLMe assays. The protamine assay proved a useful alternative to fibrinolytic and synthetic substrate.     

Absence of synergism between tissue-type plasminogen activator and urokinase on plasminogen activation rate in plasma

Effects of tissue-type plasminogen activator (t-PA), urokinase(u-PA) and their combinations on plasminogen activation rate (PAR) in plasma, were investigated. T-PA and u-PA over concentrations range of 10 U/ml to 50 U/ml induced a linear, concentration dependent increase in PAR. Combinations of t-PA and u-PA in ratios of 3/1,1/1 and 1/3 induced additive but not synergistic effect in the activation of plasminogen. We conclude, therefore, that t-PA and u-PA do not act synergistically in the activation of plasminogen in plasma .     

The behaviour of urokinase and porcine kidney cell plasminogen activator towards some synthetic peptides

The behaviour of human urokinase and porcine kidney cell plasminogen activator towards some synthetic substrates has been investigated. Although N- benzyloxycarbonylglycylglycyl -L-arginine 4-methyl-7- coumarylamide (Z-Gly-Gly-Arg-Amc) (I), glutaryl-Gly-Arg-Amc (II) and Z-Gly-Gly-Arg-Val-OMe (III) were substrates, Boc-Gly-Gly-Arg-Val-Val-Gly-Gly-OEt (IV) and Z-Ala-Pro-Gly-Arg-Val-Val-Gly-Gly-OEt (V) were neither substrates nor inhibitors. Steady-state kinetic parameters for the hydrolysis of (II) and (III) by urokinase and porcine kidney cell plasminogen activator were similar.     

Localization of urokinase-type plasminogen activator, its receptor, and inhibitors in mouse forebrain during postnatal development

Proteolytic enzymes are postulated to play a role in cell migration and synapse organization during brain development. Among these, urokinase-type plasminogen activator (uPA) has been studied in neoplastic and cultured brain cells extensively. We hypothesized that uPA, its receptor, and its inhibitors would be expressed in immature glial and neuronal cells in postnatal mouse forebrain. Immature cortical neurons were immunoreactive for uPA, its receptor, and its substrate plasminogen peaking at the end of postnatal week two, consistent with the postulated role in synaptogenesis. Immunoreactivity for uPA receptor was also observed on astroglial cells in vitro. Neither it nor uPA were convincingly detected in subventricular zone precursor cells, immature white matter or pre-labeled immature cells that had been transplanted into brain. Plasminogen activator inhibitor type 1 immunoreactivity was observed on endothelia up to 12 days age, and type 2 was observed to surround immature cells. We conclude, based on the spatial and temporal distribution of immunoreactivity, that uPA and its receptor may be relatively more important for synaptogenesis, remodeling, and reactive processes than for cell migration in developing mouse brain.