血管内皮祖细胞的体外扩增特性研究

目的:探讨血管内皮祖细胞(EPCs)在体外扩增特性。方法:利用磁性活化细胞分选系统(MACS)系统富集CD34⁺细胞,在相同条件下与同批的单个核细胞(MNC)、CD34⁺和CD34^-混和细胞进行对照培养,比较EPCs体外扩增效果。另外研究血管内皮生长因子(VEGF)和传代培养对细胞分化、扩增动力学和细胞凋亡的影响。应用细胞免疫化学和流式细胞术对细胞定性定量分析。结果:MNC培养、CD34⁺和CD34^-细胞混和培养明显高于CD34⁺细胞单独培养EPCs扩增率(P＜0.05),一旦细胞形成线索样结构行传代培养明显低于未传代的细胞凋亡(P＜0.05)。VEGF对细胞凋亡(P＞0.05)无明显影响,这些分化的EPCs免疫细胞化学染色CD34、vWF、KDR、CD31阳性,并且吞噬乙酰化低密度脂蛋白(Ac-LDL)。培养7d流式细胞检查CD34⁺、AC133^＋分别占贴壁(AT)细胞的68.2%±6.3%(n=6)、57.2%±9.8%(n=6)。结论:MNC培养、CD34⁺和CD34^-细胞混和培养提高了EPCs扩增率,早传代使凋亡率明显降低。VEGF对EPCs体外扩增无明显影响。     

Isolation of endothelial progenitor cells from cord blood and induction of differentiation by ex vivo expansion

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1 beta , fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血干/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述。     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血于/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述.     

Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization

A subset of human peripheral blood mononuclear cells (PB-MNCs) differentiate into endothelial progenitor cells (EPCs) that participate in postnatal neovascularization. Although tissue ischemia can mobilize EPCs from bone marrow, the effects of hypoxia on differentiation and angiogenic function of EPCs are little known. We examined whether hypoxic conditioning would modulate differentiation and function of human PB-MNC-derived EPCs. A subset of PB-MNCs gave rise to EPC-like attaching (AT) cells under either normoxic or hypoxic conditions. However, hypoxia much enhanced the differentiation of AT cells from PB-MNCs compared with normoxia. AT cells released vascular endothelial growth factor (VEGF) protein and expressed CD31 and kinase insert domain receptor/VEGFR-2, endothelial lineage markers, on their surface, which were also enhanced by hypoxia. Both a neutralizing anti-VEGF mAb and a KDR-specific receptor tyrosine kinase inhibitor, SU1498, suppressed PB-MNC differentiation into EPC-like AT cells in a dose-dependent manner. Migration of AT cells in response to VEGF as examined by a modified Boyden chamber apparatus was also enhanced by hypoxia. Finally, in vivo neovascularization efficacy was significantly enhanced by in vitro hypoxic conditioning of AT cells when cells were transplanted into the ischemic hindlimb of immunodeficient nude rats. In conclusion, hypoxia directly stimulated differentiation of EPC-like AT cells from human PB-MNC culture. Moreover, hypoxic preconditioning of AT cells before in vivo transplantation is a useful means to enhance therapeutic vasculogenesis.     

法舒地尔通过促进APP/PS1双转基因小鼠神经再生改善认知功能

目的:观察Rho激酶抑制剂法舒地尔(fasudil)在治疗阿尔茨海默病(Alzheimer disease,AD)模型——淀粉样前体蛋白/早老素1双转基因(amyloid precursor protein/presenilin 1 double transgenic,APP/PS1 Tg)小鼠中促进神经再生并改善认知功能的作用。方法:将8月龄雄性APP/PS1 Tg小鼠作为AD模型,随机分为fasudil组(腹腔注射fasudil 25 mg·kg~(-1)·d~(-1))和生理盐水(normal saline,NS)组(腹腔注射等体积生理盐水),取同月龄同性别野生型(wild type,WT)小鼠为WT组,连续治疗2个月;应用Morris水迷宫(Morris water maze,MWM)实验和SMART 3.0行为学记录系统检测、记录并分析各组小鼠空间认知功能;免疫荧光组织化学染色法检测小鼠脑内海马齿状回和CA3区及大脑皮层p-Tau、ChA T、BrdU和nestin的蛋白表达水平和分布;Western blot法检测小鼠脑组织p-APP(Thr668)和p-Tau的蛋白水平。结果:在MWM实验中,与WT组小鼠相比,NS组10月龄APP/PS1 Tg小鼠从入水点到达平台的时间增加,在平台所在象限活动时间占总活动时间的比例及在平台所在象限游泳距离占总路径的比例减少;而fasudil治疗明显拮抗上述行为学指标,改善APP/PS1 Tg小鼠认知功能。此外,与NS组小鼠相比,fasudil明显减少小鼠p-Tau蛋白水平,增加p-APP蛋白水平,使海马区胆碱能神经元数量增多,促进神经元的轴突再生,动员脑内海马齿状回颗粒下区和脑室下区的内源性神经干细胞增殖。结论:Fasudil通过促进APP/PS1 Tg小鼠中枢神经系统p-Tau蛋白水平下调,增加可溶性APP水平,促进胆碱能神经元再生,动员内源性神经干细胞增殖,从而改善APP/PS1双转基因小鼠空间认知功能。     

In vitro angiogenesis properties of endothelial progenitor cells: a promising tool for vascularization of ex vivo engineered tissues

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at the improvement of neovascularization of engineered tissues are a key issue. A method to enhance graft vascularization is to establish a primitive vascular plexus within the graft before transplantation by the use of cellular-based concepts. To explore the utility of endothelial progenitor cells (EPCs) for the ex vivo vascularization of tissue engineered grafts, we analyzed the in vitro angiogenic properties of this cell type in two different angiogenesis models: the 3-dimensional spheroid sprouting assay and the 2-dimensional matrigel assay. In both assays, EPCs were able to form tubelike structures, resembling early capillaries. This process was significantly enhanced by the addition of angiogenic growth factors. Direct comparison between EPCs and mature endothelial cells, represented by human umbilical vein endothelial cells (HUVECs), revealed that both cell types displayed an almost identical angiogenic potential. Other functional in vitro parameters such as angiogenic growth factor induced cell proliferation and cell survival were investigated as well, revealing a significantly decreased level of apoptosis of EPCs in relation to HUVECs under serum-deprived conditions. The observed survival advantage of EPCs along with the observation that EPCs perform very well in the above mentioned in vitro angiogenesis assays, make them an ideal autologous cell source for vascularization of ex vivo generated tissues. The attractiveness of this cell type for tissue engineering applications is strengthened further by the fact that these cells can be easily isolated from the peripheral blood of patients, thereby eliminating donor site morbidity.     

高血压前期循环内皮祖细胞数量和功能的性别差异

背景：目前对高血压前期循环内皮祖细胞数量和功能的性别差异尚不明确。 目的：探讨性别差异对高血压前期循环内皮祖细胞数量和功能的影响。 方法：选择79例年龄(46.4±4.5)岁的人群作为研究对象,其中绝经前健康女性志愿者18例、绝经前高血压前期女性患者21例、健康男性志愿者21例和高血压前期男性患者19例,取外周血用流式细胞仪测定CD34和 KDR双标阳性循环内皮祖细胞水平,ac-LDL 及 lectin 荧光标记方法评估体外培养内皮祖细胞数量, MTT法和Transwell小室评估内皮祖细胞的增殖能力和迁移能力,测定各组血浆雌激素水平。 结果与结论：与高血压前期男性患者和健康男性志愿者比较,绝经前高血压前期女性患者和绝经前健康女性志愿者的CD34+/KDR+双阳性循环内皮祖细胞数量、ac-LDL及lectin 抗体双阳性内皮祖细胞数量增加      (P均< 0.05),内皮祖细胞迁移和增殖能力明显增强(P < 0.05),血浆雌激素水平增高(P < 0.05)。绝经前高血压前期女性患者与绝经前健康女性的内皮祖细胞迁移和增殖能力无明显差异(P > 0.05),而高血压前期男性患者的内皮祖细胞迁移和增殖能力较健康男性减弱。血浆雌激素水平与循环内皮祖细胞数量及功能呈明显的直线相关关系(P < 0.05)。这些结果证实,高血压前期循环内皮祖细胞数量和功能存在性别差异,相比高血压前期男性患者,绝经前高血压前期女性患者循环内皮祖细胞数量增多,增殖和迁移能力增强,其可能与机体内较高的雌激素水平有关。     

The origin and in vivo significance of murine and human culture-expanded endothelial progenitor cells

In adults highly purified populations of early hematopoietic progenitors or cells derived from ex vivo expanded unmobilized human peripheral blood mononuclear cells contribute to new blood vessel formation. However, the source of these culture-expanded endothelial progenitor cells (CE-EPCs) remains controversial. We demonstrate that ex vivo expansion of unmobilized human peripheral blood generated CE-EPCs with similar numbers, kinetics, and antigen expression profile as compared to plating unfractionated CD34(+)/lin(-)-enriched bone marrow mononuclear cells. Both CE-EPC populations uniformly co-expressed myeloid and endothelial markers, suggesting that peripheral blood progenitor enumeration does not correlate with the numbers of early outgrowth CE-EPCs. Using purified myeloid subpopulations obtained from mice harboring the lacZ transgene driven by an endothelial-specific promoter, we showed that the immature myeloid lineage marker CD31(+) cells generated CE-EPCs with fourfold greater frequency than mature myeloid populations. Biphenotypic cells co-expressing myeloid/endothelial antigens were not detected in circulating human or murine peripheral blood or bone marrow but were associated with murine tumors. Unlike CE-EPCs, CD14(+) leukocytes admixed within tumors did not generate vWF-positive blood vessels during a similarly defined period of tumor growth, but some leukocytes up-regulated the endothelial marker VE-cadherin. Taken together, the data suggest that the local neovascular microenvironment may facilitate vasculogenesis by promoting endothelial differentiation and that CE-EPCs may accelerate such vasculo-genesis.     

外周血内皮祖细胞数量变化与颅内动脉瘤的生成

背景：动脉内膜损伤是动脉瘤发生的始动因素,内皮祖细胞能修复受损的动脉内膜。 目的：建立大鼠颅内动脉瘤模型,探讨动脉瘤大鼠内皮祖细胞数量变化及意义。 方法：40只SD大鼠随机分为两组,正常组作为正常对照,不给予任何干预。模型组大鼠手术结扎双侧肾动脉后支以及左侧颈总动脉,术后喂食含8%氯化钠鼠粮。于2周,1,2,3个月末测量大鼠内皮祖细胞变化,并与3个月末测量各组大鼠血压及动脉瘤大小,RT-PCR检测willis环相关基因表达。 结果与结论：模型组大鼠内皮祖细胞数目于2周后就开始下降,与正常组比较差异有显著性意义(P < 0.05),一直持续到3个月末(P < 0.01)。模型组大鼠动脉瘤壁基质金属蛋白酶9表达明显高于正常组正常大鼠(P < 0.01),而内皮型一氧化氮合酶明显低于正常(P < 0.05)。结果提示循环内皮祖细胞数量降低可能是动脉瘤生成的一个重要因素。     

内皮细胞Rho及Rho激酶在肉瘤细胞向血管外游走过程中的作用

目的:探讨肉瘤细胞向血管外游走过程中内皮细胞Rho及Rho激酶的作用。方法: 利用肉瘤细胞向血管外游走的体外模型,观察肉瘤细胞的血管外游走状况;并测定肉瘤细胞向血管外游走过程中血管内皮单细胞层的电阻变化。结果:肉瘤细胞可以穿过血管内皮细胞游走至血管外;肉瘤细胞向血管外的游走能够引起单层血管内皮细胞的电阻下降;而内皮细胞Rho抑制剂(C3转移酶)及Rho激酶抑制剂(Y-27632)能够抑制肉瘤细胞向血管外的游走、并抑制游走过程中引起的血管内皮单细胞层电阻下降。结论: 内皮细胞Rho及Rho激酶可以通过诱导血管内皮细胞骨架蛋白的改变,来影响肉瘤细胞穿过内皮细胞间缝隙向血管外游走。     

Rho激酶在糖尿病血管内皮功能改变中的作用

目的: 探讨Rho激酶激活在糖尿病血管内皮功能损伤中的作用。方法:利用正常SD大鼠、基因型糖尿病动物模型和高脂饮食诱发的小鼠肥胖模型,用器官浴槽和肌动描记方法对血管功能进行检测,用免疫印迹方法对血管组织中内皮型一氧化氮合酶(eNOS)磷酸化水平进行测定,用ELISA方法对血管组织中血栓烷受体(TP受体)激活物进行测量。结果: TP 受体激活可以抑制正常血管的内皮依赖性舒张,使最大舒张值(E_max)由正常组的(78.8±10.6)% 降到 (17.9±5.1)%; Rho激酶抑制剂预处理翻转了TP受体的抑制作用,E_max值为 (62.0±11.2)%;抑制eNOS抵消了Rho激酶抑制剂对血管功能的改善作用。与此相一致,TP受 体激活可以抑制正常血管eNOS磷酸化水平,并且该作用被Rho激酶抑制剂所翻转。此外,抑制Rho激酶可以使糖尿病小鼠和肥胖小鼠的血管内皮依赖性舒张作用得到改善,E_max值显著增加。并且,与对照组相比,糖尿病动物血管组织中血栓烷B₂和前列腺素F_2α水平升高。结论:Rho激酶参与了糖尿病血管内皮功能紊乱的调节,可能与TP受体激活和抑制eNOS活性有关。     

Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence

Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.     

血管内皮干/祖细胞的研究进展

Endothelial progenitor cell (EPC) is a kind of directional cell that is able to differentiate to endothelial cell. The role of EPC is associated not only with vasculogenesis during embryonic development but also with physiological organ maintenance and angiogenesis during postnatal and adult period. There is a good clinical therapeutic prospective use for EPC in the treatment of ischemia diseases and inhibition of tumor angiogenesis.     

In vitro functional testing of endothelial progenitor cells that overexpress thrombomodulin

This study investigated the augmentation of endothelial progenitor cell (EPC) thromboresistance by using gene therapy to overexpress thrombomodulin (TM), an endothelial cell membrane glycoprotein that has potent anti-coagulant properties. Late outgrowth EPCs were isolated from peripheral blood of patients with documented coronary artery disease and transfected with an adenoviral vector containing human TM. EPC transfection conditions for maximizing TM expression, transfection efficiency, and cell viability were employed. TM-overexpressing EPCs had a fivefold increase in the rate of activated protein C production over native EPCs and EPCs transfected with an adenoviral control vector expressing β-galactosidase (p<0.05). TM upregulation caused a significant threefold reduction in platelet adhesion compared to native EPCs, and a 12-fold reduction compared to collagen I-coated wells. Additionally, the clotting time of TM-transfected EPCs incubated with whole blood was significantly extended by 19% over native cells (p<0.05). These data indicate that TM-overexpression has the potential to improve the antithrombotic performance of patient-derived EPCs for endothelialization applications.     

Measuring angiogenic cytokines, circulating endothelial cells, and endothelial progenitor cells in peripheral blood and cord blood: VEGF and CXCL12 correlate with the number of circulating endothelial progenitor cells in peripheral blood

Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) are thought to play an important role in the vascularization of damaged tissues and cancers. These cells are also required for tissue-engineered blood vessels and to help skin substitutes revascularize more efficiently. A standard approach to the phenotyping and enumeration of CEC and EPC is key to the development of new therapies, and the identification of biomarkers within the blood that regulate their levels may be important for the treatment of cancer. We have devised an improved multiparameter flow cytometric assay for CEC and circulating EPC enumeration. This assay uses antibodies recognizing CD133 and CD34 to identify EPC and CEC, respectively, and incorporates specific markers CD144 and vascular endothelial growth factor receptor 2 (VEGFR-2) for both CEC and EPC cells. In peripheral blood (PB), mean CEC numbers were 55 +/- 95 mL(-1) and mean EPC numbers were 44 +/- 58 mL(-1) (n = 60). We also found a significant correlation of both plasma VEGF (r = 0.90, p < 0.001) and CXCL12 (r = 0.84, p < 0.001) with EPCs, but not CECs. The cytokines also correlated with each other (r = 0.85, p < 0.001). In umbilical cord blood (UCB) we found on average 13 times more CEC (719 +/- 338 mL(-1)) and 7 times more EPC (299 +/- 245 mL(-1)) than in PB. However, serum VEGF and CXCL12 levels in UCB did not correlate with either EPC or CEC numbers. These results suggest a major role for VEGF and CXCL12 in the control of marrow-derived EPCs in adult PB and provide normal data for comparison with patient populations.     

运动条件下健康人循环内皮祖细胞的数量和功能*****☆

背景：运动条件对内皮祖细胞数量和功能是否会产生的影响目前尚无公识。 目的：观察急性运动对健康成人循环内皮祖细胞数量和功能的影响。 方法：健康男性成人志愿者12例参加急性平板运动锻炼(9.3±2.1) min。用流式细胞仪测定运动前后CD34和KDR双标阳性循环内皮祖细胞水平,ac-LDL及lectin荧光标记方法评估体外培养内皮祖细数量,检测内皮祖细胞的黏附、迁移和增殖能力,并测定运动前后血浆和内皮祖细胞分泌一氧化氮、血管内皮生长因子和粒-巨噬细胞集落刺激因子水平变化。 结果与结论：流式细胞仪检测显示健康志愿者运动后循环内皮祖细胞水平较运动前增加(P < 0.05)。荧光标记法证实运动后ac-LDL及lectin抗体双阳性内皮祖细胞数量较运动前增加(P < 0.05),内皮祖细胞迁移和增殖能力明显增强(P < 0.05),但黏附能力无明显变化。急性运动能明显增加健康志愿者的血浆一氧化氮水平(P < 0.05),健康志愿者运动前后血浆一氧化氮水平和循环内皮祖细胞数量及功能的增加倍数呈明显的直线相关回归关系(P < 0.05)。结果证实,运动可明显增加健康成人循环内皮祖细胞的数量和功能,其机制与其诱导一氧化氮释放增多有关。