内皮祖细胞的生物学特性及其在皮肤创面修复中的作用

内皮祖细胞（endothelial progenitor cells,EPCs）主要生成和存在于骨髓中,是一种能定向分化为成熟血管内皮细胞（endothelial cells,ECs）,具有自我更新、游走、高增殖潜能和定向分化特性的前体细胞。     

流动激活血管内皮细胞中转录因子核因子-кB和激活蛋白-1的表达

目的比较血管内皮细胞(endothelial cells,ECs)在不同流场中转录因子核因子-кB(nuclear factor-кB,NF-кB)和激活蛋白-1(activator protein-1,AP-1)的表达规律,为力学信号对基因调控模式的影响提供依据。方法将培养的人脐静脉ECs加载于层流和湍流的离体流室模型中,以作用时程的不同分为6个时间点(0.5 h、1 h、2 h、3 h、4 h和5 h),运用共聚焦显微镜,从蛋白水平观察ECs中NF-кB和AP-1在不同流室中表达的时间规律和空间规律。结果层流中NF-кB的表达高峰出现在1 h段(26.49±1.63,P<0.05),随后转行向下;湍流中NF-кB表达在3 h段升至峰值(34.41±6.43,P<0.05);层流中c-Jun/AP-1表达一过性增高,0.5 h到达峰值(18.95±5.38,P<0.05),随即下降至基线水平;湍流中c-Jun/AP-1表达呈持续缓慢上升趋势(P<0.05)。结论湍流流场对血管ECs中NF-кB和AP-1表达的影响不同于层流,湍流对NF-кB和AP-1的持续激活上调作用可能引起了ECs形态结构和功能行为的病理学改变。     

犬骨髓间充质干细胞向血管内皮细胞定向分化的研究

目的：研究犬骨髓间充质干细胞（MSCs）在体外定向诱导,向血管内皮细胞（ECs）方向分化,探讨其作为组织工程血管种子细胞的可行性。方法：获取犬骨髓单个核细胞,体外分离出MSCs。将分离的MSCs接种于含EBM-2完全培养液的培养瓶,向内皮样细胞定向诱导分化;LG-DMEM完全培养液培养的MSCs作为对照组。对MSCs表型进行流式细胞仪鉴定;诱导的内皮样细胞进行CD31、Ⅷ因子免疫细胞化学鉴定和摄取Dil-Ac-LDL、结合FITC-UEA的双染色功能鉴定。结果：流式细胞术检测培养的原代MSCs表面标志CD44表达呈阳性,而CD34表达则呈阴性;诱导分化的内皮样细胞具有内皮细胞形态特征,与成体内皮细胞相似;诱导的内皮样细胞Ⅷ因子染色和CD31免疫荧光染色均呈明显阳性,对照组细胞为阴性;诱导的内皮样细胞Dil-Ac-LDL摄取实验和FITC-UEA结合实验,细胞呈阳性表现。结论：在体外用骨髓MSCs能够成功诱导分化为血管内皮样细胞,为组织工程血管内皮化提供了一种可行的种子细胞来源。     

血管内皮生长因子在人工骨血管化中的应用

目的：探讨血管内皮生长因子(vascular endothelial growth factor，VEGF)在人工骨血管化中的作用。方法：(1)将人工骨材料用鼠尾胶原预湿3d后与血管内皮细胞(endothelial cells，ECs)悬液在体外进行复合；(2)在培养液中加入VEGF，并设立对照组；(3)1周后对人工骨材料表面进行电镜扫描，观察细胞生长情况。结果：实验组内皮细胞在人工骨材料表面呈片状生长，细胞形状呈梭形，而对照组内皮细胞黏附较差，细胞形状不规则。结论：VEGF可以促进内皮细胞在胶原包埋的生物陶瓷表面黏附形成血管内皮样结构。     

组织工程血管种子细胞培养的实验研究

目的：为血管组织工程的实验研究提供细胞来源，方法：分别采用消化法和和组织块法培养血管内皮细胞和血管平滑肌细胞，并进行两种细胞的传代、纯化、鉴定以及形态学观察，结果：培养的两种细胞均符合血管内皮细胞和血管平滑肌细胞的形态特征，结论：所培养的细胞可提供作进一步深入的血管组织工程的研究。     

硝酸银染色法鉴定生物瓣支架材料体外再内皮化程度的实验研究

目的探讨生物瓣支架再内皮化程度的形态学观察方法,为体外构建心脏组织工程瓣提供手段。方法选择经液氮保存的同种主动脉带瓣管道作为生物瓣支架,0.1%十二烷基硫酸钠(sodiumdodecylsulphate,SDS)脱去表面的内皮细胞(endothelialcells,ECs),纤维连接蛋白(bironectin,FN;80μg/ml)预覆盖瓣膜;选择从正常成人骨髓间充质干细胞(mesenchymalstemcells,MSCs)经体外定向诱导分化的ECs作为种子细胞,以>1～2×105个/cm2密度种植于瓣膜支架上,采用含20%胎牛血清和血管内皮细胞生长因子等的DMEM-HG培养基静态培养,0.5%硝酸银染色,于培养的7、14和20d在立体显微镜下观察、摄片,确定其内皮化程度。结果同种生物瓣表面的ECs完全脱去,而细胞外基质成分保存良好;种植细胞与生物瓣支架复合体静态培养的第7、14和20天,其内皮化程度分别为73%、85%和92%。结论硝酸银染色鉴定生物瓣支架再内皮化的方法,简便、经济,是体外构建心脏组织工程瓣形态学观察的一种有效手段。     

细胞凋亡在下肢静脉曲张发生机理中的作用

目的　探讨细胞凋亡和血管重塑(remodeling)在静脉曲张发生机理中的作用。方法　采用凋亡细胞原位检测(TUNEL)法,V.G.胶原纤维染色,静脉图像分析和电镜形态学等方法研究83例慢性静脉功能不全(CVI)和10例对照组。结果　①曲张静脉囊性扩张型与非囊性扩张型和对照组比较,内膜内皮细胞(ECs)、平滑肌细胞(SMC)和中膜SMC凋亡及凋亡指数显著增高,差异均有显著性意义(P＜0.01);②非囊性扩张型与对照组比较,差异无显著性意义(P＞0.05);③曲张静脉囊性扩张型和非囊性扩张型胶原含量均显著高于对照组(P＜0.05和P＜0.01);④囊性扩张型内膜、中膜明显变薄(P＜0.01);⑤逐步回归和相关分析提示静脉壁胶原蛋白含量和中膜SMC凋亡对静脉壁厚度作用较大(P=0.04143和P=0.006563)。胶原含量越高,管壁厚度越大(r=0.9777,P＜0.001);中膜SMC凋亡指数越大,管壁厚度越薄(r=-0.5432,P=0.003),而内膜ECs和SMC凋亡指数与管壁厚度无相关性(r=0.1619,P=0.420);⑥电镜超微结构显示曲张静脉ECs和SMC呈典型凋亡形态学特征。结论　静脉胶原含量增加,细胞过度凋亡和细胞构成减少导致静脉血管重塑,可能是静脉曲张发病机理之一。     

血管移植物内皮种植的新证据与新希望

自从1979年Herring等提出血管移植物可以种植上内皮细胞(ECs)的观点以来,许多研究者已在寻找可资利用的复合或单一的ECs以增加小口径移植物的通畅度。然而,由于ECs在移植物表面的粘着与繁殖均较差,故其在临床上的真正应用尚受到限制。最近,一些研究者建议使用细胞外基质成分例如纤维结合素以帮助ECs在移植物表面的粘着,作者也在这方面进行了研究,本文乃报道其研究进展。从人脐静脉获取ECs,并用改良的Jafee方法进行细胞的培养与繁殖。接生时由胎盘取得脐带,24小时内对脐静脉作以下处理:外表面洗涤、修整、冲净管腔、制取含脐静脉内皮细胞的混悬液以及作细胞培养等。用相差显微镜观察细胞形态,并用兔的抗人VIII因子抗原加以确认,同时测定内皮细胞融合的百分比,当细胞融     

肝星状细胞对VEGF介导的脂肪间充质干细胞向内皮细胞分化的影响

目的通过大鼠肝星状细胞(hepatic stellate cells,HSCs)与大鼠脂肪间充质干细胞(rat adipose tissue-derived mesenchymal stem cells,r ADSCs)在内皮细胞分化诱导培养基中的共培养,观察HSCs对r ADSCs向内皮细胞(endothelial cells,ECs)分化的影响。方法共培养实验设置四组,阳性对照组、实验组、阴性对照组和空白对照组。诱导培养2周后,观察检测各组r ADSCs向ECs的分化情况。结果诱导2周后,三组r ADSCs细胞体积逐渐变小变圆,部分细胞出现典型的内皮细胞鹅卵石样改变;四组细胞v WF、VE-Cadherin和表达均阳性;Western blotting检测结果与免疫荧光结果一致,实验组e NOS蛋白表达明显高于阴性对照组和空白对照组(P0.05);内皮细胞迁移试验显示,实验组每视野下细胞迁移数目明显高于阴性对照组和空白对照组(P0.01);低密度脂蛋白吞噬试验显示,实验组细胞吞噬Di IAc-LDL的能力明显高于空白对照组(P0.05);体外成血管试验显示实验组细胞体外成血管的能力明显高于其他三组细胞—阳性对照组(P0.05)、阴性对照组和空白对照组(P0.01)。结论 HSCs与r ADSCs的间接共培养可以促进大鼠血管内皮细胞生长因子(rat vascular endothelial growth factor,r VEGF)介导的r ADSCs向ECs的分化,并增强分化后ECs的功能。利用共培养体系对多种细胞共培养时进行生长分化现象的观察及其相互影响机制的研究,可以为组织工程功能性血管化组织器官及其生物模拟物的设计和创造提供更加可靠的理论指导和更加广阔的研究策略。     

同种生物心脏组织工程瓣体外构建的初步研究

目的　在生物瓣支架上种植并静态培养自体内皮细胞 (ECs) ,形成完整的内皮细胞单层 ,为下一步脉动流培养并最终体外构建同种生物组织工程瓣 (TEHV)提供材料基础。方法　生物瓣支架选择经液氮保存的成人主动脉带瓣管道 ,用 0 1 %SDS脱去表面的ECs ;成人骨髓间充质干细胞 (MSCs)体外定向诱导分化的ECs作为种子细胞 ,高密度 (>1 0 5cell/cm2 )种植于瓣膜支架上静态培养 2 0d ,扫描电镜观察、摄片 ,以确定再内皮化程度。结果　同种生物瓣支架表面的ECs完全脱去 ,而细胞外基质成分保存良好 ;MSCs体外诱导分化的ECs与生物瓣支架复合体静态培养第 7、1 4和 2 0d ,再内皮化程度分别为 73%、85 %和 92 %。结论　静态培养条件下构建的TEHV基本上实现了体外再内皮化的预期目标。     

Functional and phenotypic differences between glioblastoma multiforme-derived and normal human brain endothelial cells

OBJECT: Glioblastomas multiforme (GBMs) are hypervascular tumors characterized by endothelial cell (EC) proliferation. There is increasing evidence that ECs that infiltrate systemic tumors are different from normal blood vessel cells; whether this difference is seen in the central nervous system between GBM and normal brain tissue is not known. The goal of this investigation was to characterize and compare the functional and phenotypic properties of GBM-associated ECs and normal brain ECs. METHODS: Human ECs were isolated from fresh tissue specimens, purified using flow cytometry, and characterized by immunostaining. Proliferation was measured by determining bromodeoxyuridine incorporation and Ki-67 staining, and by performing the monotetrazolium assay. The migration rate of the cells was determined using the modified Boyden chamber technique. Apoptosis was evaluated by performing the TUNEL assay, cell death enzyme-linked immunosorbent assay (ELISA), and annexin V staining. Growth factor production was analyzed using the ELISA technique. The brain tumor ECs differed from normal brain ECs morphologically and by their expression and distribution of specific markers (that is, vascular endothelial cadherin [VE-cadherin] and CD31). Functional differences between the two cell populations were also evident. The brain tumor ECs proliferated more slowly and underwent less apoptosis than normal brain ECs; however, the tumor ECs migrated faster than the normal ECs. The normal ECs were sensitive to growth factors such as vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1), whereas the tumor ECs were not. In addition, the brain tumor ECs constitutively produced higher levels of ET-1 and VEGF, compared with the normal ECs. CONCLUSIONS: The data demonstrated that ECs derived from normal brain and from GBMs have significant phenotypic and functional distinctions. Further characterization of brain tumor ECs is essential for efficient antiangiogenic treatment of gliomas.     

人α1,2-岩藻糖苷转移酶和衰变加速因子基因转移克服异种移植急性血管排斥反应的研究

目的 观察人α1,2-岩藻精苷转移酶(HT)和衰变加速因子(DAF)基因转移对抑制血管内皮细胞激活从而克服异种移植急性血管排斥反应的能力.方法 通过显微注射建立转基因小鼠动物模型,Southern印迹杂交和流式细胞汁数筛选出人HT和/或DAF基因整合与表达阳性的子代转基因小鼠.研究表达不同目的 基因的转基凶小鼠血管内皮细胞与15%人血清孵育后溶破细胞的百分数,免疫细胞化学染色检测细胞核因子(NF)-κB的激活,流式细胞计数检测细胞表面血管细胞黏附分子(VCAM)-1的表达.结果 子代小鼠血管内皮细胞人HT/DAF共基因表达7只,人HT与DAF单基因表达分别为6只和8只.与15%人血清孵育后,人HT/DAF共表达组细胞的溶破细胞百分数(4±2)%低于人HT表达组细胞(25±10)%、人DAF表达组细胞(31±11)%及正常组细胞(76±24)%(P均＜0.05).转双基因抑制细胞NF-κB激活的能力强于转入HT或DAF单基因,且转双基因细胞表面VCAM-1的表达较转单基因细胞显著降低,差异有统计学意义(P＜0.05).结论 人HT/DAF基因联合转移具有部分抑制血管内皮细胞激活从而克服异种移植急性血管排斥反应的能力.     

Vascular endothelial cells evade apoptosis triggered by human leukocyte antigen-DR ligation mediated by allospecific antibodies

BACKGROUND: Human leukocyte antigen (HLA)-DR ligation mediates cell death of antigen-presenting cells (APC), including mature B cells, macrophages, and dendritic cells. This study investigates the apoptotic effects of HLA class II ligation mediated by anti-HLA antibodies on activated human vascular graft endothelial cells (ECs). METHODS: HLA class II expression was examined by flow cytometry using a panel of HLA-typed vascular ECs isolated from transplant donors and compared with that of B lymphocytes. The apoptotic effects of anti-HLA-DR monoclonal antibodies (mAbs) were investigated using viability assays, DNA content analysis, and annexin-V labeling. Intracellular signaling pathways mediated by HLA-DR ligation on ECs were examined by Western blotting. RESULTS: Even with optimal stimulation, the expression of HLA-DR on interferon (IFN)-gamma-treated ECs was quantitatively lower (3-5-fold) than that on B cells. Whereas anti-HLA-DR monomorphic mAbs induced apoptosis of B cells (approximately 22%), no significant apoptosis of IFN-gamma-activated (DR-positive) ECs ( < 5%), collected from the same donor, was observed under the same conditions. Similarly, specific polymorphic anti-HLA-DR11 or -DR16 antibodies were unable to induce EC apoptosis. Nevertheless, antibody-binding to HLA-DR on ECs is sufficient to induce intracellular signaling, as evident in the modulation of tyrosine phosphorylation and protein kinase (PK)C-alpha/beta and PKB/Akt activation. Our results suggest that HLA-DR ligation induces both common and divergent signaling events in ECs and B cells. CONCLUSION: Collectively, our data suggest that, in contrast with professional APC, graft ECs evade apoptosis mediated by HLA-DR ligation, not as a result of moderate HLA-DR expression but rather as a result of a specific signaling pathway.     

双层血管细胞种植提高人工血管内皮细胞粘附性

目的　采用血管内皮细胞 (EC)和平滑肌细胞 (SMC)双层种植方法 ,提高人工血管腔面EC的保存率。　方法　聚四氟乙烯 (PTFE)人工血管经纤维连结蛋白预衬处理 ,管腔面先后种植SMC和EC ,将受试的人工血管接入脉冲式体外灌注装置灌注 1h ,计算比较灌注前后受试标本SMC和EC密度。　结果　灌注 1h后 ,单纯EC种植组 61%细胞脱落 ,单纯SMC种植组 3 6%细胞脱落 ,而双层细胞种植组仅有 2 7%EC脱落 ,双层细胞种植组细胞保存率明显高于单纯EC种植组 (P <0 0 1)。低流速预灌注未能改善EC的保存率。　结论　在EC和人工血管壁之间种植SMC可提高EC的保存率     

婴幼儿主动脉缩窄合并心内畸形的外科治疗

目的 探讨婴幼儿主动脉缩窄合并心内畸形的外科治疗经验.方法 2000年1月至2006年12月,84例主动脉缩窄合并心内畸形患儿接受了外科手术治疗,手术年龄1个月～3岁(平均13.5个月),体重3.3～15.0 kg(平均7.3 kg).12例合并复杂心内畸形,72例合并室间隔缺损和其他简单心内畸形,23例伴有主动脉弓发育不良.一期手术62例,49例正中开胸同时矫治主动脉缩窄和心内畸形,13例左侧开胸矫治主动脉缩窄,正中开胸修补心内畸形;分期手术22例.主动脉缩窄的手术方式包括补片成形42例,切除端端吻合30例,锁骨下动脉翻转6例,血管旁路3例,球囊扩张1例.在49例正中切口一期手术中,43例应用选择性脑灌注加下半身停循环,4例应用全身低流量灌注,2例应用深低温停循环.结果 围手术期死亡8例,死亡率9.5%,其中3例为术前漏诊主动脉缩窄.结论 婴幼儿主动脉缩窄合并心内畸形的外科治疗可获得良好的近期疗效,绝大部分患儿可采取正中切口一期手术.选择性脑灌注和下半身停循环可以有效地保护脑和重要脏器.     

In vivo retention of endothelial cells adenovirally transduced with tissue-type plasminogen activator and seeded onto expanded polytetrafluoroethylene

PURPOSE: Seeding a prosthetic graft with genetically engineered vascular endothelial cells (ECs) has the potential to enhance the graft's antithrombotic properties. However, it has been reported that ECs transduced with tissue-type plasminogen activator (tPA) have very low levels of retention on grafts, probably because of increased proteolytic activity. We examined the retention of human tPA (htPA)-transduced ECs after the cells were seeded onto expanded polytetrafluoroethylene (ePTFE) grafts and implanted into dogs. We also examined the function of secreted htPA in this model.Methods and Results: Canine jugular venous ECs were transduced with adenoviral vectors encoding htPA (Adex1CAhtPA) and beta-galactosidase (Adex1CALacZ). There was a positive relationship between the percentage of X-gal ECs staining and the multiplicity of infection (MOI) of Adex1CALacZ. The level of htPA production in vitro increased with the increasing MOI of Adex1CAhtPA, but decreased gradually 4 days after infection. ECs coinfected with Adex1CAhtPA and Adex1CALacZ (htPAEC) or ECs infected with Adex1CALacZ alone (LacZEC) were seeded onto ePTFE grafts at densities equivalent to confluence to visualize retained ECs in an in vivo flow study. The grafts were implanted into canine carotid arteries and harvested after 5 hours of exposure to blood flow. The harvested grafts showed patchy defects in ECs, most of which were covered with mural thrombi. There was no significant difference in retention between htPAEC (29.3% +/- 8.7%) and LacZEC (19.5% +/- 3. 6%). There was a significant negative correlation between the in vivo EC retention on the grafts and the in vitro cellular passage level of ECs (P =.041; r = -.40). htPAEC produced 210.3 +/- 22.2 ng htPA antigen/10(6) cells per 6 hours in vitro and continued to secrete htPA on the harvested graft. CONCLUSIONS: We demonstrated that a large amount of functional htPA was produced by adenovirally modified canine ECs. The results of the in vivo study may suggest that overexpression of tPA has little effect on the short-term retention of early passage ECs seeded onto ePTFE grafts.     

Isolation of endothelial cells and their progenitor cells from human peripheral blood

Purpose: We have developed techniques to isolate endothelial cell (EC) progenitors from human peripheral and umbilical cord blood. Methods: Human adult peripheral and umbilical cord blood monocytes were isolated by centrifugation, and progenitor cells were separated with the use of magnetic polystyrene beads that were coated with a monoclonal antibody specific for the CD34 cell–membrane antigen. Cells were propagated in selective media, and developing cultures were immunostained for CD31, CD34, factor VIII, and vascular endothelial growth factor cell receptors. ECs that developed were transfected with a gene for prourokinase and used to line ePTFE grafts, which were evaluated in vitro in a pulsatile flow system. Results: Umbilical cord monocyte cultures demonstrated colonies that resembled ECs at approximately 2 weeks, with growth being best supported by EC growth media plus 20% calf serum with iron. Immunostaining of colonies was positive for CD31 and factor VIII. After 18 days in culture, CD34⁺ cells from adult peripheral blood were noted, which had the typical cobblestone appearance of ECs and immunostained positively for CD31 and factor VIII–related antigens. Cultures of umbilical cord–derived cells and adult peripheral blood–derived cells developed complex line formations within 1 week in culture that stained positively for vascular endothelial growth factor receptor-2. Urokinase-transfected ECs were shown to overexpress urokinase. Prosthetic grafts lined with transfected cells showed 87.33% ± 4.97% cell adherence after 2 hours in a pulsatile flow system at clinically relevant shear stress. Conclusion: We conclude that endothelial progenitor cells can be isolated from human adult peripheral and umbilical cord blood and developed into EC cultures as a source of cells for vascular graft seeding and gene therapy. (J Vasc Surg 2000;31:181-9.)