基质细胞衍生因子1α预处理内皮祖细胞移植治疗糖尿病下肢缺血

背景：基质细胞衍生因子1α被证明可以促进内皮祖细胞归巢。 目的：观察基质细胞衍生因子1α预处理内皮祖细胞移植治疗糖尿病模型鼠下肢缺血的疗效。 方法：取雄性Wistar鼠30只,25只成功建立糖尿病模型大鼠,随机数字表法分为3组,对照组注入等量生理盐水,共培养组注射与基质细胞衍生因子1α共培养的内皮祖细胞,内皮祖细胞组注入未进行共培养的内皮祖细胞。 结果与结论：MTT检测结果显示,基质细胞衍生因子1α能显著增加内皮祖细胞的增殖能力(P < 0.01)。移植后第28天动脉造影显示共培养组缺血侧下肢动脉显影血管数多于内皮祖细胞组和对照组(P < 0.05)。提示采用基质细胞衍生因子1α预处理内皮祖细胞,有利于使糖尿病大鼠缺血下肢血供改善,主要来自于新生血管形成和/或原有细小血管的代偿性增粗。     

应用DiI标记犬脐血内皮祖细胞的实验研究

目的应用DiI标记犬脐血内皮祖细胞,为内皮祖细胞的研究提供细胞标记方法。方法取犬的脐带血,用添加有EGM-2MV的EBM-2在纤连蛋白包被的六孔板上培养。检测细胞表面抗CD31、CD34、vWF及FLK-1的表达。用DiI标记内皮祖细胞,用荧光显微镜观察内皮祖细胞的荧光情况。结果4~6d有细胞贴壁生长,7~14d可见贴壁细胞呈克隆型生长,呈典型的“铺路石”样外观。传代后,细胞生长良好。细胞表面抗体检测显示为:CD31(+),CD34(+),vW因子(+),FLK-1(+)。荧光显微镜下见内皮祖细胞的细胞浆内有红色荧光。阳性率(68.8±1.6)%。结论应用DiI标记犬脐血内皮祖细胞,方法简单,染色效果好,是内皮祖细胞示踪的好方法。     

人脐血内皮祖细胞的分离、培养及鉴定

目的探索人脐静脉血内皮祖细胞的分离培养,为内皮祖细胞的临床应用提供实验方法。方法选择脐静脉血,应用密度梯度离心法,获取单个核细胞,接种于预先包埋了人纤维连接蛋白的培养板,用加入生长因子VEGF165和bFGF的内皮细胞专用培养基EGM-2MV培养细胞,3d后,洗掉非贴壁细胞,换培养液继续培养至7d,收集贴壁细胞进行细胞分析。激光共聚焦显微镜进行细胞功能学鉴定,流式细胞术测定祖细胞和内皮细胞系标志。MTT比色法检测细胞的生长状态。结果经过梯度密度离心和贴壁法选择的细胞能特异性吸附FITC标记的荆豆凝集素并内吞DiI-acLDL,祖细胞标志CD133及内皮细胞特异性抗原CD34、KDR检测,其阳性率分别为（27．05±2．94）％、（16．37±2．69）％和（56．67±7．29）％;体外培养的内皮祖细胞具有良好的细胞增殖活性。结论人脐静脉血中可以分离培养内皮祖细胞,为内皮祖细胞的进一步研究及临床应用奠定了基础。     

血管内皮祖细胞与糖尿病血管病变

内皮祖细胞(EPC)是能直接分化成血管内皮的前体细胞,参与胚胎期及成体的血管生长。糖尿病内皮祖细胞的数量及功能均有改变,使血管阻塞事件具有更高的发病率和致死率。已有实验证实EPC存在并参与了出生后新血管形成。在动物肢体缺血模型中,EPC移植能扩大缺血组织的侧枝血管生长,恢复血流,改善缺血,这为糖尿病血管病变提供了一种可行性治疗策略。     

脐血干细胞移植及血管成形治疗糖尿病下肢缺血性疾病

背景：近年来介入治疗因其成功率高、疗效好、创伤小已被广泛认可,但由介入治疗引起的血管内膜损伤尚无有效的治疗手段,致使术后远期发生严重再狭窄。干细胞移植技术的出现为促进受损血管内皮功能修复、再内皮化及辅助恢复缺血组织血供带来了新的曙光。 目的：总结近年来有关脐血干细胞移植技术及腔内血管成形术治疗糖尿病下肢缺血性病变的研究进展。 方法：使用计算机检索 2000年1月至2015年3 月PubMed、CNKI数据库中相关文章,检索词为“human umbilical cord blood stem cell transpltation,percutaneous transluminal angioplasty,diabetic lower limb ischemia, restenosis”及“人脐血干细胞移植、血管腔内成形术、糖尿病下肢缺血、再狭窄”。将符合研究目的的文献分类筛选并进行评价,初检获得216篇相关文献,最终纳入44篇文献进行分析。 结果与结论：通过对人脐血干细胞移植及血管成形术这两种治疗方式的疗效及远期预后分析比较,得出脐血干细胞移植联合血管成形术疗效优于单纯干细胞移植或单纯介入治疗,能够促进侧支循环建立、改善受损内皮功能及降低介入治疗后再狭窄发生。 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     

内皮祖细胞在糖尿病血管病变中的作用及干预治疗

血管内皮祖细胞(endothelial progenitor cells, EPCs)是一种能直接分化为血管内皮细胞的前体细胞,它与出生后的血管形成密切相关,具有重要的病理生理学意义。近年的研究显示EPCs功能受损和数量减少在糖尿病血管病变发生的机制中扮演着重要角色。从单个核细胞移植、内源性EPCs的动员活化以及EPCs的基因修饰这几个方面进行研究,为糖尿病血管病变治疗提供了新思路。本文就EPCs的生物学特性及其与糖尿病血管病变的关系以及内皮祖细胞对糖尿病血管病变治疗的展望进行综述。     

糖尿病内皮祖细胞的研究

<正>自1997年Asahara从人外周血单个核细胞首次分离出内皮祖细胞(Endothelial Progenitor Cells,EPCs)[1]以来,人们对其进行了大量研究。结果表明,EPCs是具有迁移     

脐血血管内皮祖细胞的分离和诱导分化

目的 从脐血中分离内皮祖细胞，诱导其向内皮细胞分化，研究内皮祖细胞的生物学特性和诱导分化条件。方法 从新鲜脐血中纯化的CD133^ 细胞接种于添加了VEGF、bFGF、IGF—1的M199培养液中，观察梭形贴壁细胞的出现时间和特异性细胞标志。结果 培养3—4d可观察到梭形贴壁细胞，14d左右可形成索条状结构，贴壁细胞表达血管内皮细胞特异性标志VE-cadherin,vWF,UEA-1和VEGFR-2。结论 脐血中含有内皮祖细胞，在一定的条件下，可分化为内皮样细胞。     

下肢缺血及G-CSF促进小鼠外周血内皮祖细胞动员

目的：观察下肢缺血以及腹腔注射粒细胞集落刺激因子（G-CSF）对小鼠外周血内皮祖细胞（EPCs）的影响。 方法： 建立小鼠下肢缺血模型，双色荧光标记流式细胞技术检测缺血状态下外周血内皮祖细胞的变化，同时给予G-CSF后观察对EPCs的影响。 结果： 下肢缺血后，外周血中反映EPCs变化的CD34和VEGFR2双荧光阳性细胞比例轻度增加，在缺血基础上同时给予G-CSF，上述变化更明显。 结论： 下肢缺血刺激可以使外周血内皮祖细胞数量增加，G-CSF通过骨髓动员可增强这种效应。     

Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

Hemogenic endothelial progenitor cells isolated from human umbilical cord blood

Hemogenic endothelium has been identified in embryonic dorsal aorta and in tissues generated from mouse embryonic stem cells, but to date there is no evidence for such bipotential cells in postnatal tissues or blood. Here we identify a cell population from human umbilical cord blood that gives rise to both endothelial cells and hematopoietic progenitors in vitro. Cord blood CD34+/CD133+ cells plated at high density in an endothelial basal medium formed an endothelial monolayer and a nonadherent cell population after 14-21 days. AML-1, a factor required for definitive hematopoiesis, was detected at low levels in adherent cells and at high levels in nonadherent cells. Nonadherent cells coexpressed the endothelial marker vascular endothelial (VE)-cadherin and the hematopoietic marker CD45, whereas adherent cells were composed primarily of VE-cadherin+/CD45- cells and a smaller fraction of VE-cadherin+/CD45+ cells. Both nonadherent and adherent cells produced hematopoietic colonies in methylcellulose, with the adherent cells yielding more colony-forming units (CFU)-GEMM compared with the nonadherent cells. To determine whether the adherent endothelial cells were producing hematopoietic progenitors, single cells from the adherent population were expanded in 96-well dishes for 14 days. The clonal populations expressed VE-cadherin, and a subset expressed AML-1, epsilon-globin, and gamma-globin. Three of 17 clonal cell populations gave rise to early CFU-GEMM hematopoietic progenitors and burst-forming unit-erythroid progenitors. These results provide evidence for hemogenic endothelial cells in human umbilical cord blood.     

自体骨髓单个核细胞移植治疗糖尿病性下肢缺血83例

背景：自体骨髓单个核细胞移植治疗糖尿病周围血管病变是目前广泛开展的一项技术,对缺血性病变有较好的效果,有个案报道对周围神经损伤所致临床症状有改善作用。 目的：观察自体骨髓单个核细胞移植前后糖尿病足病患者缺血性及神经性病变指标的变化。 方法：行自体骨髓单个核细胞移植治疗的糖尿病足病患者83例153条肢体。每例患者抽取自体骨髓250~400 mL,经密度梯度法提取单个核细胞,提取的单个核细胞数量为1.01×10⁸~4.96×10⁹个,平均数量为(2.04±0.53)×10⁸个,稀释后双下肢肌肉内注射,每点1~3 mL,间隔约3 cm。移植后3,6个月进行缺血性指标和神经病变指标检测。 结果与结论：自体骨髓单个核细胞移植后增加了双下肢血流量和外周血管数量,延长了行走距离,提高了皮温,促进了溃疡的愈合。治疗3个月及6个月后均有明显效果,差异有显著性意义。治疗6个月后,还可以改善糖尿病周围神经的自觉症状,增加运动及感觉神经传导速度,差异有显著性意义。     

静脉移植脐血干细胞可抑制脑缺血大鼠的神经细胞凋亡

背景：已有实验证实脐血干细胞移植后可迁移至脑损伤区域,存活并分化为神经细胞。 目的：探讨经静脉移植脐血干细胞治疗大鼠脑缺血的疗效以及对神经细胞凋亡的影响。 方法：改良线栓法建立大鼠大脑中动脉闭塞再灌注模型,48只大鼠随机数字表法分成治疗组和对照组,分别在造模1 d后经尾静脉注射脐血干细胞和PBS进行观察。 结果与结论：治疗后3,7 d两组神经功能评分比较,差异无显著性意义(P > 0.05);14,21 d治疗组神经功能评分明显低于对照组(P < 0.05)。对照组未见BrdU阳性细胞,治疗组大鼠脑组织切片中可见BrdU阳性细胞,对侧半球也可见少量BrdU阳性细胞。对照组大鼠缺血侧凋亡细胞显著多于治疗组(P < 0.05)。提示经静脉移植的脐血干细胞可迁移至大鼠缺血侧脑组织,抑制神经细胞凋亡,并显著改善大鼠神经功能。     

Human umbilical cord blood as a source of transplantable hepatic progenitor cells

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21-day cultures expressed ALB, and the ALB(+) cells coexpressed hepatocyte lineage markers. The ALB-expressing cells were able to proliferate in the culture system. Moreover, in the cell-transplantation model into liver-injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB-derived cell transplantation as a novel therapeutic option for liver failure.     

人外周血与脐血内皮祖细胞生物学特性的比较

背景：内皮祖细胞可从外周血与脐血中获取,是修复各种疾病所致损伤的血管内皮细胞不可或缺的细胞来源。 目的：比较人外周血与脐血来源的内皮祖细胞经体外培养后生物学特性的差异。 方法：通过密度梯度离心法和6%羟乙基淀粉结合密度梯度离心法分别分离人外周血与脐血中的单个核细胞,分别设为脐血源组和外周血源组,计数各组单个核细胞数量,按1.0×106/cm2接种于大鼠尾胶包被的培养皿中,用内皮细胞培养基进行诱导,共培养7 d。 结果与结论：外周血与脐血体外培养分离出内皮祖细胞具有类似的形态学特征。光学显微镜下观察,随着培养天数的增加,大多数细胞由早期的贴壁圆形转变为梭形。外周血源组内皮祖细胞有细胞集落形成,脐血源组可见梭形细胞自行排列生长为典型的线样结构。锥虫蓝染色及绘制细胞生长曲线后发现,外周血源组单个核细胞及内皮祖细胞数量、内皮祖细胞活率及增殖能力均低于脐血源组(P < 0.05)。外周血源组和脐血源组内皮祖细胞在接种后第3天增殖速度达到峰值,在随后的培养中细胞增殖呈衰减态。流式细胞仪及免疫荧光染色检测结果显示,外周血源组和脐血源组内皮祖细胞均可表达具有内皮祖细胞表型的CD133、CD34和血管内皮细胞因子受体2表面标志物,两组既摄取Dil标记乙酰化低密度脂蛋白,也能标记体外内皮祖细胞的标志物荆豆凝集素Ⅰ。结果证实,脐血来源的内皮祖细胞与外周血来源的内皮祖细胞生物学特性相近,脐血来源的内皮祖细胞增殖能力更强。     

外周血内皮祖细胞移植对肢体缺血的改善作用

背景：内皮祖细胞移植为肢体缺血的治疗提供了新的选择。 目的：研究人外周血来源内皮祖细胞移植对改善肢体缺血的作用。 方法：采用Ficoll密度梯度离心法获取人外周血单个核细胞,体外诱导扩增6 d后,检测其内皮祖细胞特异性标志的表达,并将荧光染料标记后的贴壁细胞通过缺血局部多点注射移植到后肢缺血的裸鼠动物模型体内,以评价其治疗效果。  结果与结论：从人外周血单个核细胞诱导出的贴壁细胞可表达内皮祖细胞特异性标志物CD133、CD34和血管内皮生长因子受体2,说明从人外周血单个核细胞中可诱导出内皮祖细胞。移植内皮祖细胞后裸鼠缺血后肢的坏死情况和毛细血管密度均明显改善(P < 0.05);在缺血后肢肌肉石蜡切片中可见分散不均的红色和黄绿色荧光标记的内皮祖细胞的掺入。表明移植的内皮祖细胞可以定向整合到缺血局部,改善裸鼠的后肢缺血。     

Osmotic selection of human mesenchymal stem/progenitor cells from umbilical cord blood

The isolation of undifferentiated adult stem/progenitor cells remains a challenging task primarily due to the rare quantity of these cells in biological samples and the lack of unique markers. Herein, we report a relatively straightforward method for isolation of human mesenchymal stem cells (MSCs) based on their unusual resistance to osmotic lysis, which we term "osmotic selection" (OS). MSCs can remarkably withstand significant exposure to hypotonic conditions (> 30 min) with only a reversible impairment in cell proliferation and with no loss of stem cell potential after exposure. Comparison of MSCs to other circulating nonhematopoietic cells revealed a time regime, by which purification of these cells would be attainable without considerable cell loss. OS showed a 50-fold enrichment of fibroblast colony-forming units from umbilical cord blood samples when compared to commonly employed techniques. After upstream processing, isolated cells using OS were immunophenotyped to be CD14-, CD34-, CD45-, CD44+, CD105+, and CD106+, and displayed multipotent differentiation. Preliminary investigations to determine mechanisms responsible for osmolytic resistance revealed MSCs to have an ineffective volume of 59%, with the ability to double cell volume at infinite dilution. Disruption of filamentous actin polymerization by cytochalasin D sensitized MSCs to osmotic lysis, which suggests a cytoskeletal element involved in osmolytic resistance.     

人脐血和外周血内皮祖细胞的分离培养和鉴定

背景：国内外有关内皮祖细胞的分离培养和鉴定方面的文章很多,但是人脐血和外周血内皮祖细胞的鉴别方面文章不多。 目的：从人脐血和外周血中分离出内皮祖细胞,并对其进行培养和鉴定。 方法：选取密度梯度离心法从脐血和外周血中分离获得单个核细胞,按照1× 10⁶/cm²的浓度种植于预先铺有纤维连接蛋白的培养板中,用含血管内皮生长因子的M199培养基进行诱导培养。 结果与结论：人脐血和外周血中存在内皮祖细胞,浓度梯度离心联合贴壁筛选获得的单个核细胞在血管内皮生长因子的诱导培养下可分化成内皮祖细胞,内皮祖细胞表达CD34、CD133、CD105、KDR和CD31,能吞噬乙酰化低密度脂蛋白,结合荆豆凝集素1,它们可作为体外分选内皮祖细胞的标志。