重组血小板第4因子在甲醇酵母中的高效表达

目的 利用甲醇酵母高效表达重组人血小板第4因子（rhPF4)。方法 用PCR方法扩增出血小板第4因子（PF4）cDNA序列，插入到穿梭质粒pPIC9的MFα信号肽后，在醇氧化脱氢酶1（AOX1）启动子的下游，得到重组质粒pPIC9-PF4，转化到甲醇酵母宿主菌GS115，经筛选得到PF4高表达菌株进行表达，并测定表达产物氨基酸序列和生物学功能。结果 rhPF4氨基酸序列与天然PF4相同，可中和肝素抗凝作用，并呈剂量相关。结论 rhPF4可在甲醇酵母中获得生产规模表达，产物性质和天然的PF4相同。     

人血小板第4因子和P—选择素的纯化

目的用单克隆抗体亲和层析方法从人血小板破碎液中纯化血小板因子4和P-选择素.方法将单克隆抗体SZ-95-IgG和SZ-51-IgG分别与溴化氰活化的Sepharose4B凝胶连接成SZ-95-IgG-Sepharose4B和SZ-51-IgG-Sepharose4B亲层析柱,人血小板破碎液经此亲和层析柱上样后,再经FPLC系统获得的所要的产品,产品经SDS-聚丙烯酰胺凝胶电泳鉴定其纯度,用点杂交鉴定其生物学活性.结果SZ-95-Sepharose4B和SZ-51-Sepharose4B亲和层析柱的偶联率分别72%和68%,每1ml(约1×109个血小板)血小板破碎液中可以纯化到18μgPF4和12μgP-selectin,所得产品经SDS-PAGE,在分子量约为12kD和140KD处各显单一的蛋白区带,经点杂交印迹显示产品分别与单抗SZ-95和SZ-51反应显带.结论用单克隆抗体亲和层析柱纯化的PF4和P-selectin产品得率高、纯度高、活性好.     

天然抗菌肽分离纯化方法的建立

目的寻找高效、广谱的新型天然抗菌肽，建立稳定、有效的分离纯化方法。方法家蝇免疫诱导后制成组织匀浆，经超速离心、分子筛过滤、SephadexG50层析、反相高效液相色谱法（RP-HPLC）、透析浓缩等技术进行分离纯化，用K-B法鉴定抗菌活性。结果组织提取液经超速离心后，用pH6．8Tris-HCI尿素缓冲液作流动相，SephadexG50层析，收集到有明显抗菌活性的成分。再用pH6．8磷酸盐缓冲液作流动相，经RP-HPLC半制备柱进一步纯化，各成分分离效果良好，保留时间为18．008min处的谱峰，有明显的抗菌活性。用RP-HPLC分析柱显示其为单一成分，达到了色谱纯。该抗菌肽对大肠埃希菌ATCC25922、铜绿假单胞菌ATCC27853和耐甲氧西林金黄色葡萄球菌（methicillin-resistantS．aureus，MRSA）均有抗菌活性。结论建立了有效的家蝇抗菌肽分离纯化方法，纯化的家蝇抗菌肽有广谱抗菌活性。     

用双工位血液凝固自动测定仪测定血小板第4因子活性

<正> 血小板第4因子(简称PF4),是人体内一种重要的抗肝素因子,也是血小板激活的主要标志之一.本文主要介绍采用我所研制的“双工位血液凝固自动测定仪”测定PF4活性,报告如下.     

急性脑梗死患者止凝血相关因子变化的研究

目的探讨脑梗死患者止凝血系统的改变及其临床意义.方法检测52例脑梗死患者和55例正常人的外周血血管性血友病因子(vWF)、因子Ⅷ活性(FⅧ∶C)、血栓调节蛋白(TM)、蛋白C(PC)、游离蛋白S(FPS)、组织纤溶酶原激活物(t-PA)、组织纤溶酶原激活剂抑制剂(PAI-1)、β-TG、PF4的含量和血小板粘附试验(PAdT)及血小板聚集试验(PAgT),并进行统计学分析.结果脑梗死组vWF、FⅧC、PC、PAI-1、β-TG、PF4、PAdT及PAgT较对照组增高,t-PA下降,患者组TM及FPS含量与对照组无明显差异.结论提示脑梗死患者凝血活性增强、纤溶活性减低,血小板功能亢进.     

MB-P对血浆部分细胞因子与ABO血型抗体活性影响

目的研究亚甲蓝光化学病毒灭活法(MB-P)对血浆SCF、TPO、PF4与GPⅡb/Ⅲa活性与ABO血型抗体活性的影响。方法应用ELISA方法对MB-P制备前后血浆SCF、TPO、PF4与GPⅡb/Ⅲa活性进行检测;应用血型血清学检测方法对MB-P制备前后血浆IgM抗-A、IgM抗-B与IgG抗-A、IgG抗-B活性进行检测。结果 MB-P制备血浆SCF、TPO、PF4与GPⅡb/Ⅲa活性均有不同程度下降,分别为2.59±2.71,248.54±49.01,12.02±3.21,10.21±9.97;与制备前相互比较有统计学意义(P0.05)。另外,MB-P制备后血浆红细胞IgM抗-A、IgM抗-B与IgG抗-A、IgG抗-B效价也有不同程度下降,与制备前相互比较有统计学意义(P0.05)。结论应用MB-P制备血浆须高度重视部分细胞因子与红细胞ABO血型抗体变化情况,确保临床输血安全性与有效性。     

中药活性成分抗肺纤维化及机制研究概况

目的:中药活性成分抗肺纤维化及其机制研究进展。方法:肺纤维化(pulmonary fibrosis,PF)是一种以弥漫性、进行性肺间质纤维增生和肺泡结构紊乱并最终导致肺功能障碍为特点的呼吸系统疾病。结果:其病理特征是肺泡上皮损伤、成纤维细胞灶的形成以及细胞外基质的过度沉积,最终导致了肺组织结构的异常重塑。PF的病因复杂,预后极差,临床尚无公认有效的治疗手段。结论:近年来,随着国内外学者对PF发病机制研究的逐渐深入和治疗方法的不断探索,有关中药活性成分治疗PF的实验研究及其抗PF机制取得了较大进展。     

急性脑梗死患者止凝血相关因子变化的研究

目的　探讨脑梗死患者止凝血系统的改变及其临床意义。方法　检测 5 2例脑梗死患者和 5 5例正常人的外周血血管性血友病因子 (vWF)、因子Ⅷ活性 (FⅧ∶C)、血栓调节蛋白 (TM )、蛋白C(PC)、游离蛋白S(FPS)、组织纤溶酶原激活物 (t PA)、组织纤溶酶原激活剂抑制剂 (PAI 1)、β TG、PF4 的含量和血小板粘附试验(PAdT)及血小板聚集试验 (PAgT) ,并进行统计学分析。 结果　脑梗死组vWF、FⅧ :C、PC、PAI 1、β TG、PF4 、PAdT及PAgT较对照组增高 ,t PA下降 ,患者组TM及FPS含量与对照组无明显差异。 结论　提示脑梗死患者凝血活性增强、纤溶活性减低 ,血小板功能亢进     

凝血试验真空管"死腔"所致 APTT、PF4偏差探讨

目的　探讨“有死腔”凝血试验管收集的血样进行血小板功能和肝素治疗患者APTT结果的偏差及机制。方法　将 2 0例肝素治疗心肌梗死患者血液分别收集在无或能形成死腔的采血管中 ,分别进行APTT和PF4检测。结果　“有死腔”的采血管使7例患者APTT结果缩短 ,PF4活性增强。结论　采血管死腔增加了血小板与管壁或死腔气体的接触而激活血小板 ,释放PF4并中和肝素 ,造成APTT负偏差。建议进行血小板功能试验或APTT用于肝素治疗监测时使用“无死腔”真空采血管     

基质细胞衍生因子-1和血小板第4因子对体外扩增后脐血CD34+细胞黏附特性和趋化功能的影响

为了研究基质细胞衍生因子-1（SDF—1）和血小板第4因子（PF4）对扩增后脐血CD34^＋细胞归巢相关功能的影响，将纯化的脐血CD34^＋细胞接种入无血清培养液中，加入不同组合的细胞因子FST（FL＋SCF＋TPO）、FST＋SDF—1、FST＋PF4或FST＋SDF—1＋PF4，分别于培养第7、10、14天检测CD34^＋细胞扩增倍数、集落形成能力、细胞的黏附分子表达、总黏附性、趋化功能。结果表明：①加入SDF—1的实验组CD34^＋细胞及造血祖细胞集落扩增倍数高于对照组；②加入SDF—1明显上调扩增的CD34^＋细胞CD49e的表达，加入PF4明显上调扩增的CD34^＋细胞CD49e、CD54的表达，在扩增体系中加入SDF—1或PF4均能够明显提高扩增的CD34^＋细胞的总黏附性；③在扩增体系中加入SDF—1能够明显提高扩增的CD34^＋细胞的自发迁移率，但导致CXCR-4的表达和SDF—1诱导迁移率降低；而PF4能够明显提高扩增的CD34^＋细胞的CXCR-4的表达和SDF—1诱导迁移率；在扩增体系中同时加入SDF—1和PF4能够明显提高扩增的CD34^＋细胞自发迁移率和SDF—1诱导迁移率。结论：体外扩增体系中加入SDF—1和PF4能够上调部分归巢相关黏附分子的表达，保持扩增的CD34^＋细胞的黏附和迁移能力，有利于降低体外扩增对造血干／祖细胞（HSPC）归巢相关功能的不利影响，维持扩增的HSPC的归巢潜能。     

重组人血小板第4因子生物学活性的分析

目的：分析重组人血小板第４因子（ｒｈＰＦ４）的生物学活性。方法：采用小鼠骨髓巨核细胞以及人巨核细胞白血病细胞株，从体内和体外多方面比较天然ＰＦ４和ｒｈＰＦ４的生物学活性。结果：此ｒｈＰＦ４具有与天然ＰＦ４相同的抑制小鼠骨髓巨核细胞祖细胞生长、减少集落形成的能力，这种抑制作用能被肝素中和；ｒｈＰＦ４对人巨核细胞白血病细胞株Ｍｅｇ－０１生长也有抑制作用。结论：ｒｈＰＦ４可取代天然ＰＦ４应用于临床及科研。     

The development of a quantitative enzyme-linked immunosorbent assay to detect human platelet factor 4

BACKGROUND: Platelet factor 4 (PF4) is a marker for in vitro and in vivo tests of platelet (PLT) activation and alpha-granule secretion. PF4 is also a major CXC cytokine released during storage. Cytokines released during PLT storage are a potential cause of nonhemolytic transfusion reactions. Quantitative measurement of PF4 requires an assay that is both reliable and sensitive. To achieve this goal, a sensitive, cost-effective, sandwich enzyme-linked immunosorbent assay (ELISA) was developed with commercially available antibodies to human PF4. STUDY DESIGN AND METHODS: An ELISA was developed for measuring PF4 from whole human PLTs or secreted from activated PLTs. Optimal concentrations of capture antibody, detection antibody, and enzyme-conjugate were determined with serial twofold dilutions of recombinant PF4. This assay was used to determine the ideal sample dilutions needed for reliable quantitation of PF4 in releasates or from whole PLT extracts. RESULTS: Serial dilutions of recombinant PF4 resulted in a sigmoid titration curve with a maximal sensitivity of 10 pg and a dynamic quantitative range from 100 to 2500 pg. This ELISA was used to measure secretion from permeabilized PLTs stimulated with free calcium. In a secretion experiment with 2.5 x 10|*bsup*|8|*esup*| PLTs per mL, samples required a 1:10-fold dilution to reliably evaluate alpha-granule release. CONCLUSION: The parameters described yield an ELISA method with low background and high sensitivity over a range of PF4 concentrations. Using the commercial reagents described makes this assay cost-effective and therefore suitable for analyzing multiple samples in the research setting.     

Low-density lipoprotein apheresis reduces platelet factor 4 on the surface of platelets: a possible protective mechanism against heparin-induced thrombocytopenia and thrombosis

BACKGROUND: Heparin‐induced thrombocytopenia and thrombosis (HITT) is characterized by thrombocytopenia due to the formation of antibodies against heparin : platelet factor 4 (PF4) complexes. Despite the exposure to heparin during treatment and predisposition of patients with atherosclerosis to HITT, HITT in patients undergoing low‐density lipoprotein (LDL) apheresis is rare. We investigated the possibility that LDL apheresis decreases PF4 on platelet (PLT) surfaces and/or plasma HITT antibody levels, either of which would disfavor HITT. STUDY DESIGN AND METHODS: We enrolled 25 patients undergoing LDL apheresis at the Hospital of the University of Pennsylvania. Blood samples were drawn before and after treatment. Plasma samples were drawn proximal and distal to the LA‐15 treatment column. PF4, HITT antibodies, heparin levels, and P‐selectin were measured. RESULTS: No patient had clinical symptoms of HITT. The LA‐15 column was found to efficiently remove PF4. PF4 levels in peripheral blood plasma did not change significantly after LDL apheresis. However, PLT surface PF4 significantly decreased after treatment. HITT antibodies were found in only two patients and were nonfunctional. PLT surface P‐selectin did not change during treatment. CONCLUSIONS: We have demonstrated that LDL apheresis via dextran sulfate absorption removes plasma PF4 and reduces the amount of PF4 on the surface of circulating PLTs. Reduced surface PF4 may decrease antibody formation and/or recognition by HITT antibodies. These data provide a potential explanation for the near lack of HITT in hypercholesterolemic patients undergoing LDL apheresis. They also suggest the possibility that LDL apheresis using dextran sulfate adsorption may have therapeutic value in the treatment of HITT.     

人精子特异性乳酸脱氢酶在大肠杆菌中的表达及其初步应用

目的 构建人精子特异性乳酸脱氢酶(hLDH-CA)的原核表达载体,在大肠杆菌中进行表达,将重组hLDH-CA应用于抗精子抗体(ASA)的检测.方法 以人睾丸TripIEx cDNA文库为模板,PCR扩增hLDH-CA编码序列.PCR产物经Hind Ⅲ-Xho I酶切后,克隆至表达载体pET-28a(+)中,在E.coli BL21(DE3)中诱导His-Tag融合的重组蛋白表达.用免疫印迹、酶活性测定等方法鉴定表达产物.以纯化的重组hLDH-CA为基质,建立检测ASA的ELISA方法.结果 构建了hLDH-C4原核表达载体pET-28a(+).hLDHC.在IPTG的诱导下,重组菌可高效表达相对分子质量35 000的产物,与预期大小相符.免疫印迹显示,重组蛋白可被抗His-Tag单克隆抗体和兔抗人LDH-C4抗体识别.重组菌在IPTG诱导后,其裂菌液的乳酸脱氢酶活性是诱导前的11.2倍.用基于hLDH-C4抗原的间接ELISA法,在一组不育症患者中检测血清抗hLDH-CA抗体,阳性率达30.51%.结论 成功地克隆了hLDH-C4编码序列,并在E.coli BL21(DE3)中获得高效的表达,重组hLDH-C4在ASA检测中得到初步应用.     

血小板胶原受体糖蛋白Ⅵ体外表达及功能研究

为了深入研究血小板胶原受体糖蛋白Ⅵ(glycoproteinⅥ，GPⅥ)功能及筛选特异性抑制剂，利用基因重组技术体外表达GPⅥ胞外区片段。采用PCR方法扩增GPⅥ胞外区片段eDNA，构建表达载体pET-20b( )-GPⅥ，转化大肠杆菌BL21(DE3)pLysS，经IPTG诱导表达。表达产物经Ni—NTA Resin纯化、复性：使用Western blot方法鉴定重组蛋白性质；采用胶原结合试验测定重组蛋白的胶原结合能力。结果表明，经测序证明PCR扩增产物与GPⅥ胞外区eDNA序列完全一致；酶切分析证明成功构建了表达载体pET-20b( )-GPⅥ；原核细胞经诱导表达后出现新的相对分子量32kD蛋白条带，诱导产物以包涵体形式存在；Western blot分析表明重组蛋白可与抗Penta—His抗体和抗GPⅥ多抗特异性结合；胶原结合试验表明重组蛋白拥有较好的生物学功能。结论：正确构建了GPⅥ表达载体并成功表达重组蛋白，纯化、复性后的重组蛋白具有良好的抗原性和生物学活性。     

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.     

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.     

Sensitive enzyme immunoassay for the measurement of platelet factor 4 in blood plasma

A sandwich enzyme immunoassay method for the measurement of platelet factor 4 (PF4) was developed with the use of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from E. coli. The measurable range was 30 pg to 3 ng of PF4 per tube. Within-run and between-run coefficients of variation were less than 10%. The results obtained with the enzyme immunoassay correlated well with those of a radioimmunoassay (r = 0.952, slope = 0.954, gamma-intercept = 2.43 ng/ml). Platelets contained large amounts of PF4 (7.21 +/- 1.97 ng/10(6) cells or 2.51 +/- 1.13 ng/mg protein), whereas the PF4 levels in red blood cells and lymphocytes were negligible, confirming the specific localization of PF4 in platelets. The applicability of the immunoassay method was tested to determine the in vitro release of PF4 during preparation and storage of platelet concentrates.     

利用基因工程技术构建重组人血小板因子4真核表达载体

目的:构建携带绿色荧光蛋白标签的重组人血小板因子4基因(plateletfactor4,PF4)的真核表达载体,并在真核细胞中表达,为研究其生物学功能奠定基础。方法:人工合成PF4基因模板,通过PCR方法扩增PF4-cDNA,然后克隆至pUC19克隆载体,经序列测定后重组入pIRES2-EGFP真核表达载体并进行酶切鉴定。将鉴定正确的质粒用脂质体转染法瞬式转染至COS7细胞中。结果:获得了PF4-cDNA基因,序列分析表明,该序列与GenBank数据库中的序列一致。重组质粒酶切鉴定表明,PF4基因已与pIRES2-EGFP真核表达载体正确连接。荧光显微镜示,此载体成功转染至COS7细胞中,并获得有效表达。结论:利用基因工程技术,成功的构建了携带绿色荧光蛋白标签的PF4的真核表达载体,并在真核细胞中获得有效表达,为下一步研究其在真核细胞中的生物学功能奠定基础。