组织型纤溶酶原活化物的纯化及性质研究

新鲜猪心组织制成丙酮粉后,用0.45mol/L,pH4.2醋酸钾抽提组织型纤溶酶原活化物(t-PA)。抽提液经硫酸铵盐析,Benzamidine和血纤维蛋白亲和层析,Sephadex G-150凝胶过滤,纯化得到t-PA。比活11000IU/mg,经SDS-聚丙烯酰胺凝胶电泳鉴定,分子量为67000。 本文比较了t-PA、高分子量尿激酶(H-UK)和低分子量尿激酶(L-UK)的热稳定性及抑制剂对它们的抑制作用。结果表明,抑制剂对H-UK的抑制作用最强,L-UK次之,t-PA最弱;三者的热稳定性相似。     

从人脐带血中分离纯化血纤溶酶原

以人血清为原料 ,利用纤溶酶原对L型赖氨酸的高亲和性制备了Lysine -Sepharose4B和Lysine -Agarose ,以亲和层析法从人血浆中提取和纯化血纤溶酶原 (plasminogen ,PGn)。利用聚丙烯酰胺凝胶电泳对其纯度和分子量进行分析 ,结果表明纯化得到的为 92kDa的单一组分的人血纤溶酶原。这种纯化方法的建立为进一步大量制备血管生成抑制素 (angiostatin)奠定了基础。     

重组人尿型纤溶酶原激活剂的中试生产与性质研究

表达uPA的CHOdhfr工程细胞CL11G在30升生物反应器中高密度大规模培养65天后,获得1200余升细胞培养上清;经阳离子交换层析、凝胶过滤、苯甲眯亲和层析和阴离子交换层析四步纯化工艺,获得uPA冻干纯品40克。对产品进行性质研究和质量检定,各项指标符合理论数据和《中国生物制品规程》。     

纤溶酶原激活剂抑制物2型抑制细胞凋亡的研究进展

纤溶酶原激活剂抑制物２型（ＰＡＩ－２）是一种多功能蛋白质,除了能有效抑制尿激酶（ｕＰＡ）和双链组织型纤溶酶原激活物（ｔＰＡ）而调节纤溶活性外,还参与了很多其它的生理病理过程,例如组织重建、胚胎发育、感染、免疫系统发育、肿瘤浸润和迁移等。更有研究表明胞内型ＰＡＩ－２在抑制细胞凋亡方面也发挥着重要作用。本文就近年来ＰＡＩ－２抑制细胞凋亡的研究进展作一综述。     

人微小纤溶酶原基因的克隆和表达

用PCR方法扩增人微小纤溶酶原(Microplasmingen,mPlg)基因,再与表达载体重组．构造mPlg原核表达质粒并转化大肠杆菌。阳性克隆pSSE-mPlg经温度诱导表达,SDS-PAGE等方法证明表达产物的分子量约为29kDa。占全菌总蛋白的24％左右,并在菌内形成包涵体。经半胱氨酸再氧化法和空气氧化法复性。表达产物r-mPlg经SK作用后显示纤溶活性。同时对蛋白质浓度、复性时间等因素对复性的影响进行了初步探讨。     

蚯蚓体内一种纤溶酶原激活剂(e-PA)对BAEE的降解

以苯甲酰－Ｌ－精氨酸乙酯（ｂｅｎｚｏｙｌ－Ｌ－ａｒｇｉｎｉｎｅｅｔｈｙｌｅｓｔｅｒ,ＢＡＥＥ）为底物,研究了蚯蚓体内纤溶酶原激活剂（ｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒｆｒｏｍＥｉｓｅｎｉａｆｅｔｉｄａ,ｅ－ＰＡ）的酶学性质．酶促反应的最适ｐＨ为８．４,ｅ－ＰＡ降解ＢＡＥＥ的Ｋｍ为１．２４±０．１６×１０－５ｍｏｌ／Ｌ,Ｋｃａｔ为１３．８０±４．０２ｓ－１．测定了构成ｅ－ＰＡ的大,小亚基分别降解ＢＡＥＥ的Ｋｍ和Ｋｃａｔ．结果表明,大亚基的Ｋｍ与全酶的Ｋｍ相差不多,但比小亚基小约１０倍,即对底物的亲和力比小亚基强约一个数量级．大小亚基的Ｋｃａｔ比较接近,分别是全酶的１／６和１／３．研究了８种抑制剂对ｅ－ＰＡ降解ＢＡＥＥ活性的影响,其中ｐｅｐｓｔａｔｉｎ和Ｅ－６４（一种巯基抑制剂）对酶促反应有激活作用,ＴＰＣＫ,ＴＬ－ＣＫ,ＰＭＳＦ,ｃｈｙｍｏｓｔａｔｉｎ和ｌｅｕｐｅｐｔｉｎ对其有不同程度的抑制作用,ＥＤＴＡ对ｅ－ＰＡ的活性没有影响．对ｅ－ＰＡ的ＢＡＥＥ活性和ｅ－ＰＡ的纤溶活性之间作了比较．     

人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化

 本文报道了培养的人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化方法。Bowes株人黑色素瘤细胞的分泌产物,经CM-Sephadex C--50层析,赖氨酸-Sepharose 4B,苯甲眯-sepharose 4B亲和层析后,即可得到纯化470倍的蛋白纯品。样品经聚丙烯酰胺凝胶电泳鉴定为均一单带,测得其分子量约为72kD。纯化的t-PA与尿激酶相比较,发现前者有更高亲和纤维蛋白的能力。     

蚯蚓体内一种纤溶酶原激活剂(e-PA)对ATEE的降解

赤子爱胜蚓（Ｅｉｓｅｎｉａｆｅｔｉｄａ）体内的一种纤溶酶原激活剂（ｅ－ＰＡ）能够降解人工合成底物Ｎ－乙酰－Ｌ－酪氨酸乙酯（ＡＴＥＥ）,该降解反应的最适ｐＨ为８．５,而且在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４中的活性要强于在０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）中．分别测定了ｅ－ＰＡ的大小亚基及全酶在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４与０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）两种体系中的Ｋｍ和Ｋｃａｔ．结果表明,在０．２ｍｏｌ／ＬＮａ２ＨＰＯ４中,全酶的ＡＴＥＥ活性远远高于大小亚基单独的ＡＴＥＥ活性,而在０．０５ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．５）中则没有这种现象．从蛋白质结构的角度对这一结果作了解释．用不同抑制剂和ｅ－ＰＡ作用,结果表明,ｐｅｐｓｔａｔｉｎ,Ｅ－６４和ＥＤＴＡ对ｅ－ＰＡ的ＡＴＥＥ活性都有不同程度的抑制,这一点与ｅ－ＰＡ的ＢＡＥＥ活性不同．     

人纤溶酶原kringle 5在大肠杆菌中克隆和分泌表达

构建能够表达分泌性的人纤溶酶原kringle 5（简称hPK-5）的大肠杆菌工程菌。用PCR方法扩增获得hPK-5基因,构建原核表达载体pET-22b（＋）/hPK-5,转化大肠杆菌BL21（DE3）,经IPTG诱导表达并鉴定其免疫学活性。成功表达14kD的重组hPK-5,且约占菌体分泌性蛋白20%以上,经Western Blot分析表明其具有hPK-5的免疫抗原活性。人纤溶酶原kringle 5在大肠杆菌中BL21（DE3）获得可分泌表达。     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好     

蚯蚓体内一种纤溶酶原激活剂(e-PA)的分离纯化

为获得一种高效,低廉的溶栓药物,从赤子爱胜蚓（Ｅｉｓｅｎｉａｆａｅｔｉｄａ）体内分离纯化出一种可体外激活纤溶酶原从而间接降解纤维蛋白的酶（ｅ－ＰＡ）．纯化过程包括：粗品的盐析,离子交换层析,凝胶过滤层析及疏水相互作用层析．该组份是由二个亚基通过疏水相互作用维系在一起的．通过凝胶过滤层析,可测得全酶的分子量为４５０００;ＳＤＳ电泳显示大、小亚基的分子量分别是２６０００与１８０００;而质谱法测得的大、小亚基的分子量分别为２４５５６．７与１５５４６．６．对大小亚基进行了氨基酸组成分析,结果显示大亚基不含Ｌｙｓ而小亚基不含Ｃｙｓ．测定了大亚基Ｎ端２５个氨基酸序列：ＶＩＧＧＴＮＡＳＰＧＥＩＰＷＱＬＳＱＱＲＱＳＧＳＷ．并与部分已知蛋白质序列进行了比较．ｅ－ＰＡ在纤维蛋白平板上表现有三种不同的纤溶活性     

人纤溶酶原激活剂抑制物2型(PAI-2)在大肠杆菌中的表达和分离纯化

用ＰＣＲ方法从ｐＰＡＩＪ．７中扩增人纤溶酶原激活剂抑制物２型（ＰＡＩ－２）ｃＤＮＡ,与ｐＵＣ１８重组,经限制性内切酶片段分析与核苷酸序列分析,获得全长人ＰＡＩ－２ｃＤＮＡ．ＰＡＩ－２ｃＤＮＡ与原核表达载体重组,构建原核表达质粒并转化大肠杆菌．经温度诱导表达,重组ＰＡＩ－２占全菌总蛋白的１４％,以可溶性形式存在,具纤溶抑制活性．工程菌发酵、压榨后,用硫酸铵分级沉淀,沉淀物经分子筛、离子交换和疏水性色谱的分离,每升菌液可获得约３０ｍｇ纯度达９０％的蛋白质,比活性为１１８６６ＡＩＵ／ｍｇ蛋白质,得率为１９．２％．     

Isolation,Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation^1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low¹⁰; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust^11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro^11-22, far fewer exist for ICCs^10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue⁶. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.     

LRP1 Assembles Unique Co-receptor Systems to Initiate Cell Signaling in Response to Tissue-type Plasminogen Activator and Myelin-associated Glycoprotein

In addition to functioning as an activator of fibrinolysis, tissue-type plasminogen activator (tPA) interacts with neurons and regulates multiple aspects of neuronal cell physiology. In this study, we examined the mechanism by which tPA initiates cell signaling in PC12 and N2a neuron-like cells. We demonstrate that enzymatically active and inactive tPA (EI-tPA) activate ERK1/2 in a biphasic manner. Rapid ERK1/2 activation is dependent on LDL receptor-related protein-1 (LRP1). In the second phase, ERK1/2 is activated by tPA independently of LRP1. The length of the LRP1-dependent phase varied inversely with the tPA concentration. Rapid ERK1/2 activation in response to EI-tPA and activated α₂-macroglobulin (α₂M*) required the NMDA receptor and Trk receptors, which assemble with LRP1 into a single pathway. Assembly of this signaling system may have been facilitated by the bifunctional adapter protein, PSD-95, which associated with LRP1 selectively in cells treated with EI-tPA or α₂M*. Myelin-associated glycoprotein binds to LRP1 with high affinity but failed to induce phosphorylation of TrkA or ERK1/2. Instead, myelin-associated glycoprotein recruited p75 neurotrophin receptor (p75NTR) into a complex with LRP1 and activated RhoA. p75NTR was not recruited by other LRP1 ligands, including EI-tPA and α₂M*. Lactoferrin functioned as an LRP1 signaling antagonist, inhibiting Trk receptor phosphorylation and ERK1/2 activation in response to EI-tPA. These results demonstrate that LRP1-initiated cell signaling is ligand-dependent. Proteins that activate cell signaling by binding to LRP1 assemble different co-receptor systems. Ligand-specific co-receptor recruitment provides a mechanism by which one receptor, LRP1, may trigger different signaling responses.     

The Voltage-dependent Anion Channel (VDAC) Binds Tissue-type Plasminogen Activator and Promotes Activation of Plasminogen on the Cell Surface

The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells where in addition to its role in regulating cellular ATP release and volume control it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induced a decrease in K_m and an increase in the V_max for activation of its substrate, plasminogen (Pg). This resulted in accelerated Pg activation when VDAC, t-PA, and Pg were bound together. VDAC is also a substrate for plasmin; hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids Ile⁵ and Asn³⁷ and a VDAC region including amino acids ²⁰GYGFG²⁴. These VDAC residues correspond to a GXXXG repeat motif commonly found in amyloid β peptides that is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid β peptide deposits from the normal brain and cell debris from injured brain tissue.     

胎儿胰岛样细胞团源上皮样细胞分离、纯化和鉴定

旨在优化胰腺干细胞分离、鉴定的方法和体系,为糖尿病研究和治疗提供种子细胞。采用胶原酶消化法,分离培养出胰岛样细胞团(ICCs),对其进行贴壁培养,从中纯化出上皮样细胞。采用MTT法测定其生长情况并绘制生长曲线。采用免疫组织化学染色检测分离得到细胞的PDX-1、PCNA、CK-7、CK-19、Nestin、Glut2、Vimentin、Insulin表达情况。应用流式细胞仪检测其表面标志。由分离培养的ICCs成功纯化出上皮样细胞;传40代,每代冻存106~108个细胞;生长曲线显示其传代第3天进入对数生长期,第5天进入平台期;免疫组织化学染色显示其表达PDX-1、PCNA、CK-7、CK-19、Nestin、Glut2、Vimentin;不表达Insulin;流式细胞仪分析表明其表达CD29、CD44、CD166,不表达CD11a、CD14、CD34、CD45、CD90、CD105、CD117。由胎儿胰腺能分离出具有自我更新能力的上皮样细胞,为导管来源,具干细胞特性。     

Plasminogen activator: Isolation and purification from Lymphosarcoma of ascites bearing mice

Plasminogen activator secreted by lymphosarcoma (ascites) of mice was purified up to 163-fold by ammonium sulphate fractionation at 35% saturation and chromatography on p-aminobenzamidine-Sepharose 4B. The purified activator contained specific activity of 9980 IU/mg. The plasminogen activator displayed homogeneity by polyacrylamide slab gel electrophoresis and high performance liquid chromatography. The activator consisted of a single polypeptide chain with an apparent molecular weight of 66,000 daltons as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions as well as gel filtration on Sephadex G-100. Distinct differences between this activator and urokinase were discernible in respect of specific activities, fibrin affinity and immunochemical properties. The lymphosarcoma activator appears to be of tissue-type origin since it showed gross similarity to standard tissue plasminogen activator in terms of modes of binding to fibrin and immunological attributes.