Change of Coagulation Factor Ⅷ and Antithrombin Ⅲ Activity in Bank-Stored Blood

Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd day, and 70 % of the activity at the 7th day. FⅧ:c showed no obvious change after 24 h, until the 3rd day. It lost 40 %-60 % of the activity after 36 h and was reduced to the 30 % of the original activity at the 5th day. Our results suggested that at the 3rd day coagulation factor Ⅷ of bank stored blood can be used to replenish antithrombin Ⅲ, while bank stored blood in one day can be used to replenish FⅧ.

Construction and application of gene targeting replacement vector formouse coagulation factor IX gene

Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragment of coagulation factor IX gene from 129Sv mouse genomic DNA A phage library, PMFIXDEL plasmid was designed with positive-negative-selection (PNS) strategy, and constructed with commonmolecular cloning techniques. Structure of PMFIXDEL was identified by PCR and restriction analysis. Afterelectroporation with the linearized PMFIXDEL DNA, transfected ES cells were cultured in G418/GANC drugselection medium. The recombination efficacy of this vector was tested. Results: The main components ofPMFIXDEL were two copies of negative selection gene (HSV-tk expression cassette), a positive selectiongene (Neo expression cassette), long and short homologous fragments and plasmid backbone. The introduction of negative selection gene (HSV-tk ) into the construct resulted in 24-fold increase of selection.Conclusion: An effective replacement vector for mFIX gene targeting was constructed and applied in ES cell.

Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

Producing and secreting human factor IX with high efficiency bymurine primary and C2C12 muscle cells carrying human factor IX gene

objective:To study systematically the abilities of skeletal muscle cells in synthesizing,processing and secreting recombinant proteins in vitro.Methods:Primary murine myoblasts from SCID mice (SCID-MB) and C2C12,a muse myoblast cell line,were trans fected with muscle specific (pdLMe4bAh ff m) or non-specif ic (LIXSN) vectors encoding human factor IX (FIX). The capacities of the transgenic cells in producing re combinant FIX were compared in the levels of intracellular and secreted FIX protein as well as FIX mRNA in primary muscle cells and C2C12 cells. Results:Both primary muscle cells and C2C12 cells can efficiently trans late and process FIX protein. Myotubes derived from the SCID-MB produced 1 800~2 000 ng FIX/106 cells/ 24 h in culture with specific activity of 95% ~ 103%. C2C12 cells can synthesize and process recombinant FIX as efficiently as primary muscle cells,but their secretion ability is less efficient in comparison with primary cells. Conclusion:This observation indicates that primary cells should be used in gene therapy related research projects and care should be taken while explaining the results derived from established cell lines.

Molecular mechanism of microRNA125 regulating human coagulation factor IX gene with nonsense mutation

Objective To construct human coagulation factorⅨmini-gene(Mini-h F9)and some nonsense mutants,detect the levels of the Mini-h F9 mRNA,and analyze the molecular mechanism of microRNA125 regulating F9gene with nonsense mutation.Methods Three nonsense mutants were obtained by using PCR mutagenesis to ana-

Polymorphisms in the coagulation factor Ⅶ gene and the risk of myocardial infarction in patients undergoing coronary angiography

Objective To investigate whether coagulation factor Ⅶ (FⅦ) polymorphisms play a role in the pathogenesis of coronary artery disease (CAD) and/or myocardial infarction (MI) in a series of Hans.Methods The Arg 353 Gln and HVR4 polymorphisms of FⅦ gene were determined in 374 patients undergoing selective coronary angiography by PCR and restriction fragment length polymorphism assay.Results The FⅦ genotype distribution was in accordance with Hardy-Weinberg equilibrium. The frequencies of FⅦ genotypes or alleles did not show significant differences between the CAD group and the controls or between the males and the females. The frequencies of carriers of the Gln 353 allele and (Arg/Gln+Gln/Gln) genotypes were significantly higher in the CAD patients without MI than in those with MI ( P =0.031,odds ratio 0.37,95% CI: 0.15-0.94). However,HVR4 polymorphisms were not significantly different between the two groups ( P >0.05).Conclusion Carrying the F Ⅶ Gln 353 gene may be a protective factor against MI in the Chinese Hans.

High level expression of human Factor Ⅷ in mammalian cells after retroviral-mediated gene transfer

Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Protein trans-splicing based dual-vector factor VIII （FVIII） gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide （442± 151 ng mL i vs. 305±103 ng mL-1） and coagulation activi- ty （1.46±0.37 IU mL i vs. 0.85±0.23 IU mL-1） compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector ex- pressing FVIII （1.79_＋0.59 IU mL-l）. These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.

Original article ：Identification of seven novel mutations in the factor Ⅷ gene in 18 unrelated Chinese patients with hemophilia A

Background Hemophilia A （HA） is an X-linked inherited bleeding disorder caused by decreased activity of factor Ⅷ（FⅧ） due to heterogenous mutations in the FⅧ coding gene （F8）. The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA. Methods Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTERS database. A clotting method was used to assay the FⅧ activity level and the Bethesda assay was used to detect the FⅧ inhibitor. Results A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation （4382-3 AC deletion） was associated with inhibitor development. Conclusion These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FⅧ inhibitor.

中国血友病A患者因子Ⅷ抑制物形成特征及随访研究

目的随访研究中国血友病A患者因子Ⅷ(FⅧ)抑制物发生率和特征。方法 215例血友病A患者在24月(2007年6月至2009年6月)的连续随访中,监测FⅧ抑制物发生、变化及转归,并观察患者临床特征。结果 215例血友病A患者随访24月FⅧ抑制物累积发病率为11.6%(25/215);其中低滴度者占72%(18/25),高滴度者占28%(7/25);FⅧ抑制物阳性发生时的中位年龄为25岁(6～59岁)、累积中位暴露日为150日;15/25(60%)低滴度阳性者(中位滴度1.25BU/ml)在自然情况下于6～15月(中位10月)转为阴性,5/25(20%)高滴度抑制物者(中位滴度100BU/ml)则随访24月持续阳性,另外5/25(20%)FⅧ抑制物阳性无变化;25例FⅧ抑制物阳性者出血频率较其阴性时显著增加(P=0.025);18/25例继续应用FⅧ者,FⅧ产品用量(IU/kg·月)较前显著增加(P=0.015),但靶关节数目在24月随访期间并无增加(P=0.329)。结论我国血友病A患者FⅧ抑制物发病率和特征与欧美等国家存在差异。

B区缺失的人凝血因子Ⅷ基因在293T细胞表达

本研究目的是构建含有人凝血因子Ⅷ(FⅧ)基因的慢病毒载体,观察其在293T细胞中的表达情况。用限制性内切酶法获得B区缺失的人凝血因子Ⅷ基因(BDDhFⅧcDNA)片段,将其克隆至慢病毒载体pXZ208,构建了慢病毒表达载体pXZ208-BDDhFⅧ;用限制性内切酶法鉴定载体的连接方向,用磷酸钙共沉淀法将重组质粒pXZ208-BDDhFⅧ分别与包装质粒ΔNRF、包膜蛋白质粒VSV-G共转染293T包装细胞,包装后感染293T细胞,并以pXZ171作为对照。在感染后用逆转录-聚合酶链反应(RT-PCR)检测BDDhFⅧ基因的转录,一期法检测细胞培养上清FⅧ的活性,流式细胞仪(FCM)检测载体的感染效率,PCR检测BDDhFⅧ基因的整合。结果表明:成功构建了慢病毒表达载体pXZ208-BDDhFⅧ,其基因转导染效率达到了59.57%。RT-PCR法能够检测到BDDhFⅧ转录的mRNA。感染后24、48、72小时检测到细胞上清中FⅧ活性(FⅧ∶C)分别为12%、43%、87%。PCR法扩增出了534bp的特异性片段。结论:成功构建的慢病毒表达载体pXZ208-BDDhFⅧ,在体外可以有效感染293T细胞并表达有活性的FⅧ,提示基因治疗可应用于血友病A。

非病毒载体介导的人凝血因子Ⅷ基因在小鼠32D细胞系中的表达

为了观察非病毒载体 pRC/RSV介导的人凝血因子Ⅷ基因在小鼠 32D细胞系中的表达 ,将B结构域缺失(△ 76 0aa - 16 39aa)的人FⅧcDNA(hFⅧBDcDNA)亚克隆至质粒载体pRC/RSV ,构建重组质粒载体 pRC/RSV hFⅧBDcDNA。经SuperFectTransfectionReagent转染小鼠 32D细胞系 ,分别采用一期法、ELISA法和RT PCR检测细胞培养上清液中人FⅧ的促凝活性 (hFⅧ∶C)和抗原含量 (hFⅧ∶Ag)以及细胞中hFⅧBDcDNA的转录。结果表明 :小鼠 32D细胞系培养上清液中的人FⅧ∶Ag最高达到了 4 5 0 .0 8毫微克 /(10 6细胞·2 4小时 ) ,hFⅧ∶C最高达到了 2 .0 1单位 /(10 6细胞·2 4小时 ) ,RT PCR可检测到 32D细胞中hFⅧBDcDNA转录的mRNA。结论 :非病毒质粒载体 pRC/RSV介导的人凝血因子Ⅷ基因能够在小鼠 32D细胞系中表达人FⅧ蛋白 ,所表达的FⅧ与正常人血浆中的野生型FⅧ具有相似的凝血活性。

人凝血因子ⅧcDNA在小鼠体内的转染与表达

本研究观察逆转录病毒载体和聚酰胺 胺型 (PAMAM)树枝状聚合物介导的人凝血因子Ⅷ (FⅧ )基因在小鼠体内的转染和表达 ,并与体外转染方法作比较。应用含B区缺失 ( 76 0aa- 16 39aa)人FⅧcDNA(FⅧBDcDNA)的以逆转录病毒为框架的重组表达质粒载体 pLNC FⅧBD包装成为逆转录病毒LNC FⅧBD ,exvivo转染小鼠骨髓基质细胞 ,同时将 pLNC FⅧBD与PAMAM树枝状聚合物按照 1∶15混合以形成复合体PAMAM pLNC FⅧBD进行小鼠invivo转染。分别于注射后第 2 4 ,4 8小时 ,第 1,2 ,3,4周取血 ,分离血浆 ,采用一期法测定人FⅧ活性 (FⅧc) ,ELISA法测定人FⅧ抗原 (FⅧAg) ,并采用Bethesdainhibitorassay测定人FⅧ抗体 ;同时取脏器肝、脾、肺、肾提取RNA ,进行RT PCR ,观察各组织的转录 ,并用苏木素 伊红染色法观察各组织的形态学改变。结果表明 ,逆转录病毒转染人FⅧ基因的骨髓基质细胞输入体内后 ,可以在体内继续表达人FⅧ ,并且能有效地分泌至血液中。宿主小鼠体内可检测到的人FⅧ表达持续 3周以上 ,注射后第 2 4小时表达最高水平 ,人FⅧAg平均为 8.6ng/ml,此后人FⅧ抗体逐渐生成 ,人FⅧAg水平渐低 ,于注射后第 4周不再能测出人FⅧAg。注射复合体PAMAM pLNC FⅧBD在体内转染后 ,获得了短暂的人FⅧ生理水平的表达 ,在注射?

超大孔离子交换制备色谱分离纯化高活性凝血因子Ⅷ

建立了一条从人血浆中分离高活性凝血因子Ⅷ(FⅧ)的纯化工艺。基于FⅧ和介质孔径的尺度比及其对蛋白质活性影响的分析,设计了以超大孔离子交换制备色谱为核心步骤的新型分离纯化工艺。分别进行超大孔离子交换色谱与传统离子交换色谱的条件优化,并对优化工艺所得产品进行了活性检测(底物显色法)和纯度检测(高效凝胶过滤和凝胶电泳)。结果表明,超大孔介质结构不但可以有效地保护蛋白质大分子结构,而且能够大幅度地提高制备色谱的传质速率,从而得到具有高凝血活性的FⅧ产品。FⅧ在超大孔制备色谱过程中的回收率(85%)比传统离子交换制备色谱高4～5倍,产品比活高达154 IU/mg。此外,还研究了超大孔介质的再生程序,采用5个柱体积的1 mol/L NaOH低流速清洗色谱柱,保证了色谱工艺的稳定性。本纯化工艺步骤简单,重现性好,易于放大生产。

蛋白C、抗凝血酶及凝血因子Ⅷ活性变化与肺癌合并肺栓塞后治疗的关系

目的探讨蛋白C（PC）、抗凝血酶（AT）及凝血因子Ⅷ（FⅧ）活性变化与肺癌合并肺血栓栓塞[简称肺栓塞（PE）]后治疗的关系.方法选择肺癌合并PE患者（肺癌＋PE组）98例及肺癌患者（肺癌组）100例.记录两组性别、年龄并检测脂蛋白（a）[Lp（a）]、总胆固醇（TC）、甘油三酯（TG）、C反应蛋白（CRP）、凝血酶时间（TT）、血小板数量（PLT）、纤维蛋白原（Fbg）等指标.检测肺癌＋PE组治疗5～7d及肺癌组入院时的PC、AT、FⅧ、D-二聚体水平.应用Logistic回归分析肺癌合并PE后治疗5～7d凝血纤溶状态的影响因素,并采用受试者工作特征（ROC）曲线分析影响因素的诊断效能.结果肺癌＋PE组Lp（a）、TC、TG、TT均明显高于肺癌组（P〈0.05）.肺癌＋PE组PC、AT、FⅧ、D-二聚体水平明显高于肺癌组（P〈0.01）.Logistic逐步回归及ROC曲线分析显示,PC、AT、FⅧ是肺癌合并PE后治疗5～7d凝血纤溶状态的影响因素,ROC曲线下面积均〉0.90.结论肺癌合并PE中期治疗需关注PC、AT、FⅧ对凝血纤溶系统的影响,其活性水平可用来评估-定时期内的治疗效果和治疗方案.

凝血因子Ⅻ及Ⅷ在主动脉夹层围术期凝血功能中的作用研究

目的探讨凝血因子Ⅻ及凝血因子Ⅷ在主动脉夹层手术患者围术期的变化及对二次开胸的影响。方法选取2014年1月至2015年9月于首都医科大学附属北京安贞医院就诊的Stanford A型主动脉夹层患者88例,均接受中度低温停循环手术治疗。根据是否二次开胸将88例患者分为二次开胸组(10例)和未二次开胸组(78例),比较2组患者手术及术后重要指标,分析凝血功能障碍导致二次开胸的危险因素。选择麻醉诱导后(t1)、术中温度最低点(t2)、复温至36℃(t3)、术后4 h(t4)、术后24 h(t5)5个时点进行血液样本的抽取,应用酶联免疫吸附法进行凝血因子Ⅻ、活化的凝血因子Ⅻ、凝血因子Ⅶ、活化的凝血因子Ⅶ、凝血因子Ⅷ、活化的凝血因子Ⅷ、血红蛋白、血小板计数的检测。结果t2~t4时点凝血因子Ⅻ水平均低于t1,差异均有统计学意义(均P<0.05),t5时点与t1比较差异无统计学意义(P=0.065)。活化的凝血因子Ⅻ和活化的凝血因子Ⅷ水平由t1~t5全程呈逐渐升高趋势,与t1比较差异均有统计学意义(均P<0.05)。凝血因子Ⅷ水平各时点均低于t1,差异均有统计学意义(均P<0.05)。凝血因子Ⅶ水平于t2和t3时点均低于t1,t4和t5时点均高于t1,差异均有统计学意义(均P<0.05)。二次开胸组术中失血量、术后总引流量以及悬红细胞输注总量、冰冻血浆输注总量、纤维蛋白原输注总量均高于未二次开胸组[1 700(1 375,2 000)ml比1 250(1 000,1 600)ml;4 250(2 533,7 060)ml比2 170(1 230,3 555)ml;2 700(1 125,3 825)ml比675(600,1 424)ml;1 200(400,1 550)ml比400(50,800)ml;2.00(2.00,2.88)g比2.00(1.00,2.00)g],t2时点血小板计数水平低于未二次开胸组[(61±28)×109/L比(72±39)×109/L],差异有统计学意义(P<0.05)。在校正体质量、体外循环时间、手术时间、低温停循环时间、术前血小板水平、术前血红蛋白水平等因素的影响后,多元Logistic回归分析发现,t2时点凝血因子Ⅻ水平(比值比=1.342,95%置信区间:1.058~1.570,P=0.012)为主动脉夹层患者接受外科手术后凝血功能障碍导致二次开胸的独立危险因素。结论凝血因子Ⅻ作为内源性凝血途径的启动因子,在主动脉夹层患者围术期的凝血功能中发挥着巨大作用,其在术中最低温时的水平较低可能是术后由于凝血功能障碍导致二次开胸的独立危险因素;而凝血因子Ⅷ作为凝血共同途径的重要因子,本研究未发现其增加二次开胸的风险。

中和低剂量重组人凝血因子Ⅷ防治血友病A关节出血的疗效

目的 比较中、低剂量重组人凝血因子Ⅷ防治血友病A关节出血的临床疗效.方法将58 例血友病A患儿按预防剂量的大小分为2组：中剂量组（28例）和低剂量组（30例）.中剂量组采用重组人凝血因子Ⅷ15～25 ·kg-1静脉推注,3次周-1,连用4个月;低剂量组采用重组人凝血因子Ⅷ＜15 U·kg-1静脉推注,3次＆#183;周-1,连用4个月.观察2组防治前、防治4个月后关节出血次数的情况.结果 2组防治4个月后关节出血次数明显低于防治前（P＜0.05）,中剂量组防治4个月后关节出血次数明显低于低剂量组（P＜0.05）.结论 重组人凝血因子Ⅷ用于防治血友病A能明显改善关节出血症状,且中剂量防治优于低剂量.

215例血友病A患者因子Ⅷ抑制物形成的环境因素研究

目的研究中国血友病A患者因子Ⅷ(FⅧ)抑制物形成的环境因素。方法监测215例血友病A患者在2年(2007年6月至2009年6月)的连续随访中,FⅧ抑制物发生、变化及转归,并对FⅧ抑制物形成的环境因素进行回顾性分析。结果 215例血友病A患者随访2年FⅧ抑制物累积发生率为11.6%(25/215)。FⅧ抑制物形成的环境因素的多变量分析结果示:①输注原因中短期低剂量预防治疗比按需治疗形成FⅧ抑制物的风险低(OR=0.037,95%CI为0.002～0.616);②血友病越严重,累积暴露日越少,FⅧ抑制物形成风险越高;③发生严重出血事件是FⅧ抑制物形成的高危因素(OR=117.045,95%CI为19.333～708.617);④输注形式和发生重大感染事件与FⅧ抑制物形成无关。结论在中国目前治疗情况下,血友病患者FⅧ抑制物的形成可能与血友病严重程度、输注原因(预防治疗或按需治疗)、累积暴露日和是否发生严重出血事件有关。

尿激酶对动脉粥样硬化患者血清纤维蛋白原、凝血因子Ⅴ和凝血因子Ⅷ的影响

目的探讨尿激酶对动脉粥样硬化患者血清纤维蛋白原、凝血因子Ⅴ和凝血因子Ⅷ的影响,以评价尿激酶对动脉粥样硬化患者凝血功能的影响。方法选择2011年2月~2013年1月期间来在本院诊断为动脉粥样硬化的100例患者,并接受尿激酶静脉注射治疗。根据临床分期分成隐匿期(n=25)、缺血期(n=25)、坏死期(n=25)和纤维化期(n=25)。检测所有患者治疗前后血清纤维蛋白原、凝血因子Ⅴ和凝血因子Ⅷ浓度,并计算各指标的变化率。结果治疗后各组纤维蛋白原(Fg)、凝血因子Ⅴ(FⅤ)和凝血因子Ⅷ浓度均显著低于治疗前(P<0.05)。血清纤维蛋白原变化率隐匿组为(52.11±1.47)%,缺血组为(52.21±1.22)%,坏死组为(49.89±1.43)%,纤维化组为(50.73±1.71)%,各组间比较差异均无统计学意义。凝血因子Ⅴ变化率隐匿组为(62.83±2.25)%,缺血组为(62.63±1.92)%,坏死组为(60.89±2.11)%,纤维化组为(61.36±2.14)%,各组间比较差异均无统计学意义。凝血因子Ⅷ变化率隐匿组为(43.02±2.28)%,缺血组为(42.04±2.13)%,坏死组为(39.99±2.93)%,纤维化组为(40.93±2.94)%,各组间比较差异均无统计学意义。结论尿激酶可通过降低动血清纤维蛋白原、凝血因子Ⅴ和凝血因子Ⅷ浓度来降低动脉粥样硬化患者凝血功能,以达到抑制血栓形成的作用。

人凝血因子Ⅷ原液生产工艺放大可行性研究

目的对比3批小试（50g冷沉淀）、3批小规模放大（400g冷沉淀）和3批中试级（5-10 kg冷沉淀）试验结果探讨人凝血因子Ⅷ原液生产工艺放大的可行性。方法 Tris溶液复溶冷沉淀,经聚乙二醇沉淀后,0.3%磷酸三丁酯和1%Tween 80处理6h以灭活脂包膜病毒,然后经DEAE 650M层析纯化。绝大部分杂蛋白和磷酸三丁酯及Tween80随流穿液直接流穿,洗脱峰样品即为人凝血因子Ⅷ原液。结果 3批小试人凝血因子Ⅷ活性回收率分别为30.50%、24.40%和25.60%,平均值26.83%,回收率变异系数8.52%;3批小规模放大回收率分别为19.40%、21.70%和22.00%,平均值21.04%,回收率变异系数5.54%;3批中试级回收率分别为14.50%、16.50%、17.80%,平均值16.27%,回收率变异系数8.34%。结论对比级放大试验各级回收率及变异系数发现,工艺重复性好,放大可行。