Design and synthesis of a near-infrared fluorescent nanofiber precursor for detecting cell-secreted urokinase activity

Abnormal proteolysis is often observed during disease progression. Up-regulation of certain tumor-associated proteases such as urokinase plasminogen activator (uPA) can be a hallmark of malignant transformation. Here we report the design and synthesis of a near-infrared nanofiber precursor (NIR–NFP) for detecting uPA activity. NIR–NFP, which is optically silent in its native state, is composed of multiple self-assembled peptide units (PEG₅₄-BK(NIR664)SGRSANA-kldlkldlkldl-CONH₂). On uPA activation, NIR–NFP releases peptide fragments (PEG₅₄-BK(NIR664)SGR-CONH₂) that contribute to a significant fluorescence amplification at 684 nm. NIR–NFP was able to detect cell-secreted uPA from human cancer cells (SKBR-3, PANC-1, MCF-7, SKOV-3, MDA-MB-231, PC-3, and HT-1080) expressing various levels of uPA. Fluorescence changes were uPA dependent, as confirmed with both Western blot analysis and enzyme activity assay. Our data suggest that an optimized preparation may be useful for imaging uPA activity in vivo.     

超声剪切波弹性成像联合血清脂联素、尿激酶型纤溶酶原激活物对甲状腺结节良恶性的鉴别价值

摘要 目的：探讨超声剪切波弹性成像（SWE）联合血清脂联素（AFN）、尿激酶型纤溶酶原激活物（uPA）鉴别甲状腺结节良恶性的价值。方法：选择2020年1月至2022年12月我院收治的行甲状腺外科切除手术治疗的127例甲状腺结节患者,所有患者术前接受SWE检查,检测血清AFN、uPA水平,记录术后病理结果,并根据术后病理结果将患者分为恶性组和良性组。分析SWE参数联合血清AFN、uPA鉴别甲状腺结节良恶性的价值。结果：经术后病理检查证实,恶性结节21例（恶性组）,良性结节106例（良性组）。恶性组弹性模量最大值（Max）、弹性模量最小值（Min）、弹性模量平均值（Mean）,血清uPA水平高于良性组（P＜0.05）,血清AFN水平低于良性组（P＜0.05）。Max、Mean、Min、AFN、uPA鉴别甲状腺结节良恶性的曲线下面积分别为0.609、0.754、0.600、0.777、0.701,联合Max、Mean、Min、AFN、uPA鉴别甲状腺结节良恶性的曲线下面积为0.935,高于各指标单独鉴别。结论：恶性甲状腺结节患者SWE参数Max、Mean、Min和血清uPA水平增加,AFN水平降低,联合SWE参数和血清AFN、uPA可提高对甲状腺结节性质的鉴别效能。     

6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, K_i = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA’s S1β subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.     

尿激酶对急性脑梗死患者血管内皮功能及氧化应激的影响及疗效观察

目的:探讨尿激酶治疗对急性脑梗死(ACI)患者内皮功能及氧化应激的影响并观察其临床疗效。方法:选择2016年7月到2017年7月我院神经内科确诊收治的急性脑梗死患者110例为研究对象,依据治疗方法分为常规组和治疗组,每组各55例。常规组患者接受对症基础治疗,治疗组患者在常规治疗基础上联合尿激酶治疗。比较两组患者治疗前后神经功能、内皮功能、氧化应激指标的变化及治疗后的疗效。结果:治疗后,两组患者美国国立卫生研究院卒中量表(NIH Stroke Scale,NIHSS)、神经组织蛋白(S-100β)、神经元特异性烯醇化酶(Neuro-specific enolase,NSE),内皮素-1(Endothelin-1,ET-1)、丙二醛(Malondialdehyde,MDA)、肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)和可溶性血管细胞粘附分子1(Soluble Vascular cell adhesion molecule 1,s VCAM-)1水平均较治疗前显著降低,一氧化氮(Nitric oxide,NO)、超氧化物歧化酶(Superoxide Dismutase, SOD)和谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-PX)水平均较治疗前明显升高(均P0.05)。而治疗组NIHSS评分、S-100β、NSE、ET-1、MDA、TNF-α和s VCAM-1水平均明显低于常规组,NO、SOD和GSH-Px水平均显著高于常规组(均P0.05)。结论:尿激酶治疗ACI患者可显著提高疗效,其可能的机制是减轻患者氧化应激状态,改善血管内皮功能并促进神经功能的恢复。     

含尿激酶mRNA的人胚肾培养细胞的poly(A)+RNA的提取及鉴定

人胚肾原代培养细胞经换无血清培养液后4～6天,可产生较高的尿激酶活性,从2ml细胞沉淀中(2×10~5细胞)可提取60～100μgpoly(A)~+RNA。6M尿素酸性琼脂糖凝胶电泳表明,其大小为9S～28S,以此poly(A)~+RNA为模板可用于体外翻译及反转录实验,DNA-RNA点杂交表明,此poly(A)~+RNA含有编码尿激酶的mRNA,通过Northern blot杂交实验,估计mRNA大小在1～3 kb之间,本制品可供应反转录途径获得尿激酶基因之用。     

影响尿激酶静脉溶栓治疗急性脑梗死预后因素的初步分析

目的:对影响急性缺血性脑卒中患者应用尿激酶溶栓治疗预后的相关危险因素进行初步分析及研究,为今后选择适宜应用尿激酶溶栓的患者打下基础。方法:以2015年1月1日至2015年10月31日入院且应用尿激酶静脉溶栓的38例急性缺血性脑卒中患者,根据治疗3个月后的预后情况将患者分为A组(24例,预后良好)和B组(14例,预后不良),对两组患者的预后影响因素进行分析。结果:预后良好组患者的溶栓前NIHSS评分低于预后不良组,差异具有统计学意义(P0.05)。多因素的Logistic回归分析显示,吸烟和HINSS评分与急性脑梗死尿激酶溶栓预后不良有关,差异具有统计学意义(P0.05),且NIHSS评分是预后的独立预测因素。结论:尿激酶静脉溶栓治疗脑梗死的患者,溶栓前较高的NIHSS评分可能与溶栓预后不良有关。吸烟的患者溶栓后出现预后不良的几率是不吸烟患者溶栓后预后不良的8.83倍,溶栓前NIHSS评分每增加1分,溶栓预后不良的几率增加1.76倍。     

双专－性抗体介导的纤溶作用

用双功能团试剂将抗尿激酶单克隆抗体Ｎ３４的ＩｇＧ和抗人活化血小板α－颗粒膜蛋白ＧＭＰ－１４０单克隆抗体ＳＺ－５１的Ｆａｂ片段通过二硫键共价偶联,偶联的抗体保留了对各自抗原的亲和性。这种对尿激酶和血栓同时具有亲和活性的双专一性抗体（Ｎ３４－ＳＺ－５１）提高低分子量尿激酶的溶栓效率３８倍,且对血浆中纤维蛋白原的含量基本上不影响。     

Assessment of the Interaction Between Urokinase and Reactive Site Mutants of Protein C Inhibitor

Urokinase-type plasminogen activator (uPA) is a serine protease involved in pericellular proteolysis and tumor cell metastasis via plasmin-mediated degradation of extracellular matrix proteins. Plasma uPA is inhibited by the serine protease inhibitor protein C inhibitor (PCI) by the insertion of PCI's reactive site loop into the active site of the protease. To better understand the structural aspects of this inhibition, 15 reactive-site mutants of recombinant PCI (rPCI) were assayed for differences in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to inhibitory activity, while P3 Arg357 mutations had little effect upon the inhibition rate. However, replacement of the P2 Phe353 with small residues like Ala and Gly increased the effectiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a computer-derived molecular model of uPA was generated and docked to a model of PCI to simulate complex formation. The changes made by mutagenesis were then recreated in the model of uPA–PCI. In accordance with the kinetic data, the poor performance of P3 variants is primarily attributable to charge repulsion, while alleviation of steric hindrance at P2 produces the observed increase in uPA inhibition. In the model, residues at P3 interact with PCI rather than uPA, consistent with P3 variants demonstrating that little variation from wild-type activity. Ultimately, this combination of mutagenesis and molecular modeling will further refine our understanding of the interaction between PCI and uPA.     

Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples

AIM: To establish an easily-handled method to isolate mesenchymal stem cells (MSCs) from coagulated human bone marrow samples.METHODS: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated, treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 °C for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast (CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents.RESULTS: The average CFU-F number of urokinase-treated samples (16.85 ± 11.77/10⁶) was comparable to that of un-coagulated control samples (20.22 ± 10.65/10⁶, P = 0.293), which was significantly higher than those of mechanically-cut clots (6.5 ± 5.32/10⁶, P < 0.01) and untreated clots (1.95 ± 1.86/10⁶, P < 0.01). The CFU-F numbers decreased after samples were stored, but those of control and urokinase-treated clots remained higher than the other two groups. Consistently, the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots. The attached cells were fibroblast-like in morphology and homogenously positive for CD44, CD73 and CD90, and negative for CD31 and CD45. Also, they could be induced to differentiate into osteoblasts and adipocytes in vitro.CONCLUSION: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.