山茱萸多糖对D-半乳糖致衰老小鼠肾组织中Klotho基因表达的影响

目的研究山茱萸多糖对D-半乳糖致衰老小鼠肾组纵中Klotho基因表达变化的影响。方法昆明小鼠60只,随机分为青年对照组、衰老模型组、山茱萸多糖组,每组20只。采用D-半乳糖法制作衰老模型,以灌胃法给山茱萸多糖组小鼠灌服山茱萸多糖。采用分光光度法检测总抗氧化能力（T—AOC）和Mn—SOD活性水平,RT—PCR法检测Klotho和Mn—SOD基闪表达水平,Westernblot法检测Klotho蛋白表达最。结果与对照组比较,衰老组小鼠肾组织中Klotho基因、Mn—SOD活性和基斟表达均下降（P〈0．01）,总抗氧化能力（T—AOC）下降（P〈0．01）;与衰老模型组比较,山茱萸多糖组小鼠Klotho基困和蛋白表达明显升高（P〈0．01）、Mn-SOD基因表达和Mn-SOD活性明显增高（P〈0．01）,总抗氧化能力也显著提高（P〈0．01）。结论山茱萸多糖可能通过上调衰老小鼠肾组织中的Klotho表达和提高衰老小鼠抗氧化能力岍发挥抗衰老的作用。     

抗衰老Klotho蛋白对过氧化氢诱导的人脐静脉内皮细胞氧化损伤的保护作用及其机制

目的研究抗衰老Klotho蛋白对过氧化氢(H2O2)氧化损伤的人脐静脉内皮细胞(HUVEC)的影响及其作用机制。方法用H2O2诱导HUVEC构建氧化损伤模型。将HUVEC设置对照组、H2O2损伤组、不同浓度(1、10、100μg/L)Klotho蛋白组、丝氨酸-苏氨酸蛋白激酶(AKT)作用组。噻唑蓝法检测细胞存活率;特定试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)含量;酶联免疫吸附法检测一氧化氮(NO)、内皮素1(ET-1)、细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)、核因子κB(NF-κB)的含量;SA-β-半乳糖苷酶染色检测细胞衰老情况;流式细胞术检测活性氧(ROS)和细胞凋亡;Western blot检测Bcl-2、Bax、NF-κB p65及p-AKT的表达变化。结果与对照组相比,H2O2损伤组细胞存活率显著下降,LDH、MDA含量显著增加,SOD、GSH活性显著下降,NO含量下降,ET-1、ICAM-1、VCAM-1、NF-κB、细胞衰老率、ROS含量和细胞凋亡率显著增加,Bax蛋白表达上升而Bcl-2蛋白表达显著降低,NF-κB p65磷酸化水平显著升高而p-AKT/AKT水平下降(均P0.05)。在不同浓度Klotho蛋白组,细胞存活率逐渐上升,LDH、MDA含量逐渐下降,SOD、GSH活性随之上升,NO含量上升,ET-1、ICAM-1、VCAM-1、NF-κB、细胞衰老率、ROS含量和细胞凋亡率逐渐下降,Bax蛋白、NF-κB p65磷酸化水平逐渐下降,Bcl-2蛋白、p-AKT/AKT水平逐渐上升(均P0.05)。AKT作用组上述指标的检测结果与Klotho蛋白组相反。结论抗衰老Klotho蛋白能提升H2O2氧化损伤后HUVEC的存活率,增强细胞功能和抗氧化作用,减少细胞炎性反应、衰老和凋亡,其通过抑制Bax、NF-κB p65及促进Bcl-2、p-AKT/AKT而发挥作用。     

Implications of Klotho in vascular health and disease

Cardiovascular disease (CVD) is a prevalent condition in general population and the first cause of death overall. Klotho, a pleiotropic protein related to longevity that acts as a co-receptor of the fibroblast growth factor 23, has been proposed as a key regulator of the development of CVD. In the few clinical studies made, it has been observed a relationship between low levels of soluble Klotho and the occurrence and severity of CVD, as well as a reduction of cardiovascular risk when they are high. Also, different polymorphisms of human Klotho gene have been related to the incidence of cardiovascular events. Moreover, several experimental studies indicate that this protein acts in the maintenance of vascular homeostasis. Klotho improves endothelial dysfunction through promotion of NO production and mediates anti-inflammatory and anti-aging effects such as suppression of adhesion molecules expression, attenuation of nuclear factor-kappa B or inhibition of Wnt signaling. Furthermore, this protein is related to the attenuation of vascular calcification as well as prevention of cardiac hypertrophy. The expression of this protein in the vascular wall implies a new scenario for the treatment of vascular disorders. The purpose of this review is to provide an overview of the relationship between the Klotho protein and CVD, in addition to its role in the maintenance of functional vascular integrity.     

Vasostatin基因对胰腺癌细胞和血管内皮细胞增殖的影响

目的研究Vasostatin转基因对胰腺癌细胞、血管内皮细胞的作用，探讨其对胰腺癌生长抑制的作用机制。方法应用腺病毒载体将Vasostatin基因转入人胰腺癌细胞SW1990、人脐静脉血管内皮细胞ECV304，应用MTT方法检测转基因后细胞的生长活力。同时，应用小管形成实验研究Vasostatin对血管内皮细胞ECV304体外血管生成的影响。结果Vasostatin转基因对人胰腺癌SW1990细胞生长无显著影响。Vasostatin转基因（MOI分别为25和50）对人脐静脉血管内皮细胞ECV304作用72h后，Ad-Vasostatin组的ECV304细胞数目显著少于PBS组和Ad-lacZ组（P〈0．05）。小管形成实验结果显示，Ad-Vasostatin组内皮细胞数目较少，细胞排列连续性差，可见条索状的细胞链，但中空闭合的管状结构缺如。结论腺病毒介导的Vasostatin基因可显著抑制人脐静脉血管内皮细胞ECV304的体外细胞增殖，抑制其体外血管生成，而对人胰腺癌肿瘤细胞SW1990的体外细胞增殖无明显影响。     

西拉普利对房颤犬内皮和纤溶功能的影响

目的观察心房快速起搏诱发心房颤动犬内皮和纤溶功能的变化及西拉普利对其影响,探讨房颤血栓前状态的机制及防治对策。方法2004年1月至8月于哈尔滨医科大学第一临床医学院动物实验中心选杂种犬16只,随机分为对照组(n=8)、西拉普利组(n=8),在右心房以单心房(AOO)模式400/min起搏6周,建立房颤犬模型。西拉普利组起搏前1周开始每天服用西拉普利(1mg/kg),直至起搏结束,分别于起搏前、起搏6周后采静脉血,测血浆血管性血友病因子(vWF)、血管紧张素Ⅱ(AngⅡ)、组织型纤溶酶原激活剂(t-PA)、纤溶酶原激活剂抑制物-1(PAI-1)。结果心房快速起搏6周,对照组犬血浆AngⅡ较起搏前明显增高,[(349·9±28·3)ng/L对(198·4±19·4)ng/L,P<0·01];西拉普利组AngⅡ无明显升高(P>0·05);快速起搏后血浆vWF升高[(109·7%±19·8)%对(33·9±5·9)%,P<0·01),西拉普利起搏后血浆vWF亦升高,但却明显低于对照组(P<0·01)];起搏6周后血浆t-PA、PAI-1抗原均明显升高[t-PA:(12·7±2·1)ng/L对(8·9±1·5)ng/L,P<0·01][PAI-1:(24·4±3·9)ng/L对(15·4±2·4)ng/L,P<0·01];西拉普利组起搏后两者亦升高但两者升高程度低于对照组。结论慢性心房快速起搏导致犬肾素-血管紧张素系统(RAS)激活、内皮功能及纤溶功能失调,西拉普利可以阻断RAS激活,不同程度改善内皮和纤溶功能。血管紧张素转换酶抑制剂(ACEI)可能用于房颤血栓防治。     

High glucose induces plasminogen activator inhibitor-1 expression through Rho/Rho-kinase-mediated NF-kappaB activation in bovine aortic endothelial cells

Recently, it has become evident that elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The molecular mechanisms involved in hyperglycemia-induced PAI-1 expression in bovine aortic endothelial cells (BAEC) were investigated. PAI-1 expression in BAEC was significantly increased in accordance with the concentration of glucose in media from 5.7 mM to 23 mM. Stimulation with high glucose (23 mM) significantly increased small GTPase Rho A activation. Pretreatment with a Rho-kinase inhibitor, Y-27632 (1-10 microM), significantly blocked high glucose-induced PAI-1 expression. NF-kappaB activity determined using the luciferase reporter gene assay was significantly enhanced by high glucose, and pretreatment with Y-27632 inhibited high glucose-induced PAI-1 expression at the basal level. An inhibitor of NF-kappaB action, namely parthenolide (0.1 microM), BAY 11-7082 (5 microM) and SN50 (1 microM), significantly blocked high glucose-mediated PAI-1 expression to a level with low glucose (5.7 mM). These data suggested that high glucose-induced PAI-1 expression in endothelial cells is mediated by NF-kappaB activation through the Rho/Rho-kinase pathway. Inhibition of Rho/Rho-kinase signaling might be a novel target for diabetes and metabolic syndrome.     

人参皂苷Rb1对白消安诱导脐静脉内皮细胞凋亡的抑制作用

目的:研究白消安诱导人脐静脉内皮细胞(HUVEC)凋亡及人参皂苷Rb1对此作用的干预效应。方法:体外培养HUVEC,分别用不同浓度白消安刺激12h和24h,以Annexin-V-流式细胞术检测细胞凋亡,选定合适白消安作用浓度及时间;以人参皂苷Rb1预处理30min后,再用白消安刺激HUVEC,以流式细胞术检测凋亡。结果:高浓度白消安(0.12g/L)可显著诱导HUVEC凋亡,而人参皂苷Rb1可抑制此诱导凋亡作用。结论:白消安诱导内皮细胞凋亡可能是造血干细胞移植中内皮损伤的重要机制之一,而人参皂苷Rb1可通过抑制白消安诱导的内皮细胞凋亡发挥内皮细胞保护效应。     

Klotho protein diminishes endothelial apoptosis and senescence via a mitogen‐activated kinase pathway

Aim: Mice that carry the Klotho mutation (KL^‐/‐) manifest diverse age‐related disorders similar to those observed in humans. Thus, the Klotho protein might function as an anti‐aging hormone in mammals. Recently, we reported that Klotho recombinant protein attenuated apoptosis and cellular senescence in endothelial cells, but the mechanism remained unclear. Here, we designed an in vitro study to test whether inhibitors of extracellular signal‐regulated kinase and mitogen‐activated kinase kinase could affect Klotho regulation of apoptosis and cellular senescence. Methods: Cellular senescence was investigated in human umbilical vein endothelial cells treated with or without Klotho recombinant protein, and with or without inhibitors of mitogen‐activated kinases. Senescence was quantified by staining with senescence‐associated β‐galactosidase and by evaluating western blots probed for phosphorylation of mitogen‐activated kinases. Apoptosis was assayed on western probed for p53, p21, and caspase‐3 and ‐9. Results: The Klotho recombinant protein induced transient phosphorylation of mitogen‐activated kinases within a few minutes. Application of inhibitors of mitogen‐activated kinases attenuated the ability of Klotho to interfere with apoptosis and senescence in endothelial cells. Conclusion: This study demonstrated that Klotho attenuated cellular apoptosis and senescence in vascular cells via mitogen‐activated kinase kinase and extracellular signal‐regulated kinase pathways. Geriatr Gerontol Int 2011; 11: 510–516.     

老年人血管内皮依赖性舒张功能的变化

目的　观察老年人肱动脉内皮依赖性舒张功能的变化。方法　采用无创性超声法检测 1 4 5例受试者血流介导性肱动脉舒张 (FMD ,内皮依赖性舒张功能 )和硝酸甘油介导性肱动脉舒张 (NMD ,非内皮依赖性舒张功能 )。结果　年龄 >60岁受试者FMD较≤ 60岁者明显降低 (5 65 %± 6 1 2 % ,3 33 %± 3 72 % ,P =0 0 0 7) ;冠心病者较非冠心病者FMD(2 98%± 3 65 % ,8 37%± 6 41 % ,P <0 0 0 1和NMD(1 6 61 %± 7 2 5% ,2 2 78%±8 82 % ,P <0 0 1 )均明显降低 ,>60岁冠心病患者FMD较≤ 60岁冠心病患者降低 (1 90 %± 2 1 9% ,4 1 7%± 4 49% ,P <0 0 1 ) ;多因素逐步回归分析显示FMD与年龄 (t=- 3 92 6,P <0 0 0 1 )呈负相关 ;在单纯冠心病者仍显示FMD与年龄成负相关 (t=- 3 2 4 9,P =0 0 0 2 )。结论　老年人内皮依赖性血管舒张功能受损 ,年龄可直接损害内皮依赖性血管舒张功能 ,即使在冠心病存在时亦然 ,加重冠心病患者受损的内皮依赖性血管舒张功能。     

Klotho RNA干扰对人主动脉平滑肌细胞钙化和表型转化的影响

目的 探讨抗衰老因子(Klotho)RNA干扰对人主动脉血管平滑肌细胞钙化和表型转化的影响.方法 采用慢病毒抑制人主动脉平滑肌细胞的Klotho表达,通过荧光显微镜、荧光定量PCR及Western印迹法验证转染效率.采用微量法检测细胞内钙离子含量及碱性磷酸酶活性,酶联免疫吸附试验检测骨桥蛋白水平,采用荧光定量PCR检测...     

Klotho基因在胃癌组织中的表达及意义

目的:探讨Klotho基因在胃癌与癌旁组织中蛋白表达水平的差异及其与胃癌临床病理特征的关系.方法:采用免疫组织化学EnVision二-步法,检测Klotho蛋白在75对胃腺癌与其癌旁组织芯片中的表达情况,并结合临床病理资料进行统计分析.结果:胃癌组织中Klotho蛋白表达阳性率(76.0％)低于癌旁组织(90.7％)(...     

射频消融对血管内皮及血小板功能的影响

目的　研究射频导管消融 (RFCA)术对血管内皮和血小板功能的影响。方法　应用放射性免疫法、酶联免疫法、单克隆抗体标记及流式细胞技术 ,观察 31例心动过速患者RFCA手术前、后血浆内皮素 (ET)、血管性假血友病因子 (vWF)水平及血小板α 颗粒膜蛋白 (CD62 P)、血小板溶酶体膜蛋白 (CD63 )表达的变化。结果　RFCA术前、后血浆ET、vWF水平无明显改变 ,但术后即刻血小板膜CD62 P、CD63 表达分别由术前的 (4 .75± 2 .32 ) %和 (9.6 2± 4 .0 8) %增高至 (7.6 4± 5 .2 5 ) % (t =3.0 5 ,P <0 .0 1)和 (12 .2 3± 5 .70 ) % (t=2 .10 ,P <0 .0 5 ) ,术后 4 7～ 115h(平均 6 5h)均降至基础水平。多元相关分析结果显示CD62 P表达变化与累积放电能量呈显著性正相关 (r =0 .30 ,P <0 .0 5 )。结论　RFCA术不引起明显的内皮损伤 ,但可导致血小板膜CD62 P、CD63 表达增加 ,促进血小板活化 ,其中手术累积放电能量是重要的影响因素。     

切应力诱导人内皮细胞白细胞介素-8基因的转录活化

目的本研究检测层流低切应力诱导人脐静脉血管内皮细胞IL8基因的转录激活。方法RTPCR检测4.2dyne/cm2切应力处理0.5、1、2h人脐静脉血管内皮细胞的IL8mRNA表达。构建IL8报告基因质粒pEGFP1IL8USCS,转染脐静脉血管内皮细胞,切应力刺激3h后,流式细胞仪分析绿色荧光蛋白表达。免疫荧光细胞化学染色观察切应力处理脐静脉血管内皮细胞0.5、1、1.5、2h的NFκBp65核转移。用对照和切应力处理10、20、30、60min的脐静脉血管内皮细胞胞质蛋白,进行IκB和磷酸化IκB的免疫印迹。结果低切应力刺激可诱导脐静脉血管内皮细胞表达IL8mRNA。切应力刺激后,重组质粒转染的血管内皮细胞表达IL8绿色荧光蛋白报告基因。切应力诱导NFκB向胞核内转移,和IκB的磷酸化和降解。结论NFκB传导通路可能介导切应力诱导脐静脉血管内皮细胞IL8基因的转录活化,参与动脉粥样硬化的形成。     

Klotho基因在慢性心力衰竭患者心肌中的表达

目的探讨慢性心力衰竭(CHF)患者心肌组织中Klotho基因的表达及其与心肌纤维化的关系。方法心肌组织样本取材于2013年8月至2014年8月在成都军区昆明总医院心脏外科建立体外循环的60例CHF患者,按照纽约心脏联合会(NYHA)心功能分级将其分为:NYHAⅠ级组、Ⅱ级组、Ⅲ级组,每组患者各20例。运用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测Klotho基因表达量,同时采用酶联免疫吸附测定(ELISA)方法检测上述60例患者血清中Ⅰ型C端胶原前肽(PⅠCP)的浓度。并对正常心肌组织及CHF患者心肌组织进行HE染色。结果(1)HE染色显示CHF患者心肌胶原增生;(2)与NYHAⅠ级组比较,Klotho基因在NYHAⅡ级、NYHAⅢ级组的心肌组织中表达明显减少(均P0.05),且在NYHAⅢ级组中的表达较NYHAⅡ级组明显减少(P0.05);(3)NYHAⅢ级组患者血清中PⅠCP的含量高于Ⅱ级、Ⅰ级组(均P0.05),NYHAⅡ级组高于Ⅰ级组(P0.05)。结论 CHF患者心肌组织Klotho基因的表达减少可能与随着心功能恶化,其抗衰老作用及抗心肌纤维化作用逐渐减弱有关。     

同型半胱氨酸对人脐静脉内皮细胞纤溶系统的影响

目的探讨同型半胱氨酸（homocysteine,Hcy）对血管内皮细胞纤溶系统影响。方法（1）将体外培养的人脐静脉血管内皮细胞（HUVEC）分为10个实验组（0、10、50、200、500μmol／L Hcy组及叶酸和上述各Hcy点共同培养组）,培养24h后,酶联免疫吸附实验法（ELISA）测定各组细胞上清液中纤溶酶原激活剂（plasminogen activator,tPA）及纤溶酶原激活物抑制剂1（plasminogen activator inhibitor1,PAI-1）抗原含量,逆转录聚合酶链反应分析（RT-PCR）法分析各组tPA及PAI-1的mRNA表达水平。（2）急性心肌梗死（AMI）患者53例及健康对照组48例,ELISA测定空腹血浆tPA及PAI-1含量,高效液相色谱法测定血浆Hcy水平。结果（1）500μmoL／L Hcy组PAI-1抗原及mRNA表达水平均明显增高（P〈0．05）。（2）以单纯培养基为对照组,生理浓度Hcy组内皮细胞tPA抗原合成及mRNA表达明显增高（P〈0．05）,而以10μmoL／L Hcy组为对照组时,500μmoL／L Hcy组tPA抗原合成及mRNA表达水平则明显减少（P〈0．05）。（3）500μmoL／L Hcy与叶酸共同培养组和单纯Hcy组相比,可以明显提高内皮细胞tPA抗原的合成及mRNA表达,减少PAI-1抗原合成及mRNA表达（P〈0、05）。（4）AMI组Hcy、tPA及PAI-1均明显高于健康对照组（P〈0．05）。结论在体外细胞时,超生理浓度Hcy可以通过下调tPA、上调PAI-1的mRNA表达,减少内皮细胞tPA抗原的分泌及增加PAI-1抗原的合成,可能降低纤溶系统的活性。叶酸则可以减少Hcy引起内皮细胞纤溶系统的损害,起到保护作用。Hcy是AMI的一个独立危险因素。     

The effect of increased dietary fruit and vegetable consumption on endothelial activation, inflammation and oxidative stress in hypertensive volunteers

Background and aims

Public health campaigns recommend increased fruit and vegetable (FV) consumption as an effective means of cardiovascular risk reduction. During an 8 week randomised control trial among hypertensive volunteers, we noted significant improvements in endothelium-dependent vasodilatation with increasing FV consumption. Circulating indices of inflammation, endothelial activation and insulin resistance are often employed as alternative surrogates for systemic arterial health. The responses of several such biomarkers to our previously described FV intervention are reported here.

Methods and results

Hypertensive volunteers were recruited from medical outpatient clinics. After a common 4 week run-in period during which FV consumption was limited to 1 portion per day, participants were randomised to 1, 3 or 6 portions daily for 8 weeks. Venous blood samples for biomarker analyses were collected during the pre and post-intervention vascular assessments. A total of 117 volunteers completed the 12 week study. Intervention-related changes in circulating levels of high sensitivity C-reactive protein (hsCRP), soluble intracellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) did not differ significantly between FV groups. Similarly, there were no significant between group differences of change in homeostasis model assessment (HOMA) scores.

Conclusions

Despite mediating a significant improvement in acetylcholine induced vasodilatation, increased FV consumption did not affect a calculated measure of insulin resistance or concentrations of the circulating biomarkers measured during this study. Functional indices of arterial health such as endothelium-dependent vasomotion are likely to provide more informative cardiovascular end-points during short-term dietary intervention trials.     

Klotho在缺血再灌注损伤中的研究进展

缺血再灌注损伤在临床中十分常见且难以避免,是影响缺血性疾病治疗效果的一个重要因素。Klotho作为一种抗衰老蛋白,具有抗氧化应激、抗凋亡、抗炎、抑制钙超载等作用。本文结合国内外Klotho与缺血再灌注损伤相关疾病的最新研究报道,就Klotho与缺血再灌注损伤关系及其作用机制作一综述,为抗缺血再灌注损伤新药的后续研发提供依据。     

血脂康对高脂饮食家兔血管内皮细胞功能的保护作用

目的探讨血脂康对高胆固醇饮食家兔血管内皮细胞功能的保护作用。方法健康纯种新西兰白兔３０只，随机分层分组法分为对照组（普通饲料），高脂组（普通饲料＋１５％胆固醇），治疗组（普通饲料＋１５％胆固醇＋血脂康０８ｇ·ｋｇ－１·ｄ－１），在实验不同阶段，观察各组家兔血清脂质、一氧化氮及血浆内皮素、前列环素、血栓素含量及血管内皮细胞病理形态学改变。结果实验前各组血清总胆固醇（ＴＣ）、甘油三酯（ＴＧ）、低密度脂蛋白胆固醇（ＬＤＬＣ）、血浆内皮素（ＥＴ）１、血清一氧化氮（ＮＯ）、血浆血栓素（ＴＸ）Ｂ２及６酮前列腺素（ＰＧ）Ｆ１α等无明显差异。实验１２周，高脂组、血脂康治疗组血清ＴＣ、ＴＧ、ＬＤＬＣ及血浆ＥＴ１、ＴＸＢ２及ＴＸＢ２／６酮ＰＧＦ１α比值明显高于对照组，但治疗组明显低于高脂组（Ｐ值均＜０．０５）；血浆ＮＯ低于对照组（Ｐ＜０．０５），但治疗组高于高脂组（Ｐ＜０．０５）。病理结果显示：血脂康治疗组主动脉、冠状动脉粥样硬化病变及血管内皮细胞损伤明显轻于高脂组。结论血脂康具有明显的调整脂质代谢及保护血管内皮细胞功能的作用。     

激肽释放酶腺相关病毒载体的构建及对脐静脉内皮细胞功能的影响

目的构建激肽释放酶(HK)腺相关病毒(AAV)载体,检测重组病毒感染脐静脉内皮细胞系(HUVEC)后HK基因和内皮功能相关基因表达的变化。方法HK基因克隆人腺相关病毒载体质粒pAAV-MCS,和腺相关病毒包装质粒pAAV-RC、腺病毒辅助质粒pHe1per共转染293细胞,包装成带有HK基因的重组腺相关病毒(rAAV-HK)。以rAAV-HK感染HUVEC。RT-PCR法检测HK基因、内皮型一氧化氮合酶(eNOS)、凋亡蛋白酶(caspase-3)、内皮素-1(ET-1)、血管内皮生长因子(VEGF)、内皮素B_1受体以及缓激肽B_1受体、缓激肽B_2受体的mRNA转录变化。ELISA法测定HUVEC上清液和胞内HK的含量。结果成功构建了rAAV-HK。rAAV-HK感染HUVEC后,实验组胞内HKmRNA转录(0．59±0．12)比空白组(0．26±0．05)明显增加(P＜0．01)；实验组胞内HK含量(120．1±40．9)比空白组(30．8±12．8)显著升高(P＜0．01)；实验组eNOSmRNA转录(1．19±0．28)较空白组(0．66±0．11)增加(P＜0．05),实验组caspase-3mRNA转录(0．30±0．25)较空白组(0．67±0．27)减弱(P＜0．05)。VEGF、内皮素-1、内皮素B_1受体、缓激肽B_1受体、缓激肽B_2受体mRNA转录两组差异无统计学意义。结论rAAV-HK病毒感染HUVEC后能高效表达HK,并促进HUVEC胞内eNOSmRNA转录,抑制caspase-3mRNA转录,提示导入HK基因能够改善内皮功能。