糖尿病对内皮祖细胞移植促血管新生作用的影响

背景：内皮祖细胞为血管新生的前体细胞,通过促血管新生作用治疗糖尿病血管病变有着良好的前景。 目的：探讨糖尿病对内皮祖细胞移植治疗缺血性疾病过程中促血管新生作用的影响。 方法：制备糖尿病大鼠模型,提取糖尿病和正常大鼠供体骨髓单个核细胞体外定向培养为内皮祖细胞。同时建立糖尿病及正常大鼠下肢缺血模型,并于缺血病变部位局部移植糖尿病或正常大鼠内皮祖细胞或PBS进行对照。移植后定期应用ELISA方法检测病变部位血管内皮生长因子含量,应用免疫组化方法计数病变部位微血管密度。 结果与结论：①受体相同,移植物不同时：移植糖尿病大鼠来源或正常大鼠来源内皮组细胞后,下肢缺血组织中血管内皮生长因子水平及微血管密度比较,差异无显著性意义(P > 0.05)。②移植物相同,受体不同时：正常大鼠移植内皮祖细胞后下肢缺血组织中血管内皮细胞生长因子含量和微血管密度均高于糖尿病组。说明在体外定向培养和病变部位局部注射条件下,糖尿病对骨髓来源内皮祖细胞移植促血管新生作用无明显影响,而对血管新生所处的病变部位微环境有明显影响。     

基质细胞衍化因子1与内皮祖细胞协同促血管新生

背景：动脉硬化性疾病的发病基础是内皮功能失调,循环中的内皮祖细胞数量和功能均下降,自身血管新生能力不足,单纯干细胞移植的疗效尚不确实,应用细胞因子以及基因修饰干细胞等方法是重要的研究方向。 目的：观察基质细胞衍化因子1对内皮祖细胞移植促血管新生的影响。 方法：制备20只单侧后肢缺血裸鼠模型,随机分为4组,分别给予静脉注射内皮祖细胞和肌肉注射基质细胞衍化因子1、静脉注射内皮祖细胞、局部肌肉注射基质细胞衍化因子1、肌肉注射培养液。造模后观察动物缺血后肢的皮温及存活情况,检测毛细血管/肌纤维比值、CD31和内皮型一氧化氮合酶表达情况。 结果与结论：荧光标记内皮祖细胞移植后整合至缺血后肢肌肉。20只裸鼠死亡2只。联合治疗组、内皮祖细胞组、基质细胞衍化因子1组和空白对照组患肢保存率分别为80%,75%,20%和0。毛细血管/肌纤维比值检测结果显示,联合治疗组和内皮祖细胞组高于空白对照组(P < 0.01),联合治疗组高于内皮祖细胞组、内皮祖细胞组高于基质细胞衍化因子1组(P < 0.05)。血管密度检测结果显示,联合治疗组、内皮祖细胞组大于空白对照组(P < 0.01),基质细胞衍化因子1组大于空白对照组(P < 0.05),联合治疗组大于内皮祖细胞组、内皮祖细胞组大于基质细胞衍化因子1组(P < 0.05)。联合治疗组和内皮祖细胞组缺血肌肉内皮型一氧化氮合酶阳性率分别为73.33%和53.33%(P > 0.05)。提示内皮祖细胞可定向迁移至缺血组织,内皮祖细胞移植能促进治疗性血管新生,基质细胞衍化因子1可增强这一作用,内皮型一氧化氮合酶参与了内皮祖细胞促血管新生的过程。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

高脂喂养2型糖尿病下肢缺血模型大鼠的构建

背景：目前普遍采用结扎或切除大鼠后肢股动脉或髂动脉的方法来制备后肢缺血模型,但对于如何建立及评估糖尿病高脂高血糖慢性动脉硬化闭塞症的缺血状态,尚无一个稳定有效慢性缺血模型及方法。 目的：构建及评价高脂喂养2型糖尿病下肢缺血模型大鼠。方法：将大鼠20只随机分为2组,糖尿病组10只予以高脂喂养6个月,腹腔注射链硫脲菌素50 mg/kg诱发糖尿病模型,对照组10只予以普食喂养6个月,两组大鼠均结扎股动脉建立右下肢缺血模型。结果与结论：建模后第1天,两组大鼠多普勒血流及CT血管造影均呈显著降低提示缺血造模成功,两组建模后第7,14天行激光多普勒检测发现血流有逐渐恢复趋势,建模后第28天糖尿病组血流恢复较对照组显著迟缓(P < 0.05)。CT血管造影检测显示,建模后第28天,糖尿病组右下肢股动脉结扎处近端仅有少量血管代偿性增加,远心端仍无明显血流。病理组织及免疫组织化学染色染色显示,建模后第28天,糖尿病组缺血部位肌肉组织出现结构组织破坏,炎性细胞浸润,毛细血管密度患侧低于健侧;糖尿病组缺血肌肉组织血管内皮生长因子较对照组的蛋白表达明显增加(P < 0.05)。结果显示,长期高脂喂养糖尿病大鼠,通过股动脉结扎及CT血管造影技术可成功构建糖尿病下肢缺血模型。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

骨髓间充质干细胞移植治疗肢体缺血的机制

背景：骨髓间充质干细胞移植治疗下肢缺血疾病已取得较好的效果,但其作用机制尚无定论。 目的：探讨骨髓间充质干细胞移植治疗SD大鼠后肢缺血的机制。 方法：结扎肾动脉下腹主动脉、腰动脉和髂腰动脉制备雌性SD大鼠后肢缺血模型。将扩增、纯化的雄性SD大鼠骨髓间充质干细胞注射入大鼠缺血的右后肢股直肌中,对照组右后肢注射等量生理盐水。移植后2,4,6周,制备大鼠右股直肌切片行苏木精-伊红染色、血管内皮生长因子免疫组织化学染色、SRY基因免疫组织化学染色。 结果与结论：移植后2,4,6周移植组右股直肌毛细血管计数、血管内皮生长因子免疫组织化学染色阳性细胞计数均明显高于对照组(P < 0.01)。移植组股直肌切片中SRY免疫组织化学染色阳性细胞出现于毛细血管壁,并散在分布于肌组织中。结果表明局部注射移植骨髓间充质干细胞能促进缺血组织迅速生成大量毛细血管,改善大鼠缺血下肢的血供。骨髓间充质干细胞作为旁分泌细胞,通过增加血管内皮生长因子的分泌,促使大鼠缺血肢体的血管新生。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;组织工程全文链接：     

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。     

自体骨髓单个核细胞移植治疗大鼠后肢缺血

目的　探讨自体骨髓单个核细胞（BM-MNC）移植用于大鼠缺血后肢的治疗后实现血管再生的能力。方法　建立大鼠后肢缺血动物模型,将取于自体的BM-MNC制成细胞悬液注射于缺血部位,分别在移植后2和30d时行动脉造影,用免疫组化方法检测内皮祖细胞（EPC）,毛细血管密度以及测定血管内皮生长因子(VEGF)的表达。结果　缺血肌组织中的EPC含量增高（P<0.01）。BM-MNC移植组在移植早期VEGF表达显著增高（P<0.01）。细胞移植后30dBM-MNC移植组毛细血管密度明显高于其他组（P<0.01）,血管造影可见侧支循环建立。结论　自体骨髓单个核细胞移植于大鼠后肢缺血区能促进血管新生,改善侧支循环,可望成为一种简单有效的治疗下肢缺血的方法。     

人外周血内皮祖细胞移植促裸鼠缺血组织血管新生的初步研究

目的观察人外周血内皮祖细胞移植对下肢缺血裸鼠血管新生的影响。方法从人自愿者外周血中分离单个核细胞 ,置于EBM -2培养基内培养7d后 ,获得内皮祖细胞。将1×106个内皮祖细胞通过尾静脉注入下肢缺血裸鼠体内 (8只 ) ,对照组 (8只 )通过尾静脉注入等量的PBS。移植后第28d ,处死动物 ,进行组织学和生理学评估。结果内皮祖细胞组毛细血管密度较对照组明显增加[(136.4±40.5)/视野 ,(53.65±14.02)/视野 (P<0.01)] ;内皮祖细胞组的肢体致残率 (25 % )较对照组 (50 % )减少。结论体外扩增的内皮祖细胞移植能够促进缺血组织的血管新生 ,并能减少肢体的致残率。     

不同时间移植人脐血CD34+细胞可影响心肌梗死大鼠心功能及血管内皮细胞生长因子的分泌

背景：干细胞移植可以改善心脏功能,改善预后。 目的：观察不同时间经静脉移植人脐血CD34+细胞对心肌梗死大鼠心功能及细胞因子分泌的影响。 方法：结扎冠状动脉左前降支制备Wistar大鼠心肌梗死模型,于梗死后1,5,10 ,30 d经尾静脉注入0.5 mL人脐血CD34+细胞(实验组)或磷酸盐缓冲溶液(对照组)。 结果与结论：与对照组相比,梗死后5,10 d实验组大鼠左室射血分数明显升高(P < 0.05),左室收缩末内径明显减小(P < 0.05),左室后壁增厚率更高(P < 0.05),毛细血管密度明显增加(P < 0.05),且以梗死后10 d移植大鼠心功能改善效果最明显(P < 0.05)。梗死后10 d实验组心肌局部血管内皮细胞生长因子最高(P < 0.05)。说明大鼠心肌梗死后5,10 d经静脉移植脐血CD34⁺细胞可明显改善心功能,梗死后10 d移植血管内皮细胞生长因子分泌更多,血管生成更多,对心功能的改善更明显;同时说明脐血单个核细胞移植可能是通过增加血管内皮细胞生长因子分泌,提高毛细血管密度来改善心功能的。     

低频电刺激对坐骨神经损伤大鼠不同类型骨骼肌萎缩及内源性胰岛素样生长因子1表达的影响

背景：低频电刺激可以缓解骨骼肌的萎缩,但对肌纤维类型的影响尚不清楚,同时内源性胰岛素样生长因子1在萎缩后的肌纤维中的表达与电刺激的关系尚无公识。 目的：观察低频电刺激对坐骨神经损伤大鼠不同类型骨骼肌纤维萎缩情况及内源性胰岛素样生长因子1表达的影响。 方法：将健康雄性SD大鼠随机分为3组,切断模型组和电刺激组大鼠左侧坐骨神经制备失神经支配模型,适应5 d后,对电刺激组大鼠损伤侧腓肠肌施以2 Hz的电刺激,2次/d,每次持续20 min,正常组和模型组常规饲养。30 d后,取大鼠腓肠肌腹部,检测其肌纤维直径和数量;免疫组织化学法检测肌组织中胰岛素样生长因子1的水平。 结果与结论：失神经支配后,大鼠腓肠肌Ⅰ、Ⅱ型肌纤维直径减小,Ⅰ型肌纤维数比例增大。与模型组比较,电刺激组大鼠腓肠肌Ⅰ、Ⅱ型肌纤维直径有所增大,尤以Ⅰ型肌纤维直径增大更明显(P < 0.05)。同时,电刺激组大鼠腓肠肌中胰岛素样生长因子1的表达也明显高于模型组(P < 0.05)。提示,2 Hz的电刺激可促进胰岛素样生长因子1的表达,减轻Ⅰ型肌纤维的萎缩。     

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。     

糖尿病鼠急性下肢缺血的血流变化

目的 研究建立糖尿病足下肢急性缺血模型的方法.方法 30只雄性Wistar鼠腹腔注射STZ(50 mg/kg)诱发糖尿病模型,血糖达16.8 mmol/L后,再手术结扎股动、静脉及周围血管,造成左下肢急性缺血模型,用激光多普勒(LDPI)测定下肢血流.结果 81.5%大鼠(22只)空腹血糖水平达16.8 mmol/L,结扎后1-14 d下肢显著性缺血(P相似文献   

The role of fucosylation in the promotion of endothelial progenitor cells in neovascularization and bone repair

Bone marrow-derived endothelial progenitor cells (EPCs) are being tested as a therapy to treat a variety of ischemic diseases. Poor homing to targeted tissues is one of the major factors limiting the therapeutic efficacy of EPCs. Here, we show that human cord blood-derived EPCs expressed little sialyl Lewis X (sLe^x) antigen that is necessary for selectin-mediated cell–cell interactions. Expression of α1,3-fucosyltransferase VI (FucT VI) in the EPCs enhanced sLe^x synthesis, E- and P-selectin-binding, and EPC adhesion to tumor necrosis factor-α-stimulated human umbilical vein endothelial cells in culture. In a mouse model of hind limb ischemia, in which EPCs were injected intravenously, FucT VI expression increased EPC homing, neovascularization, and blood flow in ischemic muscles. In another mouse model of femoral fracture, FucT VI-expressing EPCs were more efficient than control EPCs in targeting to peri-fracture tissues to enhance angiogenesis, blood flow and bone repair. These results indicate that fucosylated EPCs may be used to as an improved cellular source to treat ischemic diseases.     

The use of magnetic resonance cell tracking to monitor endothelial progenitor cells in a rat hindlimb ischemic model

A water-soluble magnetic resonance imaging (MRI) contrast agent, Dextran mono-N-succinimidyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-gadolinium³⁺ (Dex-DOTA-Gd³⁺), was shown to enable monitoring of the anatomical migration and the survival period of transplanted stem cells for up to 1 month. Gadolinium molecules in the cells were rapidly eliminated from the site and excreted upon cell death. Endothelial progenitor cells (EPCs) transplanted into the inguinal femoral muscle of rats migrated distally through the knee in rats after hindlimb ischemia but did not migrate in non-ischemic rats. Interestingly, the survival period of transplanted EPCs was notably prolonged in the ischemic limb, indicating that EPCs are required by the ischemic tissues and that the fate of transplanted EPCs was affected by the disease. Compared to the commonly used particle type of MRI contrast agents, the system described in this study is expected to be invaluable to help clarifying the process of stem cell transplantation therapy.     

Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle

Trafficking of transplanted endothelial progenitor cells (EPCs) to ischemic tissue is enhanced by stromal-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF). However, it has not been studied how these cytokines modulate the local milieu to entrap EPCs. This study was performed to elucidate a molecular pathway of trafficking EPCs through Akt and to test its application as an adjuvant modality to increase EPC homing. In a mouse hind limb ischemia model, systemically administered 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled mouse EPCs showed three stages of homing to ischemic limb: adhesion to endothelial cells (ECs), incorporation to capillary, and transendothelial migration into extravascular space. As an underlying mechanism to control adhesion of EPCs to ECs, we found that Akt was activated in ECs of ischemic muscle by ischemia-induced VEGF and SDF-1. In vitro and in vivo experiments using adenoviral vector for constitutively active or dominant-negative Akt genes showed that activated Akt enhanced intercellular adhesion molecule 1 (ICAM-1) expression on ECs. Akt activation in ECs also enhanced EPC incorporation to ECs and transendothelial migration in vitro experiments. Activated Akt was sufficient for induction of EPC homing even in normal hind limb, where VEGF or SDF-1 was not increased. Finally, local Akt gene transfer to ischemic limb significantly enhanced homing of systemically administered EPCs, new vessel formation, blood flow recovery, and tissue healing. Akt plays a key role in EPC homing to ischemic limb by controlling ICAM-1 and transendothelial migration. Modulation of Akt in the target tissue may be an adjunctive measure to enhance homing of systemically administered stem cells, suggesting a possibility of cell-and-gene hybrid therapy. Disclosure of potential conflicts of interest is found at the end of this article.     

Sustained endothelial progenitor cell dysfunction after chronic hypoxia-induced pulmonary hypertension

Circulating endothelial progenitor cells (EPCs) contribute to neovascularization of ischemic tissues and repair of injured endothelium. The role of bone marrow-derived progenitor cells in hypoxia-induced pulmonary vascular remodeling and their tissue-engineering potential in pulmonary hypertension (PH) remain largely unknown. We studied endogenous mobilization and homing of EPCs in green fluorescent protein bone marrow chimeric mice exposed to chronic hypoxia, a common hallmark of PH. Despite increased peripheral mobilization, as shown by flow cytometry and EPC culture, bone marrow-derived endothelial cell recruitment in remodeling lung vessels was limited. Moreover, transfer of vascular endothelial growth factor receptor-2+/Sca-1+/CXCR-4+-cultured early-outgrowth EPCs failed to reverse PH, suggesting hypoxia-induced functional impairment of transferred EPCs. Chronic hypoxia decreased migration to stromal cell-derived factor-1alpha, adhesion to fibronectin, incorporation into a vascular network, and nitric oxide production (-41%, -29%, -30%, and -32%, respectively, vs. normoxic EPCs; p < .05 for all). The dysfunctional phenotype of hypoxic EPCs significantly impaired their neovascularization capacity in chronic hind limb ischemia, contrary to normoxic EPCs cultured in identical conditions. Mechanisms contributing to EPC dysfunction include reduced integrin alphav and beta1 expression, decreased mitochondrial membrane potential, and enhanced senescence. Novel insights from chronic hypoxia-induced EPC dysfunction may provide important cues for improved future cell repair strategies.     

细胞移植结合激光肌肉血管重建增强鼠缺血肢体新生血管形成

目的: 研究激光肌肉血管重建（TMR）和血管内皮祖细胞（EPCs）移植相结合能否增强缺血肢体新生血管形成。方法： 密度梯度法分离脐带血单个核细胞并体外培养，免疫组织化学和流式细胞术定量和定性分析。应用DiI标记注射入激光孔道和缺血区的EPCs。结扎裸鼠右侧髂外动脉造成急性肢体缺血模型，随机分为：TMR＋EPCs组：EPCs移植入缺血区肌肉的激光孔道；TMR组： 缺血区肌肉激光打孔；EPCs组：注射EPCs至缺血区肌肉；对照组：缺血肢体模型。超声多谱勒测定缺血和非缺血后肢的股动脉血流获得比值（FAFI），组织化学和免疫组织化学检查缺血肢体肌肉标本。结果： 贴壁细胞表达KDR、CD34、AC133、CD31和vWF，并且吞噬Ac-LDL，有内皮细胞功能，培养7 d的细胞流式细胞术检查CD34（62%±7%）和AC133（57.2%±9.8%）阳性。术后28 d，TMR＋EPCs、EPCs和TMR组FAFI明显高于各自基线水平；TMR＋EPCs和EPCs组FAFI明显高于对照组。TMR+EPCs组新生血管形成明显多于对照组。结论： TMR和EPCs移植结合明显改善了缺血肢体的灌注并增强了缺血肢体新生血管形成。     

Transfection of VEGF(165) genes into endothelial progenitor cells and in vivo imaging using quantum dots in an ischemia hind limb model

Endothelial progenitor cells (EPCs) were transfected with fluorescently labeled quantum dot nanoparticles (QD NPs) with or without VEGF(165) plasmid DNA (pDNA) to probe the EPCs after in?vivo transplantation and to test whether they presented as differentiated endothelial cells (ECs). Bare QD NPs and QD NPs coated with PEI or PEI?+?VEGF(165) genes were characterized by dynamic light scattering, scanning electron microscopy, and atomic force microscopy. Transfection of EPCs with VEGF(165) led to the expression of specific genes and proteins for mature ECs. A hind limb ischemia model was generated in nude mice, and VEGF(165) gene-transfected EPCs were transplanted intramuscularly into the ischemic limbs. At 28 days after transplantation, the VEGF(165) gene-transfected EPCs significantly increased the number of differentiated ECs compared with the injection of medium or bare EPCs without VEGF(165) genes. Laser Doppler imaging revealed that blood perfusion levels were increased significantly by VEGF(165) gene-transfected EPCs compared to EPCs without VEGF(165). Moreover, the transplantation of VEGF(165) gene-transfected EPCs increased the specific gene and protein expression levels of mature EC markers and angiogenic factors in the animal model.     

纤维蛋白凝胶承载内皮祖细胞移植梗死心肌后细胞自噬变化

目的 观察心肌梗死后移植内皮祖细胞（EPCs）时的细胞自噬变化，探讨自噬维持移植细胞存活和纤维蛋白凝胶保护细胞的作用。方法 通过结扎左冠状动脉的前降支建立大鼠心肌梗死模型后，在正常区、梗死边缘区和梗死区分别注射从人脐带血中分选的EPCs。2 h后取注射处组织，作半薄切片，观察纤维蛋白凝胶承载的EPCs分布。定位后作超薄切片，透射电镜下观察EPCs的自噬结构变化以及纤维蛋白凝胶与EPCs和心肌的相容性。结果 与正常区相比，梗死边缘区发生自噬的EPCs增多，细胞内自噬结构也增多。梗死区的移植细胞的自噬显著增强，有的细胞坏死或凋亡。纤维蛋白凝胶与移植细胞和心肌的相容性良好，移植的EPCs在纤维蛋白凝胶中充分伸展，有的EPCs与心肌细胞黏附。结论 在梗死边缘区移植EPCs后，轻度缺血刺激细胞发生自噬，这有利于维持移植细胞存活。纤维蛋白凝胶承载EPCs可避免细胞丢失和促进存活。