尿激酶型纤溶酶原激活因子cDNA的克隆与原核表达

目的构建尿激酶型纤溶酶原激活因子（uPA）原核表达质粒,并在大肠埃希菌BL21中表达,为进一步研究uPA奠定基础。方法应用逆转录RT—PCR,从人肝细胞cDNA中扩增人尿激酶型纤溶酶原激活因子基因序列,与原核表达质粒pET32a重组,获得表达质粒uPA—pET32a。用氨苄青霉素平板筛选转化子。双酶切与DNA测序进行鉴定,用IPTG诱导表达,并用Westernblotting进行鉴定。结果从肝细胞cDNA中扩增的uPA基因片段长1296bp,酶切及DNA测序证实uPA-pET32a重组质粒构建正确,表达融合蛋白分子量约为68900Mr,经Western blotting证实为目的蛋白。结论成功构建了重组原核表达质粒uPA—pET32a。并能在大肠埃希菌内表达,所表达的融合蛋白分子量大小与预期的相一致．为进一步的研究奠定了基础。     

稳定表达组织型纤溶酶原激活因子基因的人脐静脉内皮细胞株的建立

为建立稳定表达组织型纤溶酶原激活因子(t-PA)基因的人脐静脉内皮细胞株,为其在基因修饰的组织工程血管中的应用奠定基础。首先构建t-PA基因的真核表达载体pcDNA3.1-Myc-HisB(-)/t-PA,将该表达载体用阳离子脂质体介导,转染人脐静脉内皮细胞系细胞,经G418筛选,获得阳性细胞克隆,扩增后分别用逆转录聚合酶链反应(RT-PCR)、免疫印迹(Western-blotting)检测t-PA转录和蛋白表达水平,用底物发色法检测t-PA的活性。逆转录聚合酶链反应检测出了t-PA的稳定转染细胞克隆,免疫印迹证实该克隆细胞有含Myc标签的t-PA蛋白表达,底物发色法检测发现其表达产物的活性增加。结果证明成功建立了稳定表达t-PA基因的人脐静脉内皮细胞株。     

慢性肾脏疾病血清和尿液纤溶活性物质的改变及临床意义

目的探讨慢性肾脏疾病血清和尿液纤溶活性物质的改变及其临床意义。方法选择38例慢性肾小球肾炎(CGN),28例肾病综合征(NS),36例非透析治疗的慢性肾功能不全(CRF)和20例正常对照作为研究对象,应用ELISA法检测血清和尿液中组织型纤溶酶原激活剂(t-PA)和纤溶酶原激活物抑制剂-1(PAI-1)的浓度,同时分析尿中t-PA和PAI-1的水平与血t-PA、PAI-1、血肌酐和24h尿蛋白总量之间相关性。结果慢性肾脏疾病出现血清t-PA、PAI-1升高,尿液t-PA、PAI-1降低,其中尿液t-PA、PAI-1的改变独立于血清,不受血肌酐和24h尿蛋白定量的影响。结论慢性肾脏疾病患者存在纤溶活性物质的异常,其中尿液纤溶活性物质的改变可反应肾脏内皮细胞损伤。     

t-PA基因新型真核表达载体的构建及鉴定

目的构建人组织型纤溶酶原激活因子(tPA)基因的新型真核表达载体,为血栓性疾病的基因治疗奠定基础。方法将tPA基因克隆到真核表达载体pcDNA3.1MycHisB()中,进行酶切鉴定及DNA测序分析,并将携带有tPA基因的该真核表达载体,通过脂质体介导,转染鼠血管平滑肌细胞,底物发色法测定转基因血管平滑肌细胞tPA活性。结果经双酶切鉴定和测序证实已将tPA基因DNA片段正确插入到真核表达载体中,并能在真核细胞中表达、分泌有活性的tPA。结论成功构建pcDNA3.1MycHisB()/tPA基因新型真核表达载体。     

Circulating Levels of Urokinase-Type Plasminogen Activator (uPA) and its Soluble Receptor (suPAR) in Patients with Atopic Eczema/Dermatitis Syndrome

Accumulating evidence has indicated that different immune and inflammatory processes may be accompanied by up-regulation of the uPA/uPAR system. Moreover, it has been suggested that the fibrinolytic system participates actively in immune-mediated skin disorders, including atopic eczema/dermatitis syndrome (AEDS). To study a possible role of such uPA/uPAR system in AEDS, we investigated circulating levels of uPA and suPAR in patients at different clinical stages of AEDS. Levels of u-PA and suPAR were measured by enzyme-linked immunoassay in plasma from 13 patients (five females and eight males; median age 27 years) with moderate AEDS, eight patients (three females and five males; median age 25.5 years) with severe AEDS, and 18 age- and sex-matched healthy subjects. Plasma levels of uPA and suPAR in AEDS patients did not differ significantly when compared with those in healthy subjects. Moreover, we failed to observe any significant differences in levels of these components between patients with moderate and severe AEDS and the controls. It seems that plasma levels of uPA and suPAR are similar in patients at the different stages of AEDS and the healthy subjects. Moreover, these data suggest that the release of uPA and its soluble receptor into the bloodstream is not increased in the course of complex immune-mediated processes associated with AEDS.     

尿激酶型纤溶酶原激活物及其特异受体和抑制物与肺癌的浸润转移

研究尿激酶型纤溶酶原激活物(uPA)及其特异受体(uPA-R)和抑制物(PAI-1、PAI-2)在肺癌浸润转移中的作用.应用RIA分别对67例经组织病理确诊的各期肺癌和30例肺部相关炎症患者及30名健康献血者进行了相应的检测.结果显示,小细胞肺癌Ⅱ期和Ⅲ期患者血浆中uPA、uPA-R、PAI-1水平显著升高(P＜0.001),而PAI-2的水平逐渐降低;腺癌、鳞癌伴有浸润主支气管及肺门淋巴结者uPA、uPA-R与PAI-1水平亦显著升高(P＜0.001);周围型肺癌未见淋巴结受侵者uPA、uPA-R、PAI-1与PAI-2水平异常升高.uPA、uPA-R与PAI-1在肺癌中水平明显升高,并与肺癌的浸润转移相关密切,可作为肿瘤患者早期诊断、预后评估的有力指标.     

肝细胞生长因子激活因子抑制因子-1的表达与功能

肝细胞生长因子激活因子抑制因子-1(hepatocyte growth factor activator inhibitor type1,HAI-1)是一种Kunitz型丝氨酸蛋白酶抑制因子,定位于细胞的基底侧,具有膜型和分泌型两种形式,能有效抑制肝细胞生长因子激活因子HGFA和丝氨酸蛋白酶Matriptase的活性,参与HGF/c-Met信号传导途径调节。HAI-1在包括妊娠、再生及肿瘤等各种正常生理及病理状态下均有不同水平的表达,其表达水平的变化以及其与靶蛋白酶表达比例的变化直接影响到靶蛋白酶的活性,从而在调控个体发育、血管生成、组织损伤修复以及抑制肿瘤的侵袭性生长等生理和病理过程中发挥重要作用。     

CNSI患者血浆中uPA及其受体(uPAR)的变化和临床意义

目的：研究中枢神经系统感染（CNSI）患者血浆中尿激酶型纤溶酶原激活物（uPA）及其受体（uPAR）的变化及意义。方法：收集我院2006年10月～2008年7月CNSI住院患者69例,将CNSI病例分为病毒性脑炎组、结核性脑膜炎组、化脓性脑膜炎组,并设立对照组。用双抗体夹心酶联免疫法检测所有CNSI病例血浆中uPA及其受体（uPAR）水平。结果：化脓性脑膜炎组、结核性脑膜炎组uPA及其受体（uPAR）水平明显高于病毒组及对照组（P〈0.01）,血浆uPAR浓度改变与uPA同步,但uPAR升高水平高于uPA。结论：血浆中uPA及其受体（uPAR）水平在化脓性脑膜炎、结核性脑膜炎中升高,可为化脓性脑膜炎、结核性脑膜炎、病毒性脑炎的鉴别诊断提供更多的实验室依据。     

The Genome of Cowpox Virus Contains a Gene Related to Those Encoding the Epidermal Growth Factor,Transforming Growth Factor Alpha and Vaccinia Growth Factor

Cowpox virus (CPV) is a member of the Orthopoxvirus genus and has the genetic capacity to encode a multitude of genes that interfere with the host inflammatory and immune response or modulate the physiological state of infected and non-infected cells. Among these CPV factors are receptors homologous to interferon and tumor necrosis factor receptors and also a viral cellular serine-proteinase analog. Here we describe the detection of a CPV gene that encodes a protein homologous to epidermal growth factor, transforming growth factor alpha and poxvirus growth factors, such as the vaccinia growth factor (VGF). The VGF and other poxvirus growth factors are produced early in the infection and are secreted into the medium where they bind to the EGF receptors, generating mytotic responses. The cowpox growth factor (CGF) gene was detected in three copies on the virus genome by PCR, and by northern and southern blot hybridization using VGF nucleotide sequences as primers and probes. The CPV gene has a strong nucleotide and predicted amino acid similarity with VGF, and is also produced early in the infection.     

Modulation of glioma cell invasion and motility by adenoviral gene transfer of PAI-1

A number of studies have emphasized the role of PAI-1 as an important regulator of tumor cell invasion and metastasis. The hallmark of primary tumors of the central nervous system and glioblastomas in particular is the diffuse invasion into the normal brain tissue. Since PAI-1 is expressed in such tumors, we studied the effect of adenoviral-mediated transfer of the PAI-1 gene in regulating the in vitro invasiveness of D54Mg glioma cells into Matrigel, and into fetal rat brain aggregates. Treatment of D54Mg cells with 50 MOI (multiplicity of infection) of the replication defective vector AdCMVPAI-1 increased PAI-1 expression 23-fold compared to control vectors, and the invasion through Matrigel was reduced by 67%. The motility of the cells was reduced by 58% compared to controls (indicating that inhibition of motility was the principal effect of PAI-1 in these cells). The ability of D54Mg tumor spheroids to invade fetal rat brain aggregates was not reduced by the PAI-1 gene transfer. The results show that overexpression of PAI-1 can inhibit glioma cell motility and invasion through extracellular matrix (ECM) components, like laminin and collagen, but does not inhibit tumor cell invasion in a three-dimensional invasion assay, simulating normal brain tissue having a different ECM and interstitial composition. The different results obtained in the two invasion assays reflect the complex biological effects of the uPA/PAI-1 system, and questions a simplistic view of PAI-1 as an inhibitor of brain tumor invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plasminogen activator inhibitor-1 aids nerve growth factor-induced differentiation and survival of pheochromocytoma cells by activating both the extracellular signal-regulated kinase and c-Jun pathways

Astrocytes are thought to be critical to neurons' surviving damage caused by ischemic stroke or other injury. Plasminogen activator inhibitor-1 is one of the active soluble factors released by astrocytes and regulates plasminogen activator-plasmin proteolytic sequence in the CNS as a serpin. In this study, we show that plasminogen activator inhibitor-1 can promote neurite outgrowth and survival of rat pheochromocytoma cells in serum-deprived conditions, and that this neuroprotective activity is correlated with enhanced activation of both extracellular signal-regulated kinases following a direct phosphorylation of nerve growth factor receptor, Trk A, and of c-Jun. Our results suggest that plasminogen activator inhibitor-1 can act as a neurotrophic factor, protecting neurons from serum deprivation-induced neuron death not only by compensating for nerve growth factor functions, but also by activating the c-Jun/activating protein-1 pathway.     

Addition of a C-Terminal Extension Sequence to Transforming Growth Factor-pl Interferes with Biosynthetic Processing and Abolishes Biological Activity

Abstract

Transforming growth factor-β1 (TGF-β1) is synthesized and secreted as a biologically latent complex. It has been proposed that one role of the latent complex is to prevent premature interaction of ligand and receptor intracellularly during biosynthesis (Wakefield et al, J. Cell Biol. (1987) 105, 965-9751. To test this hypothesis, the endoplasmic reticulum retention sequence Lys-Asp-Glu-Leu (KDEL) was added to the C-terminus of the wildtype TGF-β1 coding sequence, and to a construct in which mutagenesis of two cysteine residues in the precursor pro region results in the synthesis and secretion of active, as opposed to latent, TGF-β. Addition of either SEKDEL, or the control sequence SEKDVS to the TGF-β1 protein abolished biological activity. Western blot analysis indicated that the extended gene products are synthesized, but that the extension sequence partially interferes with the normal dimerization of the protein product, and totally inhibits the normal proteolytic processing and glycosylation of the precursor protein. The data suggest that correct folding of the highly conserved C terminus of TGF-PI is critical for subsequent proteolytic cleavage and glycosylation at sites that are quite distant in the primary sequence. Thus molecular strategies for the generation of TGF-β antagonists or superagonists should avoid extensive modification of this region of the molecule. Since synthesis of the endogenous TGF-β1 is unaffected by the presence of the mutated analog, the data further indicate that transfection with the KDEL-extended TGF-β1 sequence cannot be used as a dominant negative mutation to prevent secretion of the endogenous TGF-Pβ protein.     

The Role of Estradiol,Progesterone, and Transforming Growth Factor on Human Endometrioma Cell Culture

The present study demonstrates several aspects of endometrioma cells in culture, namely, 1) cell growth, proliferation, and morphology, 2) effect of cell culture on estrogen and progesterone receptor concentration, 3) effect of estradiol, progesterone, and transforming growth factor. The tissue sample was obtained from ovarian endometriomas that were removed via laparotomy or laparoscopy. The tissue sample was digested with collagenase. After washing, the tissue was cultured in endothelial cell culture medium. Cell count was done by flow cytometry. Receptor study was done by immunohistochemistry. The results demonstrated the growth and proliferation of endometrioma cell in culture medium. Electron microscopy showed stroma-like cells. The cells lost their estrogen and progesterone receptors. Estradiol and progesterone added to these cultures did not affect the rate of growth and proliferation of the cells. Transforming growth factor significantly increased the rate of growth and proliferation of these cells.     

Transforming Growth Factor Beta Stimulates Mitogenically Mouse NIH3T3 Fibroblasts and Those Cells Transformed by the EJ-H-ras Oncogene

Abstract

TGF-β1 stimulates thymidine incorporation and the growth rate of mouse NIH3T3 fibroblasts and of those cells transformed by the EJ-H-ras oncogene (TR15 cells), in the presence and the absence of serum. Thymidine incorporation, in serum-deprived cells, is stimulated to a higher degree by 0.1-1 ng/ml of TGF-β in NIH3T3 than in TR15 cells, which have a 10-fold higher basal level of incorporation. In both cell types TGF-β1 is as active, or more active than other mitogens (TGF-α, PDGF-AB, bFGF) at the same concentration. The growth rate of NIH3T3 cells, in low serum or serum-free (S^?) medium, is stimulated by only 10 picograms/ml of TGF-β1, and that of TR15 cells, in S^? medium, by only 1 picogram/m1. In contrast, TGF-β1 inhibits mitogenically unestablished mouse embryo fibroblasts and these fibroblasts immortalized spontaneously and able to grow in S^? medium. It also inhibits the anchorage-independent growth of TR15 cells. NIH3T3 and TR15 cells respond, similarly, to TGF-β activated by acification of their culture medium. The kinetics of thymidine incorporation and of activation of the c-myc proto-oncogene, observed already after 1 hr, in treated NIH3T3 and TR15 cells, suggests a direct mitogenic stimulation. The level of activated c-myc RNA is 2-fold higher at 2 hr, and subsequently decreases relatively less in the TR15 cells.     

Initiation of Bone Development by Osteogenin and Promotion by Growth Factors

The cellular and molecular basis of bone development and its regulation by differentiation and growth factors is an exciting area of current research. This article briefly reviews the historical progress in the isolation of osteogenin, a novel bone differentiation factor, and its modulation by well known growth factors. Endochondral bone development is a multistep sequential cascade and the process must be operationally dissected. It has been accomplished with the demineralized bone matrix-induced bone formation model. The reproducible development of cartilage and bone in an extraskeletal site permits the study of the initiation of the first cycle of endochondral bone formation and mineralization. Recent progress in the isolation of osteogenin, a specific bone differentiation factor, by heparin affinity chromatography permits the further investigation of the commitment and clonal expansion of the putative osteoprogenitor stem cells. Once initiated, bone formation is promoted by growth factors such as platelet derived growth factor, fibroblast growth factor, insulin like growth factor, transforming growth factor β and a plethora of non specific cytokines. Finally bone development is further modulated by systemic hormones and nutrition and a host of physical signals including electrical, gravitational and mechanical forces.     

针刺对多囊卵巢综合征大鼠卵巢TGF-α、EGFR表达的影响

目的:研究针刺对多囊卵巢综合征(PCOS)大鼠卵巢转化生长因子α(TGF-α)、表皮生长因子受体(EGFR)表达的影响,探讨针刺促排卵的作用机制。方法:24日龄雌性大鼠颈背部皮下注射脱氢表雄酮(DHEA)的油溶液制作(PCOS)模型,对照组同期皮下注射油剂。PCOS大鼠随机分为模型组和针刺组。模型组不作处理,针刺组大鼠从80日龄起针刺关元、中极、双侧三阴交、双侧子宫穴,1次/天,15min/次,连续6周。治疗结束后各组大鼠断头处死,迅速取血并分离血清,-20℃冰箱保存,待测性激素水平。摘取双侧卵巢,称重,4%多聚甲醛固定,作HE染色和免疫组织化学染色。结果:与模型组相比,针刺组卵巢湿重、卵巢TGF-α、EGFR表达及血清睾酮(T)、雌二醇(E2)水平均显著降低(P<0.01),而卵泡刺激素(FSH)、黄体生成素(LH)、孕酮(P4)水平差异无统计学意义(P>0.05)。结论:针刺能显著降低PCOS大鼠卵巢TGF-α及EGFR的表达,抑制TGF-α对卵巢和激素合成的作用,改善PCOS大鼠多囊样变和高雄激素血症,促进排卵。     

氯沙坦对自发性高血压大鼠左心室肥厚和转化生长因子β_1的影响

目的 :探讨转化生长因子 β1 (TGFβ1 )在自发性高血压大鼠(SHR)左心室肥厚中的作用及氯沙坦对左心室肥厚和TGFβ1 水平的影响。方法 :2 0只SHR随机分成氯沙坦治疗组和未治疗组 ,每组10只 ,用免疫组织化学法检测SHR心肌TGFβ1 的表达及氯沙坦治疗后的改变 ,同时观察心肌肥厚的变化 ,并与正常WKY组对照。结果 :正常组心肌细胞胞浆内TGFβ1 表达呈弱阳性 ,SHR对照组呈强阳性 ,氯沙坦治疗组呈阳性反应。用图像分析仪计算其平均吸光度分别为 17.46± 2 .3 8、15 6.81± 2 1.75、67.19± 7.2 4,并且SHR左心室质量指数 (LVWI)高于WKY组 ,氯沙坦治疗组LVWI低于对照组。结论 :TGFβ1 在自发性高血压大鼠心肌中表达增强 ,而氯沙坦治疗可抑制其表达 ,从而可能延缓SHR左心室肥厚的病理学进展。     

Regulation of Natural Killer Cell Activity by Transforming Growth Factor-β and Prostaglandin E2

The separate and combined effects of transforming growth factor-β1 (TGF-β1) and prostaglandin E₂ on human natural killer (NK) activity were studied. Peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL, 70–90% purity) were used as effector cells and K562 as targets. Overnight incubation of the effector cells with TGF-β1 resulted in a significant inhibition of NK activity. TGF-β1 did not influence the expression of CD3, CD 16, CD 18 or CD56 antigens on PBL. Combination of TGF-βl with indomethacin gave the same NK-suppressive effect as TGE-β1 alone, showing that the inhibition of NK acuvity by TGF-β1 is not due to an increase in PGE₂ levels. TGF-β did not influence cAMP level in PBL whereas PGE₂ significantly increased it. On the other hand. TGE-β1 and PGE₂ showed an additive inhibitory effect on NK activity. TGF-β1 did not reduce the binding of PBL and LGL to K562. PGE₂ suppressed the binding and TGF-β1 did not influence this suppression. TGF-β1 also suppressed IL-2-induced activation of NK activity and increase of expression of the granule proteins granzyme A and perforin. PGE₂ did not appear to affect granzyme A and perforin contents. The results indicate that TGF-β1 and PGE₂ suppress NK activity by different mechanisms.