KINETICS OF BABOON CIRCULATING ENDOTHELIAL PROGENITOR CELLS MOBILIZED BY G-CSF

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在此期间，利用SNPRC基金支持了一项试点研究。该研究计划开发狒狒模型，以研究狒狒中循环内皮祖细胞（CEPC）的动员和成熟动力学。这项研究的结果将作为美国国立卫生研究院资助申请的试点数据，旨在确定可能影响细胞反应个体差异的因素，特别是遗传因素，这些因素反过来控制对CVD发展进展和恢复的易感性。 我们利用这笔资金完成了以下工作。 1. 鉴定与狒狒细胞表面抗原反应的小鼠单克隆抗体。由于大多数用于鉴定CEPC的单克隆抗体最初是针对相应的人抗原产生的，因此必须检测这些抗体与狒狒细胞的交叉反应性。我们用流式细胞术方法筛选了一组小鼠单克隆抗体，并确定了一个对狒狒样品具有免疫特异性的克隆。我们已经使用这种抗体来表征CEPC，并通过显示内皮谱系特征的EPCs的功能分析来验证阳性交叉反应性结果。 2. 研究G-CSF动员后循环CEPC动力学。粒细胞集落刺激因子（G-CSF）给药是CEPC动员的公认刺激剂。我们在不同时间对CEPC数量进行了定量，以记录G-CSF注射后5只狒狒中CEPC动员的时间和剂量依赖性变化。我们的结果表明，动员CEPC在外周血以及骨髓样品中的最大时间点和幅度。 3. 我们从两组中手工挑选代表性菌落，并通过Cytofuge（StatSpin）离心到载玻片上。用各种抗体进行免疫染色。这些免疫细胞化学分析的图像显示，G-CSF处理样品中占优势的集落（15个集落中的11个）高度表达广泛的造血相关蛋白（CD 45、CD 14和CD 235 a）以及内皮细胞标志物（如CD 31和CD 146），但不表达vWF。在该组的15个集落中有4个集落具有抗vWF染色阳性。然而，从动脉结扎组收获的集落（15个集落中的14个）显示出在这些样品中对CD 31、CD 146和vWF、中度CD 45阳性细胞以及阴性CD 14和CD 235 a的强烈染色。 最终，我们希望利用500只遗传特征的纯种狒狒来表征有助于基线和可动员CEPC的基因。为了达到这一目标，我们需要建立一个狒狒模型来研究CEPC在外周血中的动力学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A pilot study was supported by using SNPRC fund during this period. The study was planned to develop a baboon model to investigate circulating endothelial progenitor cell (CEPC) mobilization and maturation kinetics in baboons. The results of this study will serve as pilot data for an NIH grant application aimed at identifying the factors, particularly the genetic factors, which may influence individual variation in cellular responses which in turn control susceptibility to CVD development progression and recovery. We have completed the following works using this fund. 1. Identify mouse monoclonal antibodies reacting with baboon cell surface antigens. Because most of monoclonal antibodies to identify the CEPC are originally produced against the corresponding human antigens, these antibodies must be tested for cross-reactivity with baboon cells. We have screened a panel of mouse monoclonal antibodies by flow cytometry method; and determined a clone that has immunological specificity to the baboon sample. We have used this antibody to characterize CEPC and validated positive cross-reactivity results by functional analyses of EPCs that show features of endothelial lineage. 2. Investigate circulating CEPC kinetics after G-CSF mobilization. Granulocyte colony-stimulating factor (G-CSF) administration is a well-established stimulator for CEPC mobilization. We have quantified CEPC numbers at different times to document the time- and dose-dependent changes of CEPC mobilization in five baboons after G-CSF injection. Our results have indicated the maximal time point and magnitude of mobilized CEPC in peripheral blood as well as bone marrow samples. 3. We hand-picked representative colonies from both groups and spun down by Cytofuge (StatSpin) onto slides. Immunostaining with various antibodies were carried out. Images from these Immunocytochemical analysis revealed that the colonies dominant (11 out of 15 colonies) in G-CSF treatment samples expressed highly a wide range of hematopoietic related proteins (CD45, CD14 and CD235a), as well as endothelial cell markers like CD31 and CD146, but not vWF. 4 out of 15 colonies had positive anti-vWF staining in this group. However, colonies harvested from arterial ligation group (14 out of 15 colonies) have shown to stain strongly for CD31, CD146, and vWF, moderate CD45 positive cells, and negative CD14 and CD235a in these samples. Ultimately, we would like to utilize 500 genetically characterized pedigreed baboons to characterize genes that contribute to baseline and mobilizable CEPCs. To reach this goal, we will need to establish a baboon model to study CEPC kinetics in peripheral blood.