Kunitz抑制剂家族成员抑肽酶对纤溶酶原活化的抑制作用

抑肽酶属Kunitz抑制剂家族成员，能够抑制激肽释放酶、纤溶酶及胰蛋白酶的蛋白水解活性．研究表明，抑肽酶能够抑制尿激酶型-纤溶酶原激活刹（u—PA）和组织型纤溶酶原激活剂（t—PA）对纤溶酶原的激活，但不影响u—PA和t—PA对小分子底物的酰胺水解活性．用u—PA研究了上述作用的机制，发现抑肽酶与u—PA的丝氨酸蛋白酶功能区特异性结合，而与纤溶酶原没有相互作用．抑肽酶与uPA的结合并不阻断u—PA的活性位点，因为u—PA对小分子底物的水解活性仍然保持．上述发现提示抑肽酶可能存在另一种抑制作用模式，该模式不同于以前报道的关于Kunitz抑制剂或纤溶酶原激活酶抑制剂的作用．由于人体内的Kunitz抑制剂与抑肽酶在结构上非常相似，根据研究结果，推测体内纤溶酶原的激活作用并非仅受丝氨酸蛋白酶抑制剂的控制。     

关节内注射尿激酶型纤溶酶原激活物对关节软骨的实验研究

探讨了尿激酶型纤溶酶原激活物（urokinase-type plasminogen activator,uPA）对关节软骨的影响.选择健康新西兰大白兔42只,随机分为实验组和对照组,实验组24只,对照组18只;实验组在右膝关节内注射uPA 0.4μg/0.2mL,对照组注射等容量的生理盐水,在注射后4周、8周、12周分批取兔软骨进行组织学观察及电镜检查.结果显示：实验组关节软骨表面不规则、缺损及结构破坏,软骨细胞数目减少、纤维增生及异染性降低,潮线的完整性破坏.结果表明：尿激酶型纤溶酶原激活物可能是导致关节软骨破坏的相关因素之一.     

尿激酶型纤溶酶原激活物对关节软骨蛋白多糖的影响

观察尿激酶型纤溶酶原激活物（urokinase-type plasminogen activator,uPA）对关节软骨基质中蛋白多糖的影响.实验选择健康新西兰大白兔42只并随机分为实验组和对照组,实验组24只,对照组18只;实验组在右膝关节内注射uPA 0.4μg/0.2mL,对照组注射等容量的生理盐水,注射后在4周、8周、12周分批处死取兔软骨提取测定葡糖氨基聚糖（glycosaminoglycan,GAG）含量.结果显示,在不同时期,GAG的总含量均减少,这些改变随时间的延长而渐不明显.由此可知,尿激酶型纤溶酶原激活物能降解关节软骨蛋白多糖,进而导致关节软骨的破坏.     

用静息细胞培养法研究影响组织型纤溶酶原激活剂生物合成的因素

建立了基因工程菌株里氏木霉(Trichoderma reesei)306生物合成组织型纤溶酶原激活剂(t—PA)的静息细胞培养系统。确定静息细胞培养基的成分为：葡萄糖1．0g／L，尿素1．0g／L，K2HPO41．0g／L；静息细胞培养的条件为：选择发酵30h的菌体，10％的接种量，pH5．4，150r／min，28℃静息培养12h。并在该静息细胞培养系统中，研究了糖类、有机酸、氨基酸和金属离子等因素对里氏木霉306生物合成组织型纤溶酶原激活剂的影响。     

纤维蛋白溶解酶原的纯化及性质

人血浆组分Ⅲ经Lys－Sepharose4B和SephadexG－200柱层析分离，得到分子量为90000的纯品纤维蛋白溶解酶原（HPg）．其作用的最适PH、温度、离子强度分别为6.0，25℃和0．05mol／L．Zn2＋对HPg活性有明显抑制作用；Mg2＋，Li＋，Ca2＋等离子显示了不同程度的激活作用．     

Localization and the possible role of plasminogen activators and inhibitors in early stages of placentation

The distribution of mRNAs of tissue type (t) and urokinase type (u) plasminogen activator (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) have been studied in the tissues of human first and second trimester placentae by in situ hybridization. The results show that: (ⅰ) All the molecules, tPA, uPA, PAI-1 and PAI-2, were identified in the blood vessels, the majority of extravillous trophoblastic cells of the decidual layer between Rohr’s and Nitabuch’s stria and in the trophoblast cells lining the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (ⅱ) No expression of such probes was observed in the basal and chorionic plate, glandular cells of the decidua, the septal tissues or the villous core mesenchyme. The co-distribution of the molecules observed suggests that the co-ordinated expression of the activators and inhibitors in various cells of the placental tissue may play a role in angiogenesis related to conversion of spiral arteries into utero-placental arteries and establishment of a chorio-decidual blood flow during early stages of placentation.     

人纤溶酶原Kringle 1基因的克隆与表达

从人肝组织中抽提总RNA，通过RT-PCR方法扩增出人纤溶酶原（plasminogen)Kringle 1片段，构建pPIC9K-K1载体，电击转化酵母，成功获得pPIC9K-K1/GS115工程菌，摇瓶发酵初步结果表明，工程菌能稳定表达可溶性的重组K1蛋白，分子量为15kd，发酵液中的蛋白含量为84.17mg/L。     

Role of plasminogen activator and plasminogen activator inhibitor type-1 in luteolysis—a minireview

This minireview summarized our recent studies on the role of plasminogen activator (PA) and inhibitor type-1 (PAI-1) in luteolysis. We have demonstrated that (1) both tissue type and urokinase type plasminogen activators (tPA and uPA) and a plasminogen activator inhibitor type-1 (PAI-1) were present in the corpus luteum of rat and rhesus monkey; (2) decrease in progesterone production in corpus luteum was well correlated with a sharp increase in tPA (but not uPA) and PAI-1 secretion; (3) exogenous tPA decreased luteal progesterone synthesis while monoclonal antibodies increased progesterone production; (4) interferony inhibited luteal progesterone synthesis and stimulated tPA production while LH plus prolactin increased progesterone production and decreased tPA (hut not uPA) activity in cultured luteal cells; (5) increase in proteolysis in the corpus luteum was also correlated with decrease in progesterone production in mouse. These data suggest that local degradation of extracellular matrix controlled by plasminogen activator and inhibitor is involved in the processes of luteolysis.     

Role of plasminogen activator and plasminogen activator inhibitor type-1 in luteolysis——a minireview

This minireview summarized our recent studies on the role of plasminogen activator (PA) and inhibitor type-1 (PAI-1) in luteolysis. We have demonstrated that (1) both tissue type and urokinase type plasminogen activators (tPA and uPA) and a plasminogen activator inhibitor type-1 (PAI-1) were present in the corpus luteum of rat and rhesus monkey; (2) decrease in progesterone production in corpus luteum was well correlated with a sharp increase in tPA (but not uPA) and PAI-1 secretion; (3) exogenous tPA decreased luteal progesterone synthesis while monoclonal antibodies increased progesterone production; (4) interferon y inhibited luteal progesterone synthesis and stimulated tPA production while LH plus pro-lactin increased progesterone production and decreased tPA (but not uPA) activity in cultured luteal cells; (5) increase in proteolysis in the corpus luteum was also correlated with decrease in progesterone production in mouse. These data suggest that local degradation of extracellular matrix controlled by plasminogen activator and inhibitor is involved in the processes of luteolysis.     

组织型纤溶酶原激活剂突变体基因转染细胞株的建立

以脂质体介导法将含有t-PA突变基因的真核表达载体pCSRA及pCSRK分别转染CHO-dhfr^-,经G418及DMEM双重选择,获得两者的稳定表达细胞株。结果表明,表达产物具有良好溶纤活性;Western印迹实验证明,产物具有特异抗原性;经多次传代,细胞株具有良好的稳定性。比较了无血清培养基CHO-S-SFMⅡ与常规培养基DMEM培养条件下pCSRK细胞株产物的表达量,前者约为后者的2倍,表明无血清培养基SFM有可能用于药用性蛋白的生产。实验为t-PA新型突变体的临床应用打下一定基础。     

人纤溶酶原kringle5功能区基因在大肠杆菌中的表达

将人纤溶酶原kringle5功能区基因插入融合基因表达载体pET17bBamHⅠ位点,构建成重组质粒pETK23,转化大肠杆菌BL21(DE3),在IPTG诱导下,纤溶酶原kringle5功能区基因在重组转化菌株BL21(DE3,pETK23)中获得高效表达,表达产物以可溶性蛋白形式存在,表达量占菌体总蛋白的528%.     

鼠肾纤维蛋白溶酶原激活酶的纯化和部分鉴定

采用肝素—Sepharose亲和层析和凝胶过滤自鼠肾提取纤维蛋白溶酶原激活酶,产率5.1×10~(-5)％。在还原和非还原状态下SDS—聚丙烯酰胺凝胶电泳及电泳酶谱分析均呈单一区带,证实产物仍保持单链状态。测得其分子量为70000。纤维平板试验和无纤维蛋白溶酶原纤维平板试验证实其活性依赖于纤维蛋白溶酶原。     

溶栓剂蛋白质工程的新进展

介绍了国内外溶栓剂蛋白质工程方面的新进展,主要涉及了组织型纤溶酶原激活剂(t-PA)、尿激酶型纤溶酶原激活剂(u-PA)、链激酶(SK)、葡萄球菌激酶(SAK)、DSPA及TSV-PA的研究进展。一些PA突变体及新型溶栓剂,如K2P、TNK-PA和STAR等的临床实验结果表明它们在延长体内半寿期、增强对血纤维蛋白选择性和溶栓效率等方面有较大的改进。但溶栓治疗造成的颅内出血等局部性出血问题仍是目前的难题之一,说明新型溶栓剂的研制有很大潜力。     

尿激酶原激活是其催化纤溶酶原激活的限速步骤

尿激酶原催化纤溶酶原的激活包括3个反应：（1）尿激酶原内在酶活性催化纤溶酶原转化为纤溶酶；（2）纤溶酶激活尿激酶原变成尿激酶；（3）尿激酶激活纤溶酶原，所以一直以来人们对尿激酶原激活纤溶酶原的过程知之甚少．研究发现EACA可以抑制尿激酶原催化的纤溶酶原激活。通过EACA对上述3个反应的影响进行逐个分析，观察到在EACA浓度为2．0mmol／L时，尿激酶原内在酶活性催化纤溶酶原激活的反应速度提高了4．5倍，尿激酶激活纤溶酶原的反应速度提高了6倍．相反地，纤溶酶激活尿激酶原变成尿激酶的反直被抑制了50％，此反应决定了EACA对尿激酶原催化纤溶酶原激活反应的抑制作用．该结果提示尿激酶原催化纤溶酶原激活反应的限速步骤是纤溶酶激活尿激酶原变成尿激酶，缘于纤溶酶和尿激酶原的赖氨酸结合位点的相互作用．     

表面活性剂和促进剂对重组里氏木霉生物合成t-PA的影响

重组里氏木霉(Trichoderma reesei)是染色体上整合有组织型纤溶酶原激活剂(t-PA)cDNA的基因工程菌株.对高产t-PA的重组里氏木霉菌株进行了筛选,并研究了乙酸钠和抗坏血酸(Vc)等促进剂及曲拉通(Triton100)、吐温80(Tween80)、十二烷基磺酸钠(SDS)和蔗糖酯等表面活性剂对菌株生物合成t-PA的影响.结果表明:乙酸钠能促进t-PA的生物合成,在发酵培养基中,其浓度为0.1%时,酶活可提高36.42%;吐温80对t-PA的合成有促进作用,其浓度为0.1%时,酶活可提高82.04%;抗坏血酸也可促进t-PA的合成,其浓度为0.05%时,酶活可提高124.82%;曲拉通和十二烷基磺酸钠加入时完全抑制t-PA的合成;蔗糖酯对t-PA的合成有部分抑制作用.     

A novel method of DNA shuffling without PCR process

Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends. These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.     

rPA(K)的定点突变及其真核表达载体在COS-7细胞中的瞬时表达

采用定点突变技术,对真核表达载体pCSRK上的目的片段rPA(K)进行突变,获得了含有突变体rPA(KN)(N184→Q)的真核表达载体pCSRKN.酶切和测序结果证明,成功去除了184位的糖基化位点.用LipofectamineTM2000 Reagent将pCSRKN转染至COS-7细胞,取转染后不同培养时间上清,以FAPA法进行检测.结果表明,表达产物具有良好的溶圈活性,从而验证了pCSRKN在真核系统表达的可行性.     

人tPA基因真核表达质粒载体构建及其生物学功能研究

构建携带组织纤溶酶原激活物(tPA)基因真核表达质粒pcDNA3.1( )/tPA,并研究其有无生物学活性.用RT-PCR法从人心脏组织中克隆tPA基因并将其克隆至真核表达质粒pcDNA3.1( )中,对pcDNA3.1( )/tPA进行酶切鉴定和测序.脂质体介导pcDNA3.1( )/tPA转染血管平滑肌细胞,分别用Nothern Blot和斑点印迹法从mRNA和蛋白质水平检测tPA的表达,并用纤维蛋白板法测定表达产物的生物学活性.成功克隆了人tPA基因并构建了pcDNA3.1( )/tPA真核表达质粒,pcDNA3.1( )/tPA转染VSMC后,tPA mRNA和蛋白质表达增加,所表达的tPA蛋白质具有纤溶活性.