硬脑膜血管内皮细胞生长因子表达及增殖活性与慢性颅内静脉压增高状态

目的：探讨慢性颅内静脉压增高状态下硬脑膜血管内皮细胞血管内皮细胞生长因子(vascular endothelial growth factor，VEGF)表达及增殖活性的变化。方法：实验在解放军沈阳军区总医院动物实验中心完成。选择Wistar雄性大鼠65只，造模成功24只，随机分为实验组12只，对照组12只，右侧颈总动脉和颈外静脉端-端吻合，同时结扎上矢状窦，建立慢性颅内静脉压增高的动物模型；90d后采用免疫组化方法检测硬脑膜血管内皮细胞中VEGF及增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)的表达情况。结果：对照组的硬脑膜血管中VEGF仅呈微弱表达，其吸光度值(A)为2．12&;#177;0．59，实验组硬脑膜中血管内皮细胞VEGF表达A值为8．66&;#177;1．75，两者相比实验组微血管生成数量显著增高(P&;lt;0．05)；微血管密度(&;#215;200倍计数)对照组为(3．04&;#177;0．31)／视野，实验组为(11．70&;#177;2．36)／视野，两者相比实验组微血管生成密度显著增高(P&;lt;0．05)。对照组的硬脑膜血管中PCNA仅呈微弱表达，其A值为1．12&;#177;0．19，实验组硬脑膜血管内皮细胞中PCNA表达A值为6．46&;#177;1．02。两者相比实验组血管内皮细胞增殖活性显著增高(P&;lt;0．05)。结论：慢性静脉压增高状态下硬脑膜血管内皮细胞VEGF表达升高，PCNA增殖活跃，可能与血管生成及动静脉畸形的发生发展有关。     

大鼠脑动静脉瘘模型硬脑膜中血管内皮生长因子表达与超微结构变化

目的:探讨大鼠脑动静脉瘘模型硬脑膜中血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)表达及其与血管超微结构改变的关系。方法:选Wistar雄性大鼠24只(实验组:n=12;对照组:n=12),右侧颈总动脉和颈外静脉端-端吻合,同时结扎上矢状窦,建立大鼠脑动静脉瘘模型的动物模型;90d后采用免疫组化方法检测硬脑膜血管内皮细胞中VEGF的表达和血管生成情况,透射电镜观察血管壁超微结构变化。结果:对照组的硬脑膜血管中VEGF仅呈微弱表达,其吸光度值(A)为2.12±0.59,实验组硬脑膜中血管内皮细胞VEGF表达A值为8.66±1.75,两者相比实验组微血管生成数量显著增高(P<0.05);微血管密度(×200倍计数)对照组为(3.04±0.31)/mm2,实验组为(11.70±2.36)/mm2,两者相比实验组微血管生成密度显著增高(P<0.05)。而且实验组血管内皮细胞的间隙增宽,平滑肌数量减少,胶原组织增多。结论:大鼠脑动静脉瘘模型硬脑膜血管中VEGF表达升高,可能与血管生成及血管的超微结构改变有重要关系。     

大鼠脑动静脉瘘模型硬脑膜中血管内皮生长因子表达与超微结构变化

目的：探讨大鼠脑动静脉瘘模型硬脑膜中血管内皮细胞生长因子(vascular endothelial growth factor，VEGF)表达及其与血管超微结构改变的关系。方法：选Wistar雄性大鼠24只(实验组：n=12；对照组：n=12)，右侧颈总动脉和颈外静脉端一端吻合，同时结扎上矢状窦，建立大鼠脑动静脉瘘模型的动物模型；90d后采用免疫组化方法检测硬脑膜血管内皮细胞中VEGF的表达和血管生成情况，透射电镜观察血管壁超微结构变化。结果：对照组的硬脑膜血管中VEGF仅呈微弱表达，其吸光度值(A)为2．12&;#177;0．59，实验组硬脑膜中血管内皮细胞VEGF表达A值为8．66&;#177;1.75，两者相比实验组微血管生成数量显著增高(P＜0．05)；微血管密度(&;#215;200倍计数)对照组为(3．04&;#177;0．31)／mm^2，实验组为(11．70&;#177;2．36)／mm^2，两者相比实验组微血管生成密度显著增高(P＜0．05)。而且实验组血管内皮细胞的间隙增宽，平滑肌数量减少，胶原组织增多。结论：大鼠脑动静脉瘘模型硬脑膜血管中VEGF表达升高，可能与血管生成及血管的超微结构改变有重要关系。     

慢性颅内静脉压增高状态下脑血管Tie受体表达与血管生成

背景Tie是血管内皮细胞特异性的酪氨酸激酶受体,慢性颅内静脉压增高状态下Tie1和Tie2表达的变化可能参与硬脑膜动静脉畸形的病变过程.目的观察慢性颅内静脉压增高状态下脑血管Tie受体表达及血管生成的变化.设计单因素设计.单位解放军沈阳军区总医院神经外科.对象实验于2003-12/2004-10在沈阳军区总医院动物实验中心进行.取雄性Wistar大鼠65只,24只造模成功,随机分为模型组和正常组各12只. 方法模型组大鼠右侧颈总动脉和颈外静脉端-端吻合,同时结扎上矢状窦,建立慢性颅内静脉压增高的动物模型;正常组仅单纯结扎右侧颈总动脉.两组大鼠均于手术后90d麻醉状态下断头取右侧大脑中动脉供血区的大脑皮质,采用免疫组化方法检测Tie受体表达及微血管密度.主要结局观察两组大鼠大脑皮质中Tie1及Tie2表达和微血管密度.结果24只大鼠进入结果分析.①脑组织中Tie1表达模型组和正常组的吸光度值比较无差异(3.96±0.61,4.35±0.72,P＞0.05).②脑组织中Tie2表达模型组的吸光度值显著高于对照组(8.16±1.04,1.08±0.45,P＜0.05).③脑组织中微血管密度模型组显著高于对照组[(10.34±1.05),(2.68±0.29)个/视野],并且越接近上矢状窦处,微血管密度越大结论慢性静脉压增高状态下脑血管内皮细胞Tie2表达和微血管密度升高,提示脑微血管处于高度增殖状态,而Tie1的相对低表达导致新生血管为病理性的未成熟血管.说明颅内静脉压增高不仅开放了潜在的硬脑膜动静脉吻合和侧支循环,而且启动了病理性血管生成过程,导致硬脑膜动静脉畸形不断发展扩大.     

慢性颅内静脉压增高状态下脑血管Tie受体表达与血管生成

背景：Tie是血管内皮细胞特异性的酪氨酸激酶受体，慢性颅内静脉压增高状态下Tiel和Tie2表达的变化可能参与硬脑膜动静脉畸形的病变过程。目的：观察慢性颅内静脉压增高状态下脑血管Tie受体表达及血管生成的变化。设计：单因素设计。单位：解放军沈阳军区总医院神经外科。对象：实验于2003-12／2004-10在沈阳军区总医院动物实验中心进行。取雄性Wistar大鼠65只，24只造模成功，随机分为模型组和正常组各12只。方法：模型组大鼠右侧颈总动脉和颈外静脉端一端吻合，同时结扎上矢状窦，建立慢性颅内静脉压增高的动物模型；正常组仅单纯结扎右侧颈总动脉。两组大鼠均于手术后90d麻醉状态下断头取右侧大脑中动脉供血区的大脑皮质，采用免疫组化方法检测Tie受体表达及微血管密度。主要结局观察：两组大鼠大脑皮质中Tiel及Tie2表达和微血管密度。结果：24只大鼠进入结果分析。①脑组织中Tiel表达：模型组和正常组的吸光度值比较无差异（3．96&;#177;0．61，4．35&;#177;0．72，P〉0．05）。②脑组织中Tie2表达：模型组的吸光度值显著高于对照组（8．16&;#177;1．04，1．08&;#177;0．45，P〈0．05）。③脑组织中微血管密度：模型组显著高于对照组[（10．34&;#177;1．05），（2．68&;#177;0．29）个／视野]，并且越接近上矢状窦处，微血管密度越大结论：慢性静脉压增高状态下脑血管内皮细胞Tie2表达和微血管密度升高，提示脑微血管处于高度增殖状态，而Tie1的相对低表达导致新生血管为病理性的未成熟血管。说明颅内静脉压增高不仅开放了潜在的硬脑膜动静脉吻合和侧支循环，而且启动了病理性血管生成过程，导致硬脑膜动静脉畸形不断发展扩大。     

抗血管生成药物反应停抑制乳腺癌转移的初步研究

目的探讨人乳腺癌原位异种移植的免疫缺陷小鼠,经具有抗血管生成作用的药物反应停处理后,是否对自发性肺转移形成有所影响。方法人乳腺癌细胞系,原位移植于严重联合免疫缺陷小鼠乳腺,建成乳腺癌转移模型,分为对照组及实验组,细胞移植后第10天起实验组给予反应停每天灌胃,至第60天全部处死。观察移植瘤生长及自发性肺转移情况,并应用免疫组织化学方法检测肿瘤组织内微血管密度(MVD)及增殖细胞核抗原(PCNA)的表达。结果反应停在剂量为100mg/kg时能显著减少肺转移灶数,与对照组相比差异有显著性[(12.6±13.9)个对(46.1±35.9)个,P<0.05]。实验组MVD值为(14.5±3.4)个/cm2,与对照组(26.3±11.9)个/cm2相比显著减少(P<0.05)。二组原发瘤的PCNA表达差异无显著性。结论本实验研究初步结果表明反应停能通过抑制肿瘤血管生成抑制转移。     

锌原卟啉对种植性乳腺癌血管生成影响的实验研究

目的探讨锌原卟啉(ZnPP)对种植性乳腺癌VEGF和血管生成的影响。方法应用种植性乳腺癌模型,经过ZnPP和钴原卟啉(CoPP)处理,检测乳腺癌组织中的血红素氧合酶1(HO-1)酶活性,免疫组化、Western blot蛋白印迹杂交检测癌组织中HO-1、VEGF蛋白表达,RT-PCR检测VEGFmRNA,CD34染色肿瘤微血管内皮细胞并对其进行定量分析。结果ZnPP组肿瘤直径[(7±1.06)mm,n=7]明显小于其余各组(t=3.5334,P<0.001);CoPP组[(13±2.82)mm,n=13]大于空白对照组[(10±1.63)mm,n=8](t=3.6564,P<0.002)。CoPP组VEGFmRNA(0.3046±0.0102)明显高于空白对照(0.1524±0.0046)及ZnPP组(0.1216±0.0043)(t=8.8491,P<0.001)。ZnPP处理组HO-1及VEGF蛋白印迹杂交结果低于CoPP组和空白对照组(t=4.698,P<0.005)(t=5.5428,P<0.005)。ZnPP处理组MEA为224.81±79.4266,明显低于CoPP组(448.79±138.46)(t=5.3426,P<0.05)。结论ZnPP可能通过抑制HO-1蛋白的表达来降低VEGF表达,降低肿瘤血管生成,并可能由此降低了肿瘤细胞的增殖生长。     

色素上皮细胞衍生因子对脑胶质瘤抑制作用的研究

目的评价重组色素上皮细胞衍生因子(rPEDF)对与胶质瘤有关的血管活性因子、增殖和侵袭的影响。方法采用酶联免疫吸附测定(ELISA)法检测血管内皮细胞生长因子(VEGF)与血小板反应素-1(TSP-1)的变化;四甲基偶氮唑蓝[3-(4,5-dimthy-2-2thiazoly)2,5-dipheny-2H-tetrazoliunbromide,MTT]法检测胶质瘤细胞增殖率;细胞侵袭实验检测rPEDF抑制胶质瘤细胞侵袭性。结果实验数据显示:A172PEDF组(将rPEDF加入胶质瘤细胞A172中)中VEGF表达量为0·29±0·03,与未加PEDF蛋白的胶质瘤细胞作对照组(A172con组,1·31±0·02)相比,减少4·4倍,(P<0·05),同时TSP-1的表达量A172PEBF组为1585±105·67,与A172con组的,351±20·65相比,表达上调4·5倍(P<0·01);侵袭实验结果表明:胶质瘤细胞穿透数为88·67±3·32明显多于rPEDF处理过的细胞(44·41±13·81,2·0倍),加入促血管生成因子—VEGF后,细胞穿透数仍减少至69·4%(69·42±6·35,P<0·05),表明rPEDF能有效抑制胶质瘤细胞的体外侵袭性,至少部分是在血管生成因子调节的作用下。结论rPEDF是抑制胶质瘤增殖和侵袭的主要因素。     

血管内皮细胞生长因子反义RNA抑制人食管癌血管生成及生长和转移作用的研究

目的探讨血管内皮细胞生长因子反义RNA在调节食管癌血管生成及生长、转移中的作用。方法用脂质体法将血管内皮细胞生长因子反义RNA转染到人食管癌TE-1细胞,噻唑蓝还原法检测细胞增殖情况;免疫组织化学和逆转录聚合酶链反应技术检测血管内皮细胞生长因子蛋白和mRNA的表达水平,并将转基因肿瘤细胞接种于裸鼠体内,应用免疫组化法检测肿瘤内微血管密度,探讨微血管密度与肿瘤生长及转移的关系。结果反义血管内皮细胞生长因子基因的转染,使肿瘤细胞血管内皮细胞生长因子分泌减少,反义组裸鼠的肿瘤组织微血管密度(11.0±7.6)明显低于对照组及空载体组(50.8±11.7、48.9±7.0)(均为P<0.01)。结论血管内皮细胞生长因子反义RNA可使肿瘤细胞分泌血管内皮细胞生长因子减少,血管生成能力降低,有望成为食管癌基因治疗的靶基因之一。     

喉癌瘤内微血管密度、血管内皮细胞生长因子的表达及其临床意义

目的 :探讨肿瘤血管生成与喉癌颈淋巴结转移的相关性及血管内皮生长因子与喉癌血管生成的关系。方法 :运用CD34单抗、VEGF多抗免疫组化SABC法检测 34例喉癌中瘤内微血管密度及VEGF的表达。结果 :①颈淋巴结转移组喉癌瘤内微血管密度显著高于非转移组 ( 2 6 33± 9 70、17 6 8± 6 0 6 ,P <0 0 1) ;②喉癌微血管密度高于均数组 ,颈淋巴结转移率显著增高 ( P <0 0 5) ;③喉癌组织VEGF表达与微血管密度呈正相关。结论 :喉癌瘤内微血管密度可作为预测患者预后的一个重要指标 ;VEGF对喉癌血管生成具有促进作用     

内皮祖细胞内皮修复的影响因素

内皮祖细胞（EPC）来源于骨髓,能够定向分化为成熟有功能的血管内皮细胞,是血管内皮细胞的前体细胞,又称血管母细胞。内皮祖细胞在组织缺血和血管损伤时动员入血,促进微血管的生成和血管内皮的修复,不仅参与胚胎血管生成,也参与出生后的血管发生过程,具有保护血管内皮、减少心血管疾病的发生率和死亡率的作用。许多试验证实冠状动脉粥样硬化性心脏病（冠心病）和心力衰竭患者EPC数量减少,外周循环内皮细胞功能受损。研究发现,EPC的生物学特性受到诸多冠心病危险因素的影响,致使其功能异常。     

Circulating endothelial cells: a novel marker of endothelial damage

Circulating endothelial cells (CECs) were first described over 30 years ago in smears of peripheral blood. Since then, more sophisticated techniques for CEC isolation have become available. In particular, immunomagnetic isolation and fluorescence-activated cell sorting (FACS) have been employed with success. We provide a short historical perspective and a comprehensive review on the subject. We review isolation and enumeration of CECs with an emphasis on CD146-driven immunomagnetic isolation and FACS. We describe, in great detail, advantages and pitfalls of both approaches and compare their specificity. Moreover, we provide a comprehensive list of clinical studies in this field and describe the possible clinical use of CECs. We also describe the phenotype of these cells and list typical surface markers. In addition, we review the phenotype of CECs and discuss mechanisms of detachment. We speculate about potential interactions between CECs and other cell subsets. We also describe other serum markers of endothelial damage and compare CECs with these markers. Finally, we highlight differences between circulating endothelial cells and endothelial progenitor cells. In summary, CECs must now be regarded as a sensitive and specific marker of endothelial damage. We emphasize that use of CECs in a clinical setting is on the horizon and pathogenetic clues may also be obtained.     

Origins of circulating endothelial cells and endothelial outgrowth from blood

Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5-20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after approximately 1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only approximately 20-fold over this period, whereas donor-genotype endothelial cells expanded approximately 1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts.     

Parathyroid hormone stimulates the endothelial expression of vascular endothelial growth factor

Background We showed previously that parathyroid hormone (PTH) may stimulate the endothelial expression of pro‐atherosclerotic and pro‐inflammatory markers. Considering the impact of PTH on vasculature, we decided to evaluate its effect on mRNA and intra‐cellular protein expressions of endothelial vascular endothelial growth factor (VEGF) taking into account that VEGF may play a role in the pathogenesis of endothelial dysfunctions. Materials and methods Human umbilical vein cords endothelial cells (HUVEC) were stimulated for 24 h with 10^?12–10^?10 mol L^?1 PTH. The VEGF‐165 mRNA expression (critical in stimulating endothelial cell proliferation) was evaluated by RT/PCR and the intra‐cellular VEGF protein expression by flow cytometry. The pathways by which PTH may have an effect on VEGF expression were also evaluated. Results PTH (10^?10 mol L^?1) significantly increased VEGF‐165 mRNA expression (P < 0·05). The addition of 50 nmol L^?1 protein kinase C (PKC) and 10 µmol L^?1 protein kinase A (PKA) inhibitors significantly reduced the VEGF‐165 mRNA expression (P = 0·01). We also examined whether nitric oxide (NO) may be involved in the PTH‐induced stimulation of VEGF‐165 expression. Pre‐treatment of the cells with 200 µmol L‐nitro arginine methyl ester (L‐NAME, NO synthase inhibitor) was found to inhibit VEGF‐165 mRNA expression (P = 0·006). VEGF protein could not be detected in the medium of HUVEC but it was present in the cell cytoplasm. PTH had no significant effect on cytoplasmatic VEGF protein expression. Conclusion The stimulatory effect of PTH on endothelial VEGF‐165 mRNA expression is partly through PKC and PKA pathways and is also NO dependent.     

Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry

The finding of angiogenic and vasculogenic cells in the peripheral circulation may have profound effects on the course of a variety of diseases ranging from cancer to cardiovascular disease. These cells are ascribed to be endothelial in nature and are generally referred to as circulating endothelial cells if mature or as endothelial progenitor cells if immature. Different approaches have been used to detect these cells, including in vitro culture, magnetic bead isolation, and flow cytometry. We review flow cytometric methods for the detection and enumeration of these cells and provide technical suggestions to promote the accurate enumeration of circulating endothelial cells and endothelial progenitor cells.     

缺氧时内皮细胞中血管内皮生长因子受体的表达

目的 探讨脐带静脉内皮细胞在缺氧条件培养下血管内皮生长因子特异受体的表达，为进一步研究血管内皮生长因子及其受体在新血管生成中的作用提供理论依据。方法 体外培养脐带静脉内皮细胞，不同缺氧时间处理及血管活性因子刺激，提取组织总RNA，应用Northern杂交和逆转录聚合酶连扩增反应（RT－PCR）等方法，观察基因表达的变化。结果 内皮细胞无缺氧和缺氧3h均未见flt-1表达，缺氧6h可见flt-1摸板核糖核酸（mRNA)的表达。内皮细胞缺氧6h，内皮素（ET）、一氧化氮（NO）、LNNA对flt-1表达有影响。ET促进flt-1 mRNA的表达，NO抑制其表达，而LNNA部分恢复其表达。在无缺氧和缺氧3，6h KDR基因均有表达。内皮细胞缺氧6h，ET、NO、LNNA对KDR无影响。LNNA阻断了内源性NO后，明显促进KDR mNRA的表达，其他无作用。结论 在缺氧条件下血管内皮细胞生长因子的受体flt-1表达明显增加并受血管活性因子调节，KDR有表达且部分受血管活性因子调节。