雷帕霉素联合CD34抗体复合支架快速捕获外周血中内皮祖细胞

背景：临床用于治疗血管狭窄疾病的药物洗脱支架和单纯内皮修复型支架存在内皮化延迟和植入后再狭窄的问题。雷帕霉素联合CD34抗体复合支架可协同抵消抗增殖药物的内皮化延迟和内膜的过度增生,目前尚处于实验研究阶段。 目的：观察雷帕霉素联合CD34抗体复合支架捕获内皮祖细胞能力及其所捕获内皮祖细胞的分化特征。 方法：通过扫描电镜及间接免疫荧光观察雷帕霉素联合CD34抗体复合支架体外捕获外周血内皮祖细胞形态及其分化特征。通过荧光显微镜观察雷帕霉素联合CD34抗体复合支架植入兔耳动脉后捕获内皮祖细胞情况及支架片段的内皮化程度。 结果与结论：扫描电镜观察到CD34抗体涂层支架可捕获直径6-8 μm的纺锤状细胞,24 h时细胞变得充盈饱满。所捕获细胞具有内皮祖细胞的外形特征。间接免疫荧光观察CD34抗体支架表面可见大量血管内皮生长因子受体2阳性细胞黏附的红色荧光斑点。免疫荧光观察到CD34抗体涂层支架植入兔耳动脉24 h大部分被血管内皮细胞所覆盖,48 h达到完全覆盖,未见细胞异常聚集。结果表明雷帕霉素联合CD34抗体复合支架能特异性快速捕获外周血液中的血管内皮祖细胞,植入体内48 h即可完成血管内皮细胞覆盖,实现了支架快速内皮化,可促进内皮细胞修复。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

雷帕霉素联合CD133抗体支架预防血管再狭窄的有效性和安全性北大核心

背景:药物洗脱支架和单纯内皮修复型支架在治疗血管狭窄疾病时可见内皮化延迟以及植入后再狭窄的问题。作者既往的体外研究显示雷帕霉素联合CD133抗体支架可协同抵消抗增殖药物的内皮化延迟和内膜的过度增生。目的:在小型猪冠状动脉损伤模型中,分析雷帕霉素联合CD133抗体复合支架预防血管再狭窄的效果。方法:将冠状动脉损伤小型猪模型随机分为雷帕霉素组、CD133抗体组以及雷帕霉素/CD133抗体组,分别在损伤冠状动脉置入雷帕霉素支架、CD133抗体支架和雷帕霉素联合CD133抗体支架。动物实验于2019-03-15经沈阳医学院附属中心医院实验动物伦理委员会审批,审批号20190017。结果与结论:(1)3组支架植入后14 d和1个月时,内皮化程度存在差异,其中雷帕霉素组支架内皮覆盖程度低于CD133抗体组及雷帕霉素/CD133抗体组。(2)置入后3和6个月,雷帕霉素组和雷帕霉素联合CD133抗体组管腔狭窄率较低,但雷帕霉素支架周围组织存在明显的炎症反应,且CD133抗体支架可引起明显内膜增生及管腔狭窄。(3)提示雷帕霉素联合CD133抗体支架可在体内实现早期内皮化,促进内皮细胞修复,并在置入后降低周围组织炎症反应,且其6个月内抗增殖效果与雷帕霉素支架接近。     

造血干细胞的两种形式:CD34~+和CD34~-

造血干细胞 (HSC)的表面标记是对HSC进行深入研究的关键 ,CD34抗原是一公认的指标 ,但近来大量的资料表明尚存在CD34- 的干细胞 ,两种形式HSC共存还是CD34细胞源于CD34- 细胞引起了很大争议 ,本文对此作一综述。     

091 造血干细胞的两种形式：CD34+和CD34-

造血干细胞(HSC)的表面标记是对HSC进行深入研究的关键，CD34抗原是一公认的指标，但近来大量的资料表明尚存在CD34-的干细胞，两种形式HSC共存还是CD34细胞源于CD34-细胞引起了很大争议，本文对此作一综述。     

CD34抗原的生物学特性及其临床应用

CD34抗原是一种高度糖基化Ⅰ型跨膜蛋白,它选择性的表达于人类造血干细胞(HSC),祖细 胞(PC)和血管内皮细胞(EC)表面。CD34+细胞并非通常所指的原始细胞,而是存在于淋巴细胞群内的造血 干细胞,随干细胞的分化成熟而逐渐消失。目前,已经证实了最原始的造血干细胞是CD34-细胞。本文对 CD34抗原、CD34+细胞和CD34-细胞的生物学特性以及临床应用进行了综述。     

体外扩增中CD34+细胞CD38与HLA-DR抗原表达的变化

CD34+ CD38- HLA DR- 是目前公认的原始干 祖细胞 (HSPC)的表面抗原标记 ,本研究的目的是观察脐带血(UCB)CD34+ 细胞表面CD38和HLA DR抗原在扩增过程中的变化 ,以期探讨HSPC的免疫表型与其功能的关系。收集足月妊娠的UCB标本 11份 ,分离单个核细胞 ,再按试剂盒说明纯化CD34+ 细胞 (MiltenyibiotchInc .Auburn ,CA)。将富集的UCBCD34+ 细胞接种于已建立的无血清无基质悬浮扩增体系 ,培养 2周 ,分别于 7d、10d和 14d收获部分扩增细胞 ,再次纯化CD34+ 细胞。进行流式细胞表型分析。应用直标单抗双色荧光流式细胞术检测CD34…     

CD34基因与血管内皮细胞

D34基因有 8个外显子 ,其产物CD34抗原为 1 0 5～ 1 2 0kD的跨膜细胞表面糖蛋白 ,可选择性地在造血干 /祖细胞中表达 ,而且也在血管内皮细胞以及其它组织中表达。它具有粘附、加速血管前内皮细胞聚集形成血管、调控造血细胞的增生和分化的功能。可用来测量肿瘤内及缺血组织微血管的密度和面积检测早期的新生微血管形成及分离CD34 造血干 /祖细胞等。     

脐血CD34+造血干/祖细胞基因表达图谱的研究

目的：通过脐血CD34^＋造血干／祖细胞的基因表达分析，理解造血干／祖细胞生物学特性。方法：利用MiniMACS免疫磁珠法从脐血细胞中分离CD34^＋造血干／祖细胞，提取总RNA，用SMART-PCR技术从微量RNA中扩增产生足够量的cDNA用于高密度点阵膜分析检测CD34^＋造血干／祖细胞表达的基因。结果：在所检测的588个基因中，发现63个基因具有显著的表达水平，其中18个基因强表达。这些基因主要涉及造血干细胞增殖、分化、应激响应、凋亡、转录调节以及细胞周期等。结论：对理解脐血干／祖细胞生物学性质以及指导造血干细胞体外培养提供了分子生物学基础。     

骨髓CD34+造血干/祖细胞的分离、纯化及鉴定

造血干细胞(HSC)工程是利用HSC的生物学特征．通过细胞工程的技术,在体外模拟或部分模拟体内的造血过程,对分离纯化的造血干/祖细胞(HSC/HPC)进行体外扩增、定向诱导分化、功能激活与调控、目的基因转染等,最终将造血细胞治疗更广泛有效地应用于干细胞移植、生物免疫治疗、造血支持治疗和基因治     

CD34抗原在人体组织中的表达及临床意义

198 4年Ｃｉｖｉｎ等首先发现ＣＤ34抗原 ,早期的研究表明 ,该抗原在各系造血干 /祖细胞膜上的表达水平最高 ,随着细胞分化程度增高其表达水平急剧下降 ,最后消失。ＣＤ34可以协助骨髓的ＣＤ34阳性造血干 /祖细胞粘附于骨髓基质 ,还可抑制造血细胞分化 ,促进造血祖细胞形成 ,参与细胞内信号传导等。许多造血系统疾病如急性白血病、骨髓异常增生综合征、再生障碍性贫血等都伴有ＣＤ34的异常表达 ,因此分析这些异常之处可以帮助诊断疾病并估计预后。在临床移植中 ,ＣＤ34可用于分离纯化待移植的骨髓中的ＣＤ34阳性造血干 /祖细胞 ,检测造血干…     

Expression of CD34 in endothelial cells,hematopoietic progenitors and nervous cells in fetal and adult mouse tissues

The human cell surface molecule CD34 is selectively expressed on uncommitted and committed hematopoietic progenitor cells and on vascular endothelial cells. It has been suggested that CD34 regulates early events in blood cell migration and differentiation, possibly as a cell adhesion molecule. To characterize the patterns of expression of CD34 in the mouse embryo and in the adult, as well as to dissect the function of different portions of the extracellular domain of this molecule, we have generated the first monoclonal antibodies (mAb) specific for mouse CD34. The epitope(s) recognized by these mAb are not carbohydrate moieties, and are comprised either within the immunoglobulin-like domain or within a portion of the mucin domain, containing approximately half of the predicted O- and N-linked carbohydrate attachment sites. The specificity of the antibodies was established by ELISA and Western blotting. Western analysis revealed that these mAb recognize a protein of approximately 110 kDa in PA6 stromal cell lysates, which can be specifically blocked by the recombinant CD34 protein. To establish the reactivity of these mAb on different cell lineages, a panel of cell lines was stained. This analysis showed strong reactivities with 3T3 fibroblasts, stromal cell lines from fetal liver and with the endothelial cell line D10. Bone marrow hematopoietic progenitors were also stained by these mAb. Immunostaining of frozen sections from embryonic and adult tissues revealed a strong reactivity against vascular endothelial cells at different stages of development, including sinusoidal cells in the fetal liver, yolk sac, and in the fetal bone marrow, endothelial cells from adult lung and kidney, and neural cells, including those of the neural tube of midgestation embryos and neuronal bodies in adult brain.     

Increased number of CD34+cells in nasal mucosa of allergic rhinitis patients: inhibition by a local corticosteroid

Background Eosinophils develop from CD34⁺ haematopoietic progenitor cells. Allergen exposure in susceptible individuals is known to induce a local eosinophilic inflammation, but the effect on progenitor cells is much less understood. Objective We aimed to evaluate how allergen exposure affects the number of tissue CD34⁺ cells and CD34⁺ eosinophils in allergic rhinitis (AR) patients and whether any such effect is influenced by local corticosteroid treatment. Also, we evaluated changes in the number of CXC receptor 4‐positive cells (CXCR4⁺), since the CXCR4 ligand (stromal cell‐derived factor‐1 (SDF‐1)) is a potent chemoattractant for haematopoietic progenitors. Methods In a double‐blind, randomized study, pollen‐sensitized AR patients were treated with a nasal corticosteroid fluticasone propionate (FP, 200 μg/day) or placebo throughout the pollen season. Nasal biopsies were taken before and during the season. CD34 and CXCR4 were stained using immunohistochemistry. Results The pollen season significantly increased the number of CD34⁺ cells, CD34⁺/CXCR4⁺ cells and CD34⁺ eosinophils in placebo‐treated patients, but not in FP‐treated patients. The mean pollen season‐induced increase in CD34⁺ cells, CD34⁺/CXCR4⁺ cells and CD34⁺ eosinophils in FP‐treated patients was lower compared with placebo‐treated patients. Conclusion A pollen season increases the number of CD34⁺ cells in nasal tissue accompanied by an increase in the number of CD34⁺/CXCR4⁺ haematopoietic progenitors and also the number of CD34⁺ eosinophils in subjects with AR. Treatment with a local corticosteroid provides protection against this pollen‐induced increase in tissue CD34⁺ cells and CD34⁺ eosinophils possibly via inhibition of allergen‐induced CXCR4‐mediated recruitment of CD34⁺ haematopoietic progenitors into airways and their further differentiation into eosinophils within the tissue.     

Certain anti-CD34 monoclonal antibodies induce homotypic adhesion of leukemic cell lines in a CD18-dependent and a CD18-independent way

CD34 is a highly glycosylated type I membrane protein expressed by early hematopoietic progenitor cells as well as by endothelial cells and a subset of bone marrow stromal cells. CD34 is thought to play an important role during early hematopoiesis, although its function is unknown. We demonstrate that triggering of CD34 results in a rapid and vigorous homotypic adhesion in CD34⁺ cell lines, thereby providing evidence for a cell-cell adhesion function of CD34. The cellular adhesion event, induced by only two anti-CD34 mAb, (Immu-133 and QBend-10) was dependent on metabolic energy, an intact cytoskeleton and the presence of divalent cations. Analysis of antibody inhibition experiments indicated that the aggregation process partially involved the CD18 molecule.     

TLR agonists induce the differentiation of human bone marrow CD34+ progenitors into CD11c+ CD80/86+ DC capable of inducing a Th1-type response

We recently reported that human bone marrow hematopoietic CD34(+) progenitors express functional Toll-like receptors (TLR) and can differentiate into myeloid cells just by stimulation with resiquimod (R848), a specific agonist for TLR7/8. However, the mechanisms by which R848 induces cell differentiation, the effects of other TLR agonists and the functionality of the differentiated cells are not known. Comparable to R848, loxoribine (a TLR7 agonist) and Pam(3)CSK(4) (a TLR2 agonist) induced cytokine production and cell differentiation along the myeloid lineage. R848 and loxoribine were more effective than Pam(3)CSK(4) at inducing the lineage-negative (CD11c(+) CD14(-)) dendritic cells (DC), whereas Pam(3)CSK(4) was more effective at inducing CD11c(+) CD14(+) monocytes. Both cell subsets expressed CD80/CD86 and HLA-DR molecules; however, they showed differential expression of CD1a, CD1b, CD1c, CD11b, CD206 and CD207 markers when compared with each other. Cell differentiation into DC was significantly inhibited by an anti-TNF-alpha nonoclonal antibody. The CD11c(+) CD14(-) subset was isolated and shown to be more potent in stimulating an alloreaction than the CD11c(+) CD14(+) subset. Collectively, these data highlight the differential effects of TLR agonists on human bone marow CD34(+) progenitor cells and provide a new opportunity for generating functional DC that would be useful in cancer vaccination.     

CD34~+细胞免疫亲合柱的研制

本文建立了利用免疫亲合柱纯化CD34+细胞的方法。将亲合素交联于聚丙烯酰胺凝胶Bio Gel上 ,装于塑料柱制成免疫亲合柱 ;将脐血单个核细胞与生物素化抗CD34单克隆抗体作用后 ,加到该免疫亲合柱上进行纯化 ,得到纯化的CD34+细胞。纯化后CD34+细胞纯度达 48 8%± 18 2 % ,细胞回收率为 5 5 6 %± 14 5 % ,台盼蓝染色法显示活细胞达 98%。利用该方法纯化CD34+细胞具有纯度高、回收率高、细胞活性好等优点 ,是一种方便有效的细胞纯化方法。     

人脐带血CD34+细胞体外诱导分化为嗜酸粒细胞的研究

目的： 探讨人脐血CD34+细胞在体外培养条件下向嗜酸粒细胞分化的规律。 方法： 从人脐血中分离纯化CD34+细胞，分成阴性对照组、IL-5组和变应性鼻炎血清组。各组分别于第1 d、2 d和第7 d、14 d、28 d行CD34+流式细胞仪分析、HE染色和电镜观察。 结果： IL-5组和 血清组CD34+细胞比例在第2 d时就明显减少，至 28 d 时已剩余较低比例，其中加血清组明显低于IL-5组。在体外培养条件下加入IL-5和病人血清后，在第2 d即可见典型的嗜酸粒细胞结构，至28 d时已大部为嗜酸粒细胞，其中加病人血清组明显高于IL-5组。 结论： 人脐血CD34+细胞在体外培养条件下经过IL-5和病人血清刺激可早期分化为嗜酸粒细胞。可作为有效的相关研究模型。     

Chemoattraction of femoral CD34+ progenitor cells by tumor-derived vascular endothelial cell growth factor

Patients and animals with GM-CSF-producing tumors have an increased number of mobilized CD34⁺ progenitor cells within their peripheral blood and tumor tissue. These CD34⁺ cells are inhibitory to the activity of intratumoral T-cells. The present study used the murine Lewis lung carcinoma (LLC) model to assess mechanisms that could lead to the accumulation of CD34⁺ cells within the tumor tissue. In vitro analyses showed that LLC tumor explants released chemoattractants for normal femoral CD34⁺ cells. The LLC tumor cells contributed to the production of this activity since CD34⁺ cell chemoattractants were also released by cultured LLC cells. Antibody neutralization studies showed that most, although not all, of the chemotactic activity that was produced by LLC cells could be attributed to VEGF. In vivo studies with fluorescent-tagged CD34⁺ cells showed their accumulation within the tumor tissue, but not within the lungs, spleen or bone marrow, suggesting a selective accumulation within the tumor. Whether or not VEGF could chemoattract CD34⁺ cells in vivo was measured with a VEGF-containing Matrigel plug assay. Infusion of fluorescent-tagged CD34⁺ cells into mice after the plugs became vascularized revealed the accumulation of fluorescent-tagged cells within the plugs. However, these CD34⁺ cells failed to accumulate within the VEGF-containing Matrigel plugs when they were infused together with neutralizing anti-VEGF antibody. Through a combination of in vitro and in vivo analyses, the LLC cells were shown to be capable of chemoattracting CD34⁺ cells, with most of the tumor-derived chemotactic activity being due to tumor release of VEGF. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Murine Stromal Cell Line Promotes the Expansion of CD34hlgh+-Primitive Progenitor Cells Isolated from Human Umbilical Cord Blood in Combination with Human Cytokines

Abstract

The in vitro expansion of CD34^? cells is important for clinical applications such as transplantation and gene therapy with CD34⁺ cells isolated from human umbilical cord blood. In the present study, we developed a xenogenic coculture system involving HUCB-CD34⁺ cells and a murine stromal cell line, HESS-5 cells, in the presence of recombinant human (rh) cytokines. We examined the effects of combinations of cytokines, such as rh-IL-3, rh-SCF, rh-granulocyte colony-stimulating factor (G-CSF), rh-granulocyte-macrophage-CSF and h-erythropoietin (EPO), on the expansion of CD34^hlgh- cells and colony-forming progenitor cells (CFCs). The proliferation of CD34^high+ cells and CFCs was dramatically promoted on coculture with HESS-5 cells, and the expansion ratio of the CD34^hlgh+ cells showed good correlation with that of high-proliferative potential colony-forming cells (HPP-CFCs). The most potent combination of cytokines in this xenogenic coculture system for the expansion of CD34^high+ cells and HPP-CFCs was rh-IL-3 and rh-SCF. The proliferation of CD34^high+ cells was supported in the presence of HESS-5 cells with direct cell contact, but not observed in the indirect coculture involving a microporous membrane. Furthermore, we developed a unique coculture method, designated as the bilayer coculture method, involving CD34⁺ cells and HESS-5 cells using a microporous membrane. This expansion system will be applicable to the expansion of the primitive progenitor cells of HUCB-CD34^? cells and is worthy of consideration for the clinical application of HUCB-CD34^? cells.