下肢缺血预处理对大鼠心肌HIF-1α表达的影响

目的 观察急性心肌梗死(AMI)和经下肢缺血预处理的大鼠缺血心肌中缺氧诱导因子-1α(HIF-1α)基因的表达及其在早期心肌缺血状态下的表达规律.方法 SD大鼠随机分为3组:①对照组;②AMI组:结扎左冠状动脉前降支;③下肢缺血预处理(LIP)组:下肢动脉夹闭再开放,随后复制AMI模型.应用伊文思蓝及氯化三苯硝基四氮唑红(TTC)染色,测定心肌梗死范围;应用RT-PCR测定各组心肌缺血区HIF-1α基因动态表达情况.结果 LIP组心肌梗死面积较AMI组明显减小;HIF-1α mRNA在正常心肌有表达,心肌缺血早期相升高;经下肢缺血预处理后缺血心肌的表达降低.结论 下肢缺血预处理对急性缺血心肌有保护作用,HIF-1α基因表达减少可能是其机制之一.     

碱性成纤维细胞生长因子在鼠缺血心肌中表达及其促血管新生作用

目的 探讨腺病毒穿梭质粒介导的碱性成纤维细胞生长因子(bFGF)基因质粒在大鼠缺血心肌中的表达情况及其对缺血心肌血管新生、梗死面积的影响.方法 42只健康SD大鼠接受冠状动脉左前降支结扎术,存活30只,随后随机分为治疗组(n=15)和对照组(n=15),治疗组于心肌梗死区和正常区交界处局部注射100 μl bFGF基因(1 μg/μl),对照组以等体积生理盐水处理.4 w后观察红色荧光蛋白(RFP)和bFGF 表达情况.微血管计数和氯化三苯四氮唑(TTC)分别检测血管新生情况和梗死面积.结果 ①基因转染后4 w的心肌冰冻切片中均可见RFP表达;治疗组心肌组织bFGF mRNA表达水平较对照组明显增高[1.238±0.323 vs 0.771±0.102(P＜0.05)],仅在治疗组检测到bFGF 蛋白表达.②治疗组微血管密度明显高于对照组[141.13±17.65 vs 75.58±6.59/视野(P＜0.05)];治疗组梗死面积小于对照组[16.32±0.43 vs 17.00±0.57%(P＜0.05)].结论 重组bFGF裸质粒经心肌内直接注射后能有效表达并发挥促进梗死后心肌血管新生和缩小梗死面积的作用.     

超声介导载三突变型人低氧诱导因子-1a基因微泡靶向传输

目的血管新生疗法有望成为治疗缺血性心血管疾病的新方法。然而,基因传输的有创性阻碍了血管新生疗法的深入发展,超声介导的基因转移技术为基因传输带来了希望。因此,本研究探讨超声介导载人三突变型人低氧诱导因子-1a(Hypoxia inducible factor-1a,HIF-1a)基因微泡靶向传输促缺血下肢血管新生的可行性。方法 24只实验小鼠予结扎一侧下肢股动脉制备下肢缺血模型,另一侧下肢作为对照组。术后1周所有小鼠随机等分为3组,A组:尾静脉注入HIF-1a基因微泡+超声照射;小鼠仰卧位,通过尾静脉10 min内恒速输注载基因微泡,同时将超声探头垂直固定于缺血下肢,照射10 min(探头17L5,频率7.0 MHz,机械指数1.9);B组:尾静脉注入HIF-1a基因微泡;C组:超声照射(同A);采用Western Blot检测基因转染后第5天人HIF-1a蛋白表达情况,转染后第2周利用超声灌注成像评价缺血下肢血流情况,免疫荧光染色检测微血管密度。结果 WesternBlot显示人HIF-1a蛋白的表达在A组显著高于B组,差异有统计学意义(P<0.05),两者均较C组显著增加。血流灌注成像显示转染后第2周中A组缺血下肢的血流量达到对侧非缺血下肢血流量的79.3%±6.4%,显著高于B组60.0%±4.2%和C组52.3%±3.8%,差异有统计学意义(P<0.05)。免疫荧光染色显示A组的毛细血管密度和小动脉密度均显著高于B组和C组,差异有统计学意义(P<0.05)。结论超声介导的载人三突变型HIF-1a基因微泡可实现向小鼠缺血下肢的靶向传输,并产生促血管新生效应。     

缺氧诱导因子-lα对糖尿病心肌病大鼠心肌超微结构和血管生成的影响

目的:探讨缺氧诱导因子-lα(HIF-1α)对糖尿病心肌病大鼠心肌超微结构、血管生成的影响。方法:将24只雄性Wister大鼠随机分为对照组和实验组。两组动物均采用高糖高脂饲料喂养,在喂养6周后给予链脲佐菌素腹腔注射建立糖尿病心肌病模型。分别于第8周、第10周,给予对照组大鼠尾静脉注射100μg空载体质粒(pShuttle),给予实验组大鼠尾静脉注射100μg重组HIF-1α过表达质粒(pShuttle-HIF-1α)。每2周采集大鼠静脉血,检测血糖、血脂。第12周时处死动物,留取血液和心肌组织。采用透射电镜观察心肌细胞超微结构,苏木素-伊红染色检测心肌血管密度,Western blot检测心肌组织中HIF-1α和血管内皮生长因子(VEGF)的蛋白表达水平。结果:与对照组相比,实验组大鼠心肌组织中HIF-1α和VEGF表达明显增高,心肌毛细血管数目明显增多,心肌细胞超微结构损伤明显减轻。结论:HIF-1α可能通过诱导VEGF表达,减轻糖尿病心肌病大鼠心肌超微结构损伤并促进心肌血管形成。     

HIF-1α在压力过负荷大鼠心肌组织中的表达

目的 探讨压力过负荷时大鼠心肌组织低氧诱导因子(HIF)-1α、血管内皮生长因子（VEGF）表达的变化。方法 将45只雄性Wistar大鼠随机分为两组,即假手术组（15只）和模型组（30只）。采用腹主动脉缩窄法建立压力过负荷大鼠模型。分别在6周、12周后,制作两组大鼠的心肌组织切片,检测血流动力学指标以及心肌组织中HIF-1α及其靶基因VEGF的表达。结果 与假手术相比,模型组大鼠心肌组织第6周出现明显肥厚(P<0.05);心肌组织HIF-1α及靶基因VEGF的表达明显增加,并于第12周出现心力衰竭(P<0.05);心肌组织中HIF-1α和VEGF的表达仍较假手术组增加(P<0.05),但较本组第6周大鼠表达明显减少(P<0.05)。结论 压力过负荷造成的大鼠心肌肥厚组织,HIF-1α及靶基因VEGF的表达明显增加,但当出现心力衰竭时,HIF-1α和VEGF的表达则明显减少。     

低氧诱导因子修饰内皮祖细胞促血管新生的研究

目的观察低氧诱导因子（hypoxia inducible factor-1α,HIF-1α）转染内皮祖细胞（endothelial progenitor cells,EPCs）后,EPCs在体外的扩增及迁移功能改变及对体内缺血下肢血管新生的影响。方法在人外周血EPCs中导入人HIF-1α基因。结果转染后的EPCs中HIF-1α、血管内皮生长因子（vascular endothelial growth factor,VEGF）mRNA及蛋白表达升高,并被特异性siRNA-HIF-1α中度抑制;功能学检测示HIF-1α过表达增加了EPC在体外的分化、扩增、迁移;内皮细胞（EC）标记物CD31、VEGFR2、eNOS及VEGF、NO分泌增加;移植HIF-1α-EPCs至缺血下肢后可见外源性EPCs存在于缺血部位;HIF-1α-EPCs较对照组进一步促进体内毛细血管数目增加。结论在体外,HIF-1α转染后的EPCs扩增及迁移功能改善;在体内,可促进缺血下肢的局部血管新生。     

重组腺病毒相关病毒携带人血管内皮生长因子165诱导家兔缺血心肌血管生成的研究

目的　将携带人血管内皮生长因子 16 5 (hVEGF 16 5 )cDNA的重组腺病毒相关病毒 (rAAV)载体注入家兔缺血心肌 ,观察血管内皮生长因子 (VEGF)的血管生成效应。方法　制作家兔急性心肌梗死模型 ,将rAAV hVEGF 16 5注入缺血心肌。 4周后取注射部位心肌 ,用逆转录聚合酶链式反应 (RT PCR)和免疫组织化学、Westernblot方法检测目的基因的表达 ,并计数注射部位心肌毛细血管数目 ,评价外源VEGF的生物学活性。结果　注射rAAV VEGF部位心肌VEGFmRNA及其蛋白的表达可持续 8周 ,而且比对照组增加。远隔脏器未见外源VEGF的播散和表达。注射rAAV VEGF部位毛细血管密度比对照组增加。结论　rAAV hVEGF 16 5可以有效地转染成年家兔心肌组织 ,外源基因稳定长期表达 ,同时能够刺激新血管生成 ,并具有良好的安全性。     

HIF-1对大鼠急性缺血心肌的保护作用

目的研究HIF-1对大鼠急性缺血心肌的保护作用及其作用机制。方法应用HIF-1活性诱导剂氯化钴预处理大鼠,观察预处理对大鼠急性心肌梗死面积和心肌VEGF、iNOS蛋白表达的影响。结果氯化钴预处理组大鼠较对照组和假手术组急性心肌梗死面积显著减少(P<0.01),VEGF、iNOS蛋白表达显著增高(P<0.01)。结论HIF-1可以减少大鼠急性心肌梗死面积,对大鼠急性缺血心肌具有保护作用,其机制可能与HIF-1促进VEGF、iNOS蛋白表达增加有关。     

缺氧诱导因子α亚型基因在急性心肌梗死大鼠缺血心肌表达的变化

目的 通过观察急性心肌梗死(AMI)大鼠心肌缺氧诱导因子 (hypoia-inducible factor,HIF) 3个α亚型mRNA表达的动态变化,探讨其在早期缺血心肌中的作用.方法 将大鼠随机分为对照组和AMI组.用HE染色观察心肌形态学变化, RT-PCR检测术后1、2、4、6、12 h缺血心肌HIF 3个α亚型mRNA表达情况.结果 HIF-1α、2α、3α mRNA在正常心肌中均有表达.术后1 h,缺血心肌HIF-1α mRNA表达明显上调(P<0.05),2 h达高峰(P<0.01),4 h开始下降(P＜0.05),6 h降至正常水平,12 h维持在正常水平;HIF-2α、3α mRNA在急性缺血心肌表达无明显变化.结论 早期急性心肌缺血可诱导HIF-1α基因表达改变,与HIF-2α、3α基因表达无关.     

心肌内注射碱性成纤维细胞生长因子基因对兔缺血心肌血管新生的促进作用

目的 :探讨心肌内注射碱性成纤维细胞生长因子基因 (b FGF基因 )治疗兔急性心肌梗死模型的效果。方法　碱裂解法大量制备质粒 ;采用开胸结扎兔冠状动脉左前降支 (L AD)法 ,建立兔急性前壁心肌梗死模型。模型制备成功后将动物分为治疗组 (n=19)和对照组 (n=18) ,并于心肌内分别注射 pc DNA3- b FGF10 0 μg和 pc DNA310 0 μg,饲养至 2 ,6 ,12周处死 ;免疫组化观察蛋白表达 ;行病理切片观察心肌梗死组织学变化和缺血心肌内血管新生的情况。结果　 (1)术前术后描记的心电图证实兔急性心肌梗死模型制作成功 ;(2 )免疫组化观察注射 pc DNA 3- b FGF处心肌组织在 6周内有 b FGF蛋白的表达 ;(3)病理切片行图像分析计算血管密度发现 ,治疗组毛细血管密度和小动脉密度显著高于对照组。结论　心肌内注射 b FGF基因能促进缺血心肌内血管新生 ,有可能成为一种新的冠心病治疗方法     

Current therapy for rare factor deficiencies

Haemophilia A and B and von Willebrand disease account for 80–85% of all inherited bleeding disorders. The other 15% are represented by deficiencies of fibrinogen, prothrombin, or factors V, VII, X, XI, or XIII. In addition, acquired factor deficiencies are seen in a variety of conditions ranging from malignancies to autoimmune disorders. The spectrum of symptoms in these conditions varies from severe and life-threatening haemorrhage to a mild bleeding diathesis. The diagnosis depends on demonstration of decreased activity of one of the clotting factors. Due to the rarity of each of the individual factor deficiencies, purified factor concentrates are not as readily available as they are for haemophilia A and B. Treatment of rare clotting factor deficiencies consists of the most purified blood product available that contains the missing factor. Depending on which factor is deficient, either purified concentrates, prothrombin complex concentrates, cryoprecipitate, or fresh frozen plasma can be used. In addition, recombinant factor VIIa is available for treating factor VII deficient patients.     

Bleeding symptoms in 27 Iranian patients with the combined deficiency of factor V and factor VIII

Inherited deficiency of factors V and VIII is the most frequent combined coagulation defect. The cases reported so far are mostly single cases or small series from different centres, making it difficult to evaluate the overall pattern of clinical manifestations of the combined defect. We examined at a single institution 27 Iranian patients. Mucocutaneous and post-surgical bleeding were the most frequent clinical manifestations. The presence of two defects did not make the severity of bleeding greater than that expected in patients with single coagulation defects of similar degrees.     

Treatment of factor XI inhibitor using recombinant activated factor VIIa

A 30-year-old female with severe factor XI deficiency of 0-2% acquired factor XI inhibitor following many infusions for fresh frozen plasma (FFP) for surgical procedures starting at 4 years of age. Seven months before this inhibitor was diagnosed, surgery was complicated by prolonged bleeding resistant to FFP, requiring epsilon aminocaproic acid (EACA) and surgical packing. The inhibitor was measured at 2.2 Bethesda units, 7 months since the last FFP. The inhibitor was confirmed as specific anti-XI and anti-XIa binding by patient's IgG to immobilized factor XI and factor XIa from whole plasma and purified IgG. For repair of a painful anterior cruciate ligament (ACL) defect she was given recombinant factor VIIa (rVIIa) at 90 mug kg(-1), starting one-half hour preoperatively and continued every 2 h for 8 h when haemostasis was complete. Thereafter the rVIIa was given every 3 h for two doses, and then every 4 h for four doses at which time she was discharged on EACA which was continued for 6 days. There was excellent haemostasis during and following the surgery. There was no evidence of consumptive coagulopathy, with no change in the fibrinogen, platelet count, or D-D dimer; and no increase of platelet factor 4, beta-thromboglobulin, or prothrombin fragment F 1.2. The thrombin-antithrombin complex increased over baseline after 24 h. There was no postoperative deep vein thrombosis or pulmonary embolus. In this patient with a factor XI inhibitor, the recombinant factor VIIa was effective and safe, ensuring adequate haemostasis with no thrombotic complications. This product which was designed for patients with inhibitors to factor VIII or factor IX, and factor VII deficiency, has now been given successfully to four patients with factor XI inhibitors.     

Activation of a recombinant human factor VII structural analogue alters its affinity of binding to tissue factor

A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC₅₀) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor. © 1996 Wiley-Liss, Inc.     

Free factor XIII activation peptide (fAP‐FXIII) is a regulator of factor XIII activity via factor XIII‐B

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII‐A₂ (rFXIII‐A₂) free FXIII activation peptide (fAP‐FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII‐B₂ (rFXIII‐B₂) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII‐A₂B₂). FXIII‐A₂B₂ reconstituted from rFXIII‐A₂ and rFXIII‐B₂ showed a similar effect on AUC, TTP and CP in the presence of fAP‐FXII as observed for plasma FXIII‐A₂B₂, indicating a role for FXIII‐B in this observation. An effect of fAP‐FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP‐FXIII did not interfere with the FXIIIa activity development of thrombin‐cleaved rFXIII‐A₂ (rFXIII‐A₂′) also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII‐A₂′B₂ was inhibited in a concentration‐dependent manner by fAP‐FXIII, indicating that an interaction between AP‐FXIII and FXIII‐B₂ contributes to the overall stability of FXIII‐A₂′B₂. In addition to its well‐known role, FXIII‐B also contributes to FXIII‐A₂B₂ stability or dissociation depending on fAP‐FXIII and calcium concentrations.     

Congenital factor XIII deficiency associated with von Willebrand disease

A boy with umbilical bleeding and severe hemorrhages after minor trauma, without family bleeding history, was studied. Coagulation tests showed abnormalities in FXIII subunits and FVIII/vWF complex. Both parents presented results compatible with a heterozygote state for FXIII deficiency and the father had abnormalities of FVIII/vWF. The propositus was diagnosed as congenital FXIII deficiency associated with vWD. No severe hemorrhagic complication was observed after a prophylactic regimen with cryoprecipitates.     

Potency and mass of factor VIII in FVIII products

Summary.  Several factor (F) VIII products of different origin and structure are being used for haemophilia A treatment worldwide. The assessment of FVIII concentration in these products is done using activity assays, which are dependent upon the assay and its modifications. To evaluate FVIII products for potency and for FVIII concentration and specific activity, three activity-based assays [activated partial thromboplastin time (APTT), intrinsic FXase and synthetic coagulation proteome] and two immunoassays (ELISA and western blotting) were used in this study with albumin-free full-length recombinant (r) FVIII as a standard. In all activity assays, products A and B (both contain full-length rFVIII) at 1 U mL⁻¹ showed potency similar to that of the 0.7 n m (1 U mL⁻¹) rFVIII standard. Product E (contains truncated rFVIII) was less potent in the APTT (83% of standard) and product C (contains plasma FVIII) was less potent in FXase assays (66%). The ELISA immunoassay revealed that the specific activity of FVIII proteins in products A–C and E varied over a wide range (3900–13 200 U mg⁻¹) and was higher for most lots when compared with the standard (5000 U mg⁻¹), whereas the specific activity of product D (contains plasma FVIII) was lower than expected (3200–4800 U mg⁻¹). (i) FVIII potency estimated in different assays gives dissimilar results; (ii) the specific activity of FVIII in various FVIII products is different and inconsistent. Thus, the administration of an equal FVIII potency in units means the administration of different amounts of FVIII protein, which may partly explain apparent discrepancies in product performance.     

Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function

The functional defect caused by substitution of Arg527 (--> Trp) and Arg531 (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527.