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首页> 外文学位 >Examining phosphatidylethanolamine externalization on cells and microparticles in hemolysis and hemolytic anemia.
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Examining phosphatidylethanolamine externalization on cells and microparticles in hemolysis and hemolytic anemia.

机译：检查溶血和溶血性贫血中细胞和微粒上磷脂酰乙醇胺的外在化作用。

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摘要

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are aminophospholipids located primarily on the inside face of healthy human cell membranes. The overarching hypothesis of this proposal is that the aminophospholipid probe, duramycin, is a useful biological tool for examining PE exposure in hemolysis seen in diverse biological systems and models.;Tis dissertation demonstrated the overlap between calcium-phosphate precipitates and MPs in size when examined by flow cytometry. The microprecipitates bound non-specifically to fluorescently-labeled antibodies, further mimicking membrane vesicles. A solution to this problem was proffered by the addition of a calcium chelator. Also, contrary to the common practice of using the slowest possible cytometer flow rate while examining MPs, increasing the flow cytometer flow rate was shown to enhance detection of membrane vesicles.;We investigated the utility of duramycin, a PE-binding peptide, for the direct labeling and detection of MPs. Duramycin was also compared to other aminophospholipid probes in the labeling of RBCs, RBC-derived MPs, and MPs from numerous cellular sources. Duramycin was shown to be equivalent, if not superior in the case of cancer MPs, to labeling MPs at optimal conditions with other aminophospholipid probes.;Also detailed were the effects of dilution and centrifugation on MP concentrations in the resulting supernatant, suggesting that RBC MPs are much more abundant than previously thought. A novel approach to gently isolating MPs from their parent RBCs was used to characterize large vesicles (greater than 3im in diameter), suggesting there exists a size continuum between RBC fragments and RBC MPs in hemolysis. This discovery challenges the current practice of excluding RBC vesicles from analysis based on their size being larger than 1mum.;This dissertation also demonstrated the exposure of PE on RBC units over the duration of their storage. Previous studies using the calcium-dependent aminophospholipid probe, annexin V, demonstrated less than 1% to 6% of donor unit RBCs have deranged membrane bilayers at the time of expiration. Duramycin use demonstrated that nearly 18% of RBCs exposed appreciable PE at the time of expiration. This was roughly the same percentage of RBCs that are rapidly cleared after transfusion of blood at the end of its shelf-life, providing new insights into the possible mechanism of RBC clearance and transfusion reactions.;Also demonstrated, using duramycin to probe PE at the cellular level, was the damage done to RBCs following routine laboratory manipulations, including washing and centrifugation/resuspension. Washing RBCs from donor units near their expiration date resulted in significantly elevated PE exposure. Centrifugation alone damaged both aged and fresh RBCs sufficient to cause cell-free hemoglobin release. The results from this may have implication on the standard operating procedures in blood banks world-wide.;Lastly, radiolabeled duramycin was used to examine PE exposure on the tissue level in murine models of hemolytic anemia. Mice expressing human sickle hemoglobin were injected, and the areas of duramycin uptake examined using dual 3D imaging technologies: SPECT, to determine the radiotracer distribution, and microCT to co-localize anatomical locations. This provided a novel approach to objectively identifying ischemic injury in sickle cell disease. Additionally, mice exhibiting severe hereditary spherocytosis and spontaneous thrombosis were also examined as an alternative (nonsickling) hemolytic anemia model. Duramycin localized to areas of known thrombi, highlighting radiolabeled duramycin as a means to assess upstream occlusions (thrombi, as seen in the spherocytosis mice) or downstream ischemic damage (as demonstrated in the sickle cell mice). (Abstract shortened by UMI.).
机译：磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE）是主要位于健康人细胞膜内表面的氨基磷脂。该建议的主要假设是，氨基磷脂探针杜拉霉素是一种有用的生物学工具，可用于检查在各种生物学系统和模型中溶血中的PE暴露。该论文证明了在检查时磷酸钙沉淀物和MP大小之间存在重叠通过流式细胞仪。微沉淀物非特异性地与荧光标记的抗体结合，进一步模仿膜囊泡。通过添加钙螯合剂可以解决该问题。同样，与在检查MP时使用尽可能慢的流式细胞仪流速的惯常做法相反，增加流式细胞仪流速被证明可以增强膜囊泡的检测。我们研究了PE结合肽杜拉霉素在细胞膜中的应用。直接标记和检测MP。在标记RBC，RBC衍生的MP和来自许多细胞来源的MP方面，也将杜拉霉素与其他氨基磷脂探针进行了比较。已证明杜拉霉素在最佳条件下与其他氨基磷脂探针在最佳条件下标记MP的作用相同，即使在癌症MP的情况下也不如。;还详细说明了稀释和离心对所得上清液中MP浓度的影响，表明RBC MP比以前想象的要丰富得多。一种从母RBC中轻柔分离MP的新颖方法用于表征大囊泡（直径大于3im），表明溶血过程中RBC片段和RBC MP之间存在大小连续体。这一发现对目前基于大小大于1μm的分析将RBC囊泡排除在研究范围之外的做法提出了挑战。本论文还证明了PE在RBC储存期间会暴露于PE。以前使用钙依赖性氨基磷脂探针Annexin V进行的研究表明，不到1％至6％的供体单位RBC在到期时已使双层膜变形。使用杜拉霉素表明，在过期时，将近18％的RBC暴露出明显的PE。这与在保质期结束后输血后迅速清除的RBC的百分比大致相同，这为了解RBC清除和输血反应的可能机制提供了新的见解。细胞水平是常规实验室操作（包括洗涤和离心/重悬）对红细胞造成的损害。在捐赠者的有效期限临近时从供体单位中冲洗RBC会显着增加PE暴露。单独的离心作用足以破坏陈旧的RBC和新鲜的RBC，足以引起无细胞血红蛋白的释放。由此产生的结果可能对全世界血库的标准操作程序都有影响。最后，放射性标记的杜拉霉素被用于在溶血性贫血的小鼠模型中检测组织水平的PE暴露。注射了表达人类镰刀血红蛋白的小鼠，并使用双重3D成像技术（SPECT，以确定放射性示踪剂的分布）和microCT共同定位解剖位置，检查了杜拉霉素的摄取区域。这提供了一种新颖的方法来客观地鉴定镰状细胞疾病中的缺血性损伤。此外，还对表现出严重的遗传性球血细胞增多和自发性血栓形成的小鼠进行了检查，以作为另一种（非溶血性）溶血性贫血模型。 Duramycin定位于已知血栓形成区域，突出显示了放射性标记的Duramycin作为评估上游阻塞（血栓形成小鼠中见血栓）或下游缺血性损伤（如镰状细胞小鼠中所示）的方法。 （摘要由UMI缩短。）。

著录项

  • 作者

    Larson, Michael Craig.;

  • 作者单位

    The Medical College of Wisconsin.;

  • 授予单位 The Medical College of Wisconsin.;
  • 学科 Biophysics General.;Biology Molecular.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 357 p.
  • 总页数 357
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 高分子化学（高聚物）;
  • 关键词

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