尿激酶型纤溶酶原激活因子对小鼠精子体外授精能力的影响

尿激酶型纤溶酶原激活因子（uPA）是一种丝氨酸蛋白水解酶，分子量约55×10^3，能广泛水解细胞外基质，与其抑制剂以及受体（uPAR）一起参与体内多项生理与病理过程。其中uPA与生殖系统的关系日益受关注。熊承良等^[1,2]报道人精子膜表面存在uPA和uPAR，受精卵培养液内的uPA含量明显高于未受精卵培养液内的uPA含量。[第一段]     

尿激酶型纤溶酶原激活因子在精子趋化运动中的作用

目的:研究尿激酶型纤溶酶原激活因子(uPA)是否能诱导精子产生趋化性运动,从而探讨uPA在治疗男性不育中的可能作用机制。方法:采用精子聚集毛细管内的方法来检测精子趋化性。根据毛细管内自下而上uPA浓度梯度的方向将实验分为递增浓度A组、递减浓度B组和对照C组。A组毛细管内的趋化液以及精子培养皿内的处理液分别是不同浓度的uPA和Ham'sF-10;B组相反,分别为Ham'sF-10和uPA;C组毛细管和精子培养皿内的液体均为Ham'sF-10。然后检测在不同时间点不同组毛细管内聚集的精子密度。结果:①精子顺递增的uPA浓度梯度进行趋化运动。A组毛细管内液体对精子的聚集作用明显大于B组和C组(P<0.05)。②20IU/ml的uPA对精子趋化作用最强。③3组中的精子密度随着时间的延长趋于增加,但在20、30min2个时间点,3组的精子密度之间差异有显著性(P<0.05)。④uPA除了对精子有趋化作用外,还能增加精子活力,促进精子运动。结论:uPA在体外既能诱导精子产生趋化性运动,又能增加精子的活力,推测此为uPA治疗男性不育的作用机制之一。     

尿激酶型纤溶酶原激活因子对雄性大鼠生育力影响的实验研究

目的:探讨尿激酶型纤溶酶原激活因子(uPA)对于奥硝唑所致的雄性不育大鼠生育力的影响。方法:10~12周龄成年雄性SD大鼠50只,随机分成5组,每组10只:奥硝唑模型组、uPA高剂量组、中剂量组、低剂量组和空白对照组。奥硝唑模型组和uPA各剂量组给予奥硝唑400 mg/(kg.d)灌胃,同时uPA高剂量组、中剂量组、低剂量组分别腹腔注射uPA 3 000 IU/(kg.d)、1 000 IU/(kg.d)、334 IU/(kg.d)。空白对照组给予等体积0.5%羧甲基纤维素钠灌胃,同时腹腔注射等量生理盐水。各组动物连续处理20 d后,观察动物的精液常规、睾丸和附睾功能以及与雌鼠交配情况。结果:①与空白对照组相比,奥硝唑模型组精子活动力和平均胚胎数显著降低(P<0.01)。②与模型组相比,uPA高剂量组精子活动力和平均胚胎数显著增加(P<0.01),而uPA中、低剂量组虽有所改善但无统计学意义。③uPA高剂量组精子日产量与模型组及空白对照组相比差异有显著性(P<0.05)。结论:uPA对奥硝唑引起的雄性大鼠不育起到改善作用。     

尿激酶型纤溶酶原激活因子及其受体在神经母细胞瘤中的表达及其意义

目的：研究尿激酶型纤溶酶原活因子（uPA)及其受体（uPAR）在神经母细胞瘤（NB）中的表达和意义。方法：应用免疫组织化学方法研究uPA及uPAR在42例神经母细胞瘤中的表达，并应用逆转录-聚合酶链式反应（RT－PCR）方法检测患儿骨髓和外周血中的神经蛋白基因产物9．5（PGP9．5）。结果：uPA及uPAR阳性表达主在进展期肿瘤（均为85．7％）高于局灶期肿瘤（42．9％，28．6％）；预后不良型患儿（91．7％，83．3％）高于预后良好型患儿（均为44．4％），且差异均具有非常显著性（P＜0．01）。患儿骨髓和外周血中PGP9．5阳性检出率在uPA阳性患儿组（60．0％）显著高于uPA阴性患儿组（8．3％，P＜0．01）；uPAR阳性组（57．1％）高于uPAR阴性组（21．4％，P＜0．05）。uPA和uPAR同时阳性的10例患儿，骨髓和外周血中均有PGP9．5阳性表达，而同时阴性的5例患儿中，均未检测到PGP9．5。结论：uPA和uPAR在NB的浸润转移过程中发挥重要的作用。     

组织型纤溶酶原激活因子基因对移植静脉再狭窄的影响

目的探讨组织型纤溶酶原激活因子(t-PA)局部转基因表达对移植静脉血栓形成和内膜增生的影响。方法 72只兔建立颈外静脉-颈总动脉移植模型,并随机分为基因治疗组(n= 24)、载体对照组(n=24)和空白对照组(n=24),进行局部基因转染,于术后不同时点取材,逆转录 -聚合酶链反应(RT-PCR)、免疫印迹(Western blot)和底物发色法分别检测移植静脉t-PA基因表达和活性变化,放射标记计数观察其对血小板沉积的影响,病理形态学观察移植血管新生内膜增生情况。结果术后2、5、14、28 d,转基因组检测到移植静脉外源性t-PA mRNA的转录和蛋白表达,其活性分别为(370．63±59．44)、(344．13±48．47)、(252．87±51．80)和(161．75±68．94)U／g,对照组各时点则未检测到纤溶酶活性。术后2 d,转基因组、载体组和空白对照组平均血小板沉积数分别为(85．04±21．58)×10 6,(225．87±85．13)×10 6和(211．57±78．02)×10 6,转基因组明显少于载体组和空白对照组(P<0．05)。术后5、14、28、60 d,转基因组新生内膜面积、内膜中膜面积比均明显小于对照组。结论局部转染t-PA基因,能抑制移植静脉血栓形成,减轻新内膜的增殖,从而有效预防再狭窄的发生。     

弱精子症动物模型中尿纤溶酶原激活因子的含量及表达情况研究

目的:利用奥硝唑所致弱精子症动物模型,采用免疫组化方法和RT-PCR技术,了解尿纤溶酶原激活因子(uPA)在弱精子症动物模型中的含量及表达情况,初步探讨奥硝唑所致弱精子症动物模型的机制及uPA作为预防或治疗弱精子症药物的可能性。方法:48只雄性SD大鼠随机分为1d用药组,5d用药组,10d用药组,15d用药组,20d用药组和对照组,每组8只,用药组每天给予奥硝唑200mg/kg,对照组给予0.5%羧甲基维素钠连续灌胃,用药组分别在给药第1、5、10、15、20d后24h内,麻醉处死动物取附睾和睾丸。低渗肿胀试验检测精子细胞膜完整性,免疫组化方法动态观察睾丸与附睾组织中uPA蛋白表达情况,RT-PCR检测睾丸组织中uPAmRNA含量。结果:奥硝唑持续给药构建弱精子症时,精子膜完整性下降发生在给药的第10d,并一直维持低值。与建立弱精子症模型同步的uPA在睾丸和附睾组织蛋白表达及mRNA含量下降在用药15d,下降趋势上是平行的,而显效性稍滞后。用药15d组与用药20d组uPA蛋白表达、mRNA水平分别与对照组比较有统计学意义(P<0.05)。结论:精子细胞膜受损、运动能力下降与uPA表达及含量下降平行,奥硝唑所致弱精子症模型形成可能是由于uPA含量及表达下降所致。弱精子症形成原因较复杂,uPA含量及表达的下降可认为是弱精子症形成因素之一,检测uPA含量可能有助于弱精子症的诊断和预防。     

慢性前列腺炎对纤溶酶原激活因子系统影响的初步研究

目的 :分析慢性前列腺炎对纤溶酶原激活因子 (PA)系统表达和活性的影响。　方法 :选取正常男性 2 3例 ,慢性前列腺炎患者 80例 (不育组和可生育组各 4 0例 )。采用纤维蛋白 琼脂糖PA指示胶打孔法和SDS PAGE电泳后指示胶铺盖法 ,测定精浆中总PA及组织型纤溶酶原激活因子 (tPA)和尿激酶型纤溶酶原激活因子 (uPA)的表达和活性。结果 :在正常人精浆中有高表达的总PA活性 ;而且同时表达tPA和uPA。慢性前列腺炎患者的精浆中总PA活性降低 ,tPA活性明显减低 ,uPA活性减低。慢性前列腺炎的可生育组和不育组精浆中总PA活性均降低 ,但两组间无明显差别。　结论 :患慢性前列腺炎时前列腺分泌功能降低 ,合成分泌PA减低。PA有可能在今后作为临床中判断前列腺功能检测指标之一     

纤溶酶原激活因子及其抑制因子与睾丸支持细胞

纤溶酶原激活因子 (PA)及其抑制因子 (PAI)参与机体的多种生理、病理活动。支持细胞是睾丸精曲小管的重要组成部分 ,其正常功能的发挥对精子发生等生物学行为的正常进行具有非常重要的意义。睾丸支持细胞在激素及其他因子的作用下 ,分泌PA及PAI,发挥生物学作用 ,维持正常的精子发生、精子活力及受精过程 ,两者之间的关系正日益受到人们的重视     

纤维蛋白对肾小球内皮细胞表达纤溶酶原激活物及纤溶酶原激活物抑制物的作用

目的：观察体外培养人肾小球内皮细胞（GEC）表面原位形成的纤维蛋白对GEC表达纤溶酶原激活物及纤溶酶原激活物抑制物（PA／PAI）的影响。方法：应用逆转录聚合酶链反应（RT－PCR）,酶谱分析法与反向酶谱法分别在基因转录水平与蛋白质活性水平上检测纤维蛋白对GEC表达tPA,uPA gn PAI-1r 作用,纤维蛋白平板法检测纤维蛋白对GEC PA／PAI系统的综合效应,结果：纤维蛋白能够明显促进tPA,uPA与PAI－1的mRNA表达上调,无血清RPMI 1640培养下的GEC几乎检测不到PAI知性,但可检测到PAI－1的活性。纤维蛋白能够浓度依赖性刺激GEC tPA与uPA活性增加以及PAI01的活性增加,呈浓度依赖性与时间依赖性,相同剂量的纤维蛋白原与纤维蛋白的作用相似,放线菌酮与放线菌素D均可抑制纤维蛋白上调GEC表达tPA,uPA与PAI的作用,纤维蛋白平板法显示,纤维蛋白对GEC PA／PAI系统的综合效应是以升高PA活性为主,其活性能够被抑肽酶完全阻断。结论：肾脏局部毛细血[管内沉积的纤维蛋白可能通过对GEC PA／PAI系统的调节发挥其病理作用。     

骨肉瘤中尿激酶型纤溶酶原激活因子和受体的表达

目的研究尿激酶型纤溶酶原激活因子(uPA)及其受体(uPAR)在不同外科分期骨肉瘤中的表达及意义.方法采用荧光定量RT-PCR方法检测32例骨肉瘤组织uPA和uPAR表达情况.结果肿瘤组织与癌旁组织和正常组织比较,uPA和uPAR表达的阳性率明显增高(P＜0.01).骨肉瘤Ⅰ、Ⅱ、Ⅲ期表达uPA的阳性率分别为22.2%(2/9)、76.9%(10/13)、90.0%(9/10),表达uPAR的阳性率分别为33.3%(3/9)、84.6%(11/13)、90.0%(9/10).uPA和uPAR在Ⅰ期和Ⅱ期、Ⅰ期和Ⅲ期骨肉瘤中表达差异均有显著性(P＜0.05、P＜0.01).结论uPA/uPAR在骨肉瘤组织中表达明显升高.肿瘤恶性程度越高,uPA/uPAR表达阳性率越高,提示uPA/uPAR与骨肉瘤恶性进展有高度相关性.     

少弱精子症和正常生育男性精液中尿激酶及其受体含量的研究

目的:通过研究精子正常和异常男性精浆和精子中尿激酶及受体含量差异,以了解尿激酶及受体与男性生育力的关系。方法:采用双抗体夹心ELISA法测定22例正常生育男性和44例少弱精子症男性精浆和精子中尿激酶及受体的含量。结果:①正常男性精浆尿激酶平均含量为(4 803.69±602.78)mU/L,与少弱精子症组[(4 061.35±736.23)mU/L]相比,差异有显著性(P<0.01)。正常生育男性精子尿激酶平均含量为(30.29±3.16)mU/106个精子,与少弱精子症组[(20.51±4.2)mU/106个精子],差异有显著性(P<0.01)。②正常生育男性精子尿激酶受体平均含量为(12.97±3.11)mU/106个精子相比,与少弱精子症组[(6.09±1.45)mU/106个精子]相比,差异有显著性(P<0.01)。③精子和精浆中尿激酶含量和精子活率和活力呈显著正相关。结论:尿激酶和男性生育力相关,少弱精子症和正常生育男性精液中尿激酶及其受体含量存在差异。     

尿激酶型纤溶酶原激活剂mRNA在胃癌组织中的表达及其意义

目的 研究尿激酶型纤溶酶原激活剂(uPA)mRNA在胃癌组织中的表达及其意义。方法 应用荧光定量RT-PCR方法，分别检测48例胃癌及其癌旁组织中uPA mRNA的表达，并结合其临床病理特征进行分析。结果 48例胃癌组织及癌旁组织中，uPA mRNA表达阳性率分别为83．3％和25.0％,胃癌伴有淋巴结转移和无淋巴结转移者中，uPA mRNA表达阳性率分别为93．8％和62．5％．且uPA mRNA表达与胃癌浸润程度呈正相关。结论 uPA mRNA表达可作为判断胃癌恶性程度及预后的指标之一。     

Production of Urokinase-Type Plasminogen Activator (u-PA) and Plasminogen Activator Inhibitor-1 (PAI-1) in Human Brain Tumours

Summary We investigated the role of plasminogen activators (PAs) and their inhibitor (plasminogen activator inhibitor-1, PAI-1) in human brain tumours. The amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1), and the activity of u-PA and t-PA were determined by enzyme-linked immunosorbent assay (ELISA), and u-PA and PAI-1 were immunolocalized using monoclonal antibodies in human brain tumours and normal brain tissues. The tissues were surgically removed from 64 patients; normal brain tissue (5 cases), low-grade glioma (4 cases), high-grade glioma (17 cases), metastatic tumour (9 cases), meningioma (benign 12 cases, malignant 6 cases), acoustic schwannoma (11 cases). u-PA activity and u-PA and PAI-1 antigen levels were significantly elevated in malignant brain tumours (malignant meningiomas, high-grade gliomas, and metastatic tumours) and acoustic schwannomas but very low in benign meningiomas, low-grade gliomas and normal brain. There was no difference in t-PA antigen levels among normal and malignant tissues, however levels of t-PA activity were markedly decreased in metastastic tumours. All malignant brain tumour tissues showed positive immunostaining for u-PA and PAI-1, however, some tumour cells showed negative intensity while others showed strong intensity for these antibodies. This contrasts to the homogeneous staining pattern found in acoustic schwannoma. These findings indicate that malignancy in human brain tumours is associated with elevated levels of u-PA and PAI-1 and that an imbalance between these proteins in a micro-enviroment contributes (ascribes) to tumour cell invasion.     

血尿激酶型纤溶酶原激活剂mRNA水平表达与乳腺癌及其淋巴结转移的关系

目的　研究血尿激酶型纤溶酶原激活剂(uPA)mRNA水平表达与乳腺癌及淋巴结转移的关系及其临床意义。方法　应用荧光定量RT PCR方法,检测60例乳腺良、恶性肿瘤患者血中uPA mRNA水平的表达,分析其与乳腺癌及其淋巴结转移的关系。结果　uPA mRNA表达在18例良性肿瘤患者中16例为阴性,2 例为低表达。在42例乳腺癌患者中,无淋巴结转移的20例中18例为阳性,其中1例为高表达, 5例为中度表达,12 例为低表达,另2例为阴性; 有淋巴结转移的22例均为阳性,其中16 例为高表达,5 例为中度表达,1 例为低表达。乳腺良、恶性肿瘤患者血中uPA mRNA表达差异有显著性意义(P<0.05); 乳腺癌患者中无淋巴结转移者uPA mRNA的表达强度明显低于有淋巴结转移者(P<0.05)。结论　血中uPA mRNA在乳腺癌中的表达明显高于良性肿瘤,其表达强度与乳腺癌淋巴结转移明显相关,可为临床分期及后续治疗提供依据。     

Effects of uPA on mesangial matrix changes in the kidney of diabetic rats

Objective: To investigate the effect of urokinase-type plasminogen activator (uPA) on mesangial matrix in the kidney of diabetic rats and its related mechanisms. Methods: Diabetic Sprague-Dawley (SD) rats induced by intraperitoneal injection of streptozotocin (STZ) were randomly and evenly divided into two groups: DM?+?vehicle, and DM?+?uPA (2500?U?kg^?1 uPA via tail vein once a day for four weeks). The normal SD rats without diabetes were considered as control group. Rats in the three groups were executed and the heart blood was sampled for determination of blood glucose and serum creatinine. Meanwhile, kidney tissues of rats were also harvest for measurement of glomerular area, volume, and mesangial area by periodic acid silver methenamine (PASA) staining. The expression of urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and collagen IV in renal tissues was tested with immunohistochemistry. Results: Compared with control, the DM rats had obvious albuminuria, significantly (p?<?0.01) increased glomerular volume and mesangial matrix area, and significantly (p?<?0.05) higher expression of uPAR, PAI-1 and collagen IV in mesangial matrix, significantly up-regulated (p?<?0.05) glomerular uPAR, PAI-1, and collagen IV expression. After treated with uPA, the diabetic rats had significantly (p?<?0.05) reduced albuminuria, significantly (p?<?0.01) improved glomerular volume and mesangial matrix, significantly (p?<?0.05) down-regulated PAI-1 and collagen IV expression in mesangial matrix. However, the uPAR expression in renal tissues were unchangeable (p?>?0.05) and PAI-1 and collagen IV expression were significantly (p?<?0.05) reduced when diabetic rats were treated with uPA. Conclusion: uPA can down-regulate glomerular PAI-1 expression in the DM rats but not significantly influence uPAR expression, suggesting that uPA might regulate the mesangial cell (MC) and its matrix expression and improve diseased diabetic mesangial matrix via its combination with uPAR to uptake PAI-1 and accelerate its degradation.     

JC-1单标法检测精索静脉曲张患者精子线粒体膜电位的研究

目的:检测精索静脉曲张患者精子线粒体膜电位并探讨其临床意义。方法:将67例精索静脉曲张患者分为VC1组(精索静脉曲张1度,n=26)、VC2组(精索静脉曲张2度,n=21)和VC3组(精索静脉曲张3度,n=20),以正常生育男性为正常对照组(n=29)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后用荧光染料JC-1染色后上流式细胞仪分析,检测精子线粒体膜电位(JC-1+%)。结果:VC1、VC2、VC3组精子线粒体膜电位[(56.29±16.32)%,P<0.05;(45.04±13.21)%,P<0.01;(31.63±12.91)%,P<0.01]均显著低于正常对照组[(76.21±13.96)%]。96例标本中,JC-1+%与(a+b)级精子百分率呈显著正相关(r=0.693,P=0.000)。结论:精索静脉曲张可引起精子线粒体膜电位降低,可能是导致男性不育的重要原因之一。     

Urokinase Plasminogen Activator Receptor (uPAR): A Potential Indicator of Invasion for In Situ Breast Cancer

Abstract: Urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) are important mediators in the cellular process of cancer invasion and metastasis. Ductal carcinoma in situ (DCIS) is classified by lack of invasion into the adjacent stroma, yet definitive histologic features have not been identified to indicate the propensity for cellular invasion. Therapy for DCIS remains controversial because of the probability for recurrence. We hypothesized that uPA and uPAR may represent new predictors for recurrence of DCIS. Tissue specimens were obtained from 10 normal, 10 hyperplasia, and 70 patients with DCIS. Representative sections of the regions were mounted and stained by immunohistologic techniques using mouse anti-human uPA and uPAR antibodies. Stain intensities were assessed by densitometry image analysis. Gray scale values for in situ patients were compared to normal averages to determine whether staining intensities were normal or significantly higher (p < 0.05) than normal. DCIS tissues were heterogeneous for stain intensities of uPA and uPAR. Patients with high stain intensities for uPAR (28/70 = 40%) correlated with a higher recurrence rate (15/28 = 54%) than with patients having high stains for uPA (19/70 = 28% with 17/50 = 34% recurrence). In addition, patients with combined high stains for uPA and uPAR (11/19 = 60%) showed a recurrence rate of 55% compared to high uPA/normal uPAR with 0% recurrence. Immunohistologic evaluation of DCIS for uPAR, alone and in combination with uPA, significantly correlates with recurrence of invasive breast carcinoma. Evaluation of uPAR among DCIS lesions may provide a new prognostic indicator for recurrence of breast carcinoma.