纤溶酶原激活因子与猕猴精子运动力的关系及其在附睾和附性腺中的表达调节

用11酸睾酮诱导猕猴少精子症和弱精子症及单侧隐睾手术诱导单侧少精子症和弱精子症模型,观察对附睾头、附睾体、附睾尾、前列腺和精囊组织型PA（tPA）、尿激酶型PA（uPA）及抑制因子-1（PAI-1）mRNA表达的影响。原位杂交的结果表明11酸睾酮诱导少精子症和弱精子症,tPA mRNA的表达在附睾头、精囊及前列腺减少,而在附睾体升高,附睾尾表达基本无变化;uPA mRNA的表达在附睾头、附睾体、前列腺减少,而在精囊升高,附睾尾表达基本无变化;PAI-1 mRNA的表达在附睾头、附睾体、精囊下降,而在前列腺升高,附睾尾表达无显著变化。单侧隐睾手术不影响tPA、uPA和PAI-1 mRNA的表达。这些结果提示附睾头和附睾体分泌的uPA可能与精子前向运动能力的获得相关。tPA、uPA和PAI-1 mRNA在猕猴附睾头部和体部、前列腺和精囊中的表达可能受睾酮的调节,但不受睾丸分泌因子及温度的影响,且在不同部位睾酮的调节具不同的特征,而附睾尾tPA、uPA和PAI-1的表达则可能是组成性表达。     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子(PAI-1)的激素调节

哺乳动物卵巢合成两类纤溶酶原激活因子(PA),即组织型PA(tPA)和尿激酶型PA(uPA)。大鼠卵巢除主要合成tPA外,还分泌一种纤溶酶激活因子的抑制因子(PAI-1)。在促性腺激素作用下,卵巢tPA和PAI-1两种基因的协调表达是导致滤泡破裂的原因。本实验进一步证实,卵巢中的PAI-1主要由膜-间质细胞分泌,可能作为一种屏障限制颗粒细胞tPA分泌到滤泡外间质。当排卵来临时,两种细胞所分泌的tPA和PAI-1相互作用后仍发现有大量tPA活性。这可能是引起排卵的主要原因。因为成熟的卵丘细胞除分泌高量tPA外,还分泌大量PAI-1,在人工授精中两者有可能作为鉴定卵子优劣的可靠指标。     

三七皂苷Rg1对tPA和PAI-1活性的调节作用

探讨三七皂苷Rg1对组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物(PAI-1)活性的调节作用。运用发色底物方法测定三七皂苷Rg1在体外和静脉注射对家兔血浆纤溶酶原激活物(tPA)和血浆或血小板释放的纤溶酶原激活物抑制物(PAI-1)水平的影响。结果表明,三七皂苷Rg1在体外呈浓度依赖性明显抑制血浆PAI-1活性,同时提高血浆tPA活性;30和60 mg/kg的三七皂苷Rg1静脉注射显著抑制血浆PAI-1活性,提高血浆tPA活性,同时降低凝血酶激活的血小板所释放的PAI-1水平。本实验提示三七皂苷Rg1能抑制PAI-1活性,同时升高tPA活性可能是其抗血栓作用的分子机制之一。     

卵巢纤蛋白溶酶原激活因子及其抑制因子的研究

本文综述了近年来作者在研究卵巢纤蛋白溶酶原激活因子(PA)及其抑制因子(PAI)的某些成果。PA是一种高效能蛋白水解酶激活因子,它激活纤蛋白溶酶原成为纤蛋白溶酶,此酶在纤蛋白水解过程中起重要作用。已有证据表明,PA与排卵有关。我们进一步研究发现:(1)在大鼠卵巢体细胞中存在两种PA,即组织型PA(tPA)和尿激酶型PA(uPA);而在卵细胞中只发现tPA;(2)大鼠卵巢tPA明显受促性腺激素和其他激素调节,并在排卵前达到高峰,而uPA没有明显变化;(3)在卵巢体细胞中还发现一种PA的抑制因子(PAI),它与PA结合形成复合体,能部分或完全消除PA活性;(4)只有tPA与大鼠排卵有关;PA和PAI间的相互作用和随激素的波动而引起的动态变化可能对维持卵巢正常生理功能和排卵起重要作用。     

uPA、PAI-1的表达和MVD计数与浸润性乳腺癌侵袭性的关系

目的：探讨尿激酶型纤溶酶原激活剂（uPA）、纤溶酶原激活物抑制剂（PAI-1）的表达和血管形成与浸润性乳腺癌侵袭性的关系。方法：应用免疫组化SP法检测80例浸润性乳腺癌、20例良性乳腺肿瘤组织中uPA、PAI-1的表达情况并进行微血管计数。结果：uPA和PAI-1在浸润性乳腺癌组的阳性表达显著高于良性肿瘤组,且其高表达率与乳腺癌的临床病理参数密切相关（P〈0.05）;微血管计数在乳腺癌组和良性肿瘤组分别为30.87±7.64、20.28±8.72,两组比较有显著性差异（P〈0.01）。uPA与PAI-1在浸润性乳腺癌中的表达呈正相关（P〈0.05）。结论：uPA、PAI-1的高表达与浸润性乳腺癌的浸润、转移相关,uPA、PAI-1的高表达和MVD计数可作为评价浸润性乳腺癌侵袭性、评估预后和确定治疗方案的生物学指标。     

靶向纤溶酶原激活物抑制剂1的肿瘤治疗研究进展

肿瘤是严重危害人类健康的疾病.研究表明,实体瘤周围环境中的胞外基质蛋白、浸润性免疫细胞和间充质细胞分泌的蛋白质组等均与肿瘤的发生、发展以及肿瘤治疗的耐受性等密切相关.肿瘤微环境中一个重要调控因子,纤溶酶原激活物抑制剂1 (plasminogen activator inhibitor-1,PAI-1),不仅与组织型纤溶酶原激活物(tissue-type plasminogen activators,tPA)构成调节纤溶活性的一对关键物质,而且参与肿瘤的侵袭、浸润和转移等多个环节并扮演重要角色.本文针对近年来PAI-1的结构和功能方面研究新进展及其与肿瘤微环境的相关性进行综述,并提出PAI-1可作为抗肿瘤治疗的重要靶点.同时,本文也分析了PAI-1抑制剂对肿瘤干预的研究现状,指出PAI-1抑制剂对肿瘤治疗的潜在应用价值.     

表达人PAI-1的重组腺病毒的制备及检测

人纤溶酶原激活剂抑制物1（PAI-1）基因与复制缺陷型腺病毒载体AdCMVHSgD重组,与pJM17质粒共转染293细胞,采用不铺琼脂的方法产生重组病毒.PCR证实PAI-1基因重组进入腺病毒.进一步感染B16（F10）细胞,细胞表面洗提物和上清分别经纤维显示胶和反向纤维蛋白显示胶检测PAI-1抑制纤溶酶原激活剂（PA）的活性.     

人精液的生物化学研究

人精液的生物化学研究段于峰（湖南省怀化地区卫生学校,怀化４１８０００）关键词人精液,凝固和液化,精浆,精子外套抗原射精液是灰黄色粘稠的液体,由含有精子的附睾液、精囊液和前列腺液在射精时混合而成。精囊液占总量的５５％～６０％,其中主要含高分子碱性蛋白质...     

人组织性纤溶酶原激活物衍生物在大肠杆菌硫氧化还原蛋白融合表达系统中的表达

目的:克隆含tPA中355个氨基酸密码子(1-3和176-527氨基酸)的cDNA序列(tPA355),将其在大肠杆菌融合蛋白表达系统中表达,并在体外复性使其具有激活纤溶酶原的生物活性。方法:采用RT-PCR技术从人黑色素瘤细胞Bowes中克隆出tPA355cDNA,然后在pET32a(+)BL21(DE3)大肠杆菌表达系统中表达,将表达出的融合蛋白Trx-tPA355(Thioredoxin,Trx)包涵体在体外进行变性、复性和纯化以使其具有激活纤溶酶原的生物活性。结果:测序结果表明本研究克隆的编码tPA中355个氨基酸密码子的cDNA序列与美国专利(公开号:5,587,159)中对应的序列完全一致,将其在pET32a(+)/BL21(DE3)大肠杆菌表达系统中表达可获得稳定表达的融合蛋白Trx-tPA355包涵体,该包涵体占菌体总蛋白的30%左右,此融合蛋白经变性、复性后具有激活纤溶酶原的生物活性。结论:含tPA中355个氨基酸密码子(1-3和176-527氨基酸)的cDNA在大肠杆菌Trx融合蛋白表达系统中可获得稳定表达,表达的融合蛋白产物在体外经变性、复性后具有激活纤溶酶原的生物活性。     

小鼠排卵前后卵巢纤蛋白溶酶原激活因子活性的变化

给幼龄小鼠注射PMSG刺激滤泡生长,随后注射hCG以诱发排卵。在激素处理的不同时间取出卵巢,制备卵巢匀浆液或从卵巢中分离颗粒细胞和卵丘-卵母细胞复合体,并做离体培养。样品中组织型(tPA)和尿激酶型(uPA)纤蛋白溶酶原激活因子经SDS-凝胶电泳分离,用纤蛋白铺盖技术测定。实验结果表明,注射hCG 8h后15％的受试动物排卵,而卵巢匀浆液和颗粒细胞中tPA和uPA活性分别也在hCG注射后4和8h达到高峰。排卵后酶活性下降。卵丘-卵母细胞复合体主要含tPA,注射hCG 12—24h达到高峰。上述资料证明,tPA和uPA都参入小鼠排卵过程。因为排出的卵子中仍含有大量tPA,卵细胞的tPA除参与排卵外,可能对排卵后的一些生理过程也起重要作用。     

SERPINE2, a serine protease inhibitor extensively expressed in adult male mouse reproductive tissues, may serve as a murine sperm decapacitation factor

SERPINE2, one of the potent serine protease inhibitors that modulates the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the present study, we purified SERPINE2 from mouse seminal vesicle secretion (SVS), using liquid chromatography and identified it by liquid chromatography/tandem mass spectrometry, and it showed potent inhibitory activity against the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was immunolocalized to the SVS and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testes, SERPINE2 was immunostained in spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa. SERPINE2 was also detected on the acrosomal cap of testicular and epididymal sperm and was suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind to epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was detected predominantly on uncapacitated sperm, indicating that SERPINE2 is lost before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.     

Electron Microscopic (Energy Dispersive X-ray Analysis) Study of Human Male Reproductive Organs and Semen

Recently, technological advancement helped to improve our knowledge on trace elements in human male reproductive organs and its secretion, semen. In this study, employing energy dispersive x-ray analysis facilities on electron microscope, presence of different elements in human male reproductive organs-??testis, epididymis, caput, corpus and cauda, prostate gland, seminal vesicle, Cowper??s gland and vas deferens??seminal plasma and spermatozoa pellet was studied. Several elements were observed. Gold was one among them that was present in seminal plasma and spermatozoa. It was also present in epididymis caput. Authors consider epididymis caput as the source of gold in semen.     

促性腺激素诱导猕猴排卵周期中卵巢纤溶酶...

Changes of plasminogen activator (PA) and its inhibitor (PAI-1) activity and antigen have been investigated during PMSG/hCG induced ovulation in rhesus monkeys. It has been demonstrated that the ovarian tissue type PA (tPA) activity, which reaches maximum prior to ovulation and declines thereafter, is closely related to follicular rupture; significant increases in urokinase type PA (uPA) only occurs in granulosa cells after ovulation. Since the secretory activity of ovarian PAI-1 reaches its peak level 12-24 h earlier than tPA the rapid decrease in PAI-1 activity in the approach of ovulation is correlated with the elevation of tPA activity. It is, therefore, suggested that a counterbalance of tPA and PAI-1 activity within the ovary may play an important role in the ovulation mechanism, whereas uPA may be involved in the regulation of corpus luteum formation.     

The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells.

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.     

Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation

HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA) levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta) that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1–9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA) in the axillary lymph node (6.48±0.50) were significantly higher than in the genital tract tissues: testis (3.67±2.16; p<0.05), epididymis (3.08±1.19; p<0.0001), prostate (3.36±1.30; p<0.01), and seminal vesicle (2.67±1.50; p<0.0001). Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.     

Gelatinolytic proteinase activities in human seminal plasma

Proteinase activities in human seminal plasma were detected using gelatin-containing sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. Three prominent bands of activity of Mr 60,000, 66,000 and 90,000 were observed as well as 9 other bands of less intensity (34,000-158,000). These proteinases were dependent upon calcium for optimal activity, did not hydrolyse casein, and were predominantly in the soluble portion of seminal plasma. Examination of seminal plasma of men with different sperm concentrations, split ejaculates, and prostatic secretions indicated that the prostate gland was a source of most of these activities. Proteinase activities of Mr 34,000, 37,000, 82,000 and 120,000 were expressed more frequently in seminal plasma from normozoospermic men than from seminal plasma of oligo- or azoospermic men, indicating that they may also arise from spermatozoa in the semen sample. The proteinases of Mr 60,000 and 66,000 were found in all seminal plasmas whereas there was variation in the expression of the other molecular forms of enzyme, even in the normozoospermic samples. There are multiple forms of gelatinolytic proteinase activities in human seminal plasma which appear to arise from multiple sources in the reproductive tract including the Cowper's/urethral glands, the prostate gland, seminal vesicle and/or spermatozoa. Their function(s) in semen remains to be established.     

Suppression effect of seminal vesicle autoantigen on platelet-activating factor-induced mouse sperm capacitation

Mammalian sperm gain the ability to fertilize an egg successfully by the capacitation process. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction (AR) and resulting in loss of their fertilization activity. Thus, functional sperm activation is tightly regulated by a capacitation and suppression (decapacitation) mechanism. Factors, such as platelet-activating factor (PAF) present in both sperm and the female genital tract, are able to stimulate sperm capacitation. Seminal plasma is thought to have the ability to suppress sperm capacitation; however, the regulatory mechanisms of seminal plasma protein on sperm capacitation are not well understood. Recently, we demonstrated that seminal vesicle autoantigen (SVA), a major seminal vesicle secretory protein, is able to suppress mouse sperm capacitation. To further study the suppression spectra of SVA on sperm capacitation, we investigated the effect of SVA on PAF-induced mouse sperm capacitation-related signals. Here, we demonstrate that SVA decreases the [Ca(2+)](i) to suppress the PAF's effects on [Ca(2+)](i), the cAMP level, protein tyrosine phosphorylation, and capacitation. The inhibition of PAF-induced protein tyrosine phosphorylation and capacitation by SVA can be reversed by cAMP agonists. Characterization of the interactions of SVA with PAF by TLC overlay and tryptophan fluorescence spectrum analyses indicates that SVA is capable of binding PAF with an apparent dissociation constant K(d) > 50 microM. Together with these results, we demonstrate that SVA deceases [Ca(2+)](i) and cross-talks with PAF-induced intracellular signals to regulate mouse sperm capacitation.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.