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。     

高糖刺激对人THP-1巨噬细胞肝X受体和三磷酸腺苷结合盒转运子A1表达的作用

目的 探讨高糖对人THP-1巨噬细胞肝X受体（LXR）和三磷酸腺苷结合盒转运子A1（ABCA1）表达的影响。 方法 人THP-1单核细胞经佛波脂（PMA）诱导转化成巨噬细胞。用CD68检测巨噬细胞表面标志物表达。以不同浓度葡萄糖（5.6、11.1、22.2、33.3 mmol/L）和不同时间（0、0.5、2、6、12、24、48、72 h）刺激巨噬细胞,用实时荧光定量PCR法检测其LXRα、LXRβ、ABCA1 mRNA表达;Western 印迹法检测LXRα、LXRβ和ABCA1蛋白的表达,并分析其相互间作用。 结果 PMA诱导72 h后,TPH-1细胞CD68呈阳性表达。与正常浓度糖（5.6 mmol/L）刺激比较,高糖刺激24 h后,TPH-1巨噬细胞LXR和ABCA1 mRNA和蛋白表达均下调。高糖刺激30 min后, LXRα、LXRβ和ABCA1 mRNA和蛋白表达上调;2 h后开始下调,随着时间延长而逐渐减少。 结论 高糖可能通过抑制LXR和ABCA1的表达,参与了动脉粥样硬化的发生和发展。     

ABCA1在胆固醇结石患者胆囊黏膜上皮的表达

胆石病是一种常见病、多发病,受多种因素影响.腺苷三磷酸结合盒转运体A1(ATP binding cassette transport A1, ABCA1)是细胞膜脂质转运调节蛋白,促进细胞内游离胆固醇和磷脂的流出,在胆固醇逆转运(RCT)和高密度脂蛋白(HDL)生成的起始步骤中起重要作用[1]. 本研究通过检测胆囊黏膜中ABCA1的表达水平,以进一步探讨胆石病的发病机理.[通讯作者] 周启胜, E-mail: zhouqisheng56@163.com     

缺氧诱导因子-1α和血管内皮细胞生长因子在前列腺癌组织中的表达

目的：探讨缺氧诱导因子-1α(HIF-1α)和血管内皮细胞生长因子(VEGF)在前列腺癌中的表达及其与病理分级、临床分期的关系。方法：采用免疫组化方法检测前列腺癌和良性前列腺增生(BPH)组织中HIF-1α和VEGF的表达。结果：前列腺癌中HIF-1α和VEGF的表达均明显高于BPH;HIF-1α和VEGF在前列腺癌中的表达水平与其病理分级和临床分期均呈正相关;前列腺癌中的HIF-1α表达水平和VEGF的表达水平呈正相关。结论：HIF-1α和VEGF是检测前列腺癌的较好分子标志物,可望用于前列腺癌的辅助诊断和预后判断,HIF-1α是VEGF表达的调控因子之一。     

人脑膜瘤血管内皮细胞生长因子表达的临床意义

目的　探讨脑膜瘤血管内皮生长因子 (VEGF)基因表达与血管生成和脑水肿的关系。方法　应用免疫组织化学方法检测 3 5例脑膜瘤组织中VEGF基因表达 ;Ⅷ因子相关抗原单克隆抗体组织化学染色显示微血管 ,用微血管记数 (MVC)测定血管生成 ;从脑水肿与肿瘤本身的体积之比估计脑水肿程度。结果　 3 5例脑膜瘤VEGF表达率 77% (2 7/3 5 ) ,4例可疑染色 ,其中恶性脑膜瘤和血管母细胞型脑膜瘤呈现高表达 (10 0 % ) ;VEGF表达与脑膜瘤血管生成 ,瘤周脑水肿呈显著正相关 (r =0 .682 3 ,P <0 .0 1;r =0 .765 3 ,P <0 .0 1)。结论　VEGF存在于脑膜瘤组织中 ,在脑膜瘤血管生成和瘤周水肿中发挥重要作用。     

人衰变加速因子基因在猪血管内皮细胞中的表达

目的 通过转基因方法使猪的血管内皮细胞表达人DAF蛋白，抑制猪供人移植超急性排斥反应。方法 体外培养猪腹主动脉内皮细胞，用透射电镜方法进行鉴定。脂质体转染法将含有人DAF基因的真核表达载体PSFFV－DAV转染进猪的血管内皮细胞，经G418筛选（浓度为100mg/L)12d，获得具有抗性的克隆，进而增殖，传代，保种。通过流式细胞仪检测转染细胞DAF的表达情况。结果 转染了基因的细胞与未转染基因的细胞在流式细胞仪检测中其莹光强度分别为82.18和1.69。转染的细胞5代后其荧光强度为71.96。结论 转染了人DAF基因的细胞荧光强度明显高于对照组。导入的外源基因在细胞内能高效、稳定地表达，为转基因猪的建立奠定一定的基础。     

桃核承气汤对LPS诱导血管内皮细胞炎症因子及TLR4、TRIB3表达的影响

目的:观察桃核承气汤对脂多糖(LPS)致体外培养血管内皮细胞炎症因子及TLR4、TRIB3表达的影响。方法:SD大鼠桃核承气汤灌胃制备含药血清。LPS诱导主动脉血管内皮细胞损伤制备损伤模型,分为正常组(生理盐水灌胃血清)、正常加药组(桃核承气汤含药血清)、模型组(LPS+生理盐水灌胃血清)、模型加药组(LPS+桃核承气汤含药血清),ELISA检测比较各组IL-1β、IL-6、TGF-β蛋白表达水平,qPCR检测各组IL-1β、IL-6、TGF-β、TLR4、TRIB3的mRNA相对表达量。结果:与正常组比较,模型加药组IL-1β表达显著升高、IL-6、TGF-β表达升高,与模型组比较,其余三组IL-1β、IL-6蛋白明显抑制,模型加药组TGF-β蛋白表达显著上升,差异均有统计学意义(P<0.05)。与模型组比较,模型加药组IL-1βmRNA表达抑制,其余三组IL-6、TLR4的mRNA表达下降,而TGF-βmRNA表达升高,正常组和正常加药组TGF-βmRNA的降低,差异均有统计学意义(P<0.05)。结论:桃核承气汤可抑制炎症因子IL-1β、IL-6表达,促进抗炎因子TGF-β表达,发挥对血管内皮细胞的保护作用,TLR4信号通路可能参与其中。     

乌司他丁对促炎症因子诱导的肺微血管内皮细胞通透性和细胞因子产生的影响

目的:研究乌司他丁对炎症因子诱导的体外培养的大鼠肺微血管内皮细胞通透性和细胞因子产生的影响.方法:体外培养大鼠肺微血管内皮细胞分组进行实验:①对照组:内皮细胞培养液中加入生理盐水或乌司他丁;②炎症因子刺激组:因培养液中分别含烫伤血清、胰蛋白酶、前列腺素E2(PGE2)、缓激肽、组胺而分为5个炎症因子刺激亚组;③乌司他丁...     

白细胞介素1β对人肾小球系膜细胞LOX-1和ABCA1表达的影响

目的 研究白细胞介素1β（IL-1β）对人肾小球系膜细胞株（HMCL）植物血凝素样氧化低密度脂蛋白受体1（LOX-1）以及腺苷三磷酸结合盒转运体A1（ABCA1）表达的影响，及其与细胞胆固醇稳态的关系。 方法 实时定量PCR和Western印迹法检测IL-1β对人肾小球系膜细胞LOX-1、ABCA1表达的影响。 结果 人肾小球系膜细胞表达LOX-1 mRNA和蛋白。IL-1β促进人肾小球系膜细胞LOX-1 mRNA和蛋白表达，5 μg/L IL-1β刺激细胞0~24 h，LOX-1 mRNA表达于6 h达高峰，为对照的6.87倍；LOX-1蛋白24 h达高峰，为对照的1.88倍。IL-1β降低脂质负荷的人肾小球系膜细胞 ABCA1 mRNA和蛋白表达。5 μg/L IL-1β刺激细胞0~48 h，48 h时ABCA1 mRNA和蛋白下降最明显，分别为对照的19.0％和50.62％。 结论 IL-1β促进人肾小球系膜细胞LOX-1表达，抑制ABCA1的表达，导致细胞内胆固醇的失衡，促使其变成泡沫细胞，可能加重肾小球硬化和肾病的进展。     

肾细胞癌VEGF表达和MVD检测的临床意义

目的 探讨肾细胞癌（RCC）组织中血管内皮细胞生长因子（VEGF）的表达与肿瘤间质微血管密度（MVD）检测的临床意义。方法 采用免疫组化方法对65例肾细胞癌（RCC）及10例正常肾组织进行VEGF单克隆抗体及CD34单克隆抗体染色，观察RCC的VEGF表达与MVD之间的相关性。结果 VEGF的表达与肿瘤间质微血管密度之间存在正相关性，二者均与RCC的病理分级、临床分期及远处转移显著相关（P〈0.05，P〈O．001）。结论 VEGF与MVD可客观准确反映RCC的生物学行为，上述二项指标可作为评估RCC恶性程度、转移及预后的重要指标。     

Effects of cyclophilin A on cell proliferation and gene expressions in human vascular smooth muscle cells and endothelial cells

BACKGROUND: Cyclophilin A (CypA) is a cytosolic protein which involves many biological functions including immune modulation, cell growth, tumorigenesis, and vascular disease. The objective of this study was to determine the effect of CypA on cell proliferation and several gene expressions in human endothelial cells and vascular smooth muscle cells. METHODS: Human coronary artery endothelial cells (HCAEC), human lung microvascular endothelial cells (HMVEC-L), and human aorta smooth muscle cells (HAoSMC) were used in this study. Cells were treated with 10 nM CypA for 24 h. The cell proliferation was determined by [3H]thymidine incorporation. The mRNA levels of 13 genes including CD147 (receptor for CypA), PDGF-BB, endothelin-1 (ET-1), vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, neuropilin-1 (NRP-1), NRP-2, eNOS, iNOS, nNOS, ICAM-1, and PECAM-1 were semiquantitatively determined by real time RT-PCR as standardized with a house keeping gene beta-actin. RESULTS: CypA significantly increased cell proliferation of HAoSMC and HMVEC-L by 31% and 45%, respectively, as compared to controls, but had no effect on HCAEC. Blocking CD147 did not affect the mitogenic action of CypA. In addition, CypA also significantly increased the mRNA expression of CD147 by 43% and VEGFR-2 by 65% in HAoSMCs (P < 0.05, t test). HAoSMCs expressed much higher CD147 and neuropilin-1 (NRP-1) mRNA than HMVECs-L and HCAECs (P < 0.017, ANOVA). Furthermore, CypA increased ET-1 mRNA by 22% and VEGFR-1 mRNA by 23% in HMVECs-L, but had limited effects on HCAECs. HMVECs-L had much higher expressions of PDGF-BB, ET-1, VEGFR-2, VEGFR-1, VEGFR-3, and NRP-2 than HAoSMCs and HCAECs (P < 0.017, ANOVA). By contrast, HCAECs had much higher ICAM-1 mRNA levels than HMVECs-L and HAoSMCs (P < 0.017, ANOVA). CONCLUSIONS: These data demonstrate that CypA has a mitogenic effect on HAoSMCs and HMVECs-L, but not HCAECs. CD147 may not mediate the action of CypA. In addition, CypA substantially alters the mRNA levels of several key genes in human vascular cells, indicating potential multifunctional roles of CypA in vascular system. Furthermore, this study provides several new aspects of gene expressions in vascular cells.     

肾细胞癌中血管内皮生长因子的表达

目的 探讨肾癌组织中血管内皮生长因子的表达及其与肾癌生物学行为的关系。方法 应用单克隆鼠抗-VEGF抗体通过免疫组化SP法研究肾癌中血管内皮生长因子的表达。结果 60例肾癌组织中35例(58.3％)血管内皮生长因子阳性。血表达与组织学分级(P<0.05)和TNM分期(P<0.05)均明显相关。 结论 血管内皮生长因子表达对肾癌生物学行为有重要影响,设法抑制血管内皮生长因子有望成为肾癌治疗的另一有效方法。     

内皮抑素和平阳霉素对血管内皮细胞的作用

目的：比较重组人血管内皮抑制素（rHES）、平阳霉素（PYM）对血管内皮细胞（HUVEC）的作用效果,探讨相关作用机制,以揭示rHES应用于增生期血管瘤治疗的可行性。方法：将不同浓度rHES、PYM分别作用于HUVEC细胞,以MTT法比较两药作用24h、48h后的细胞抑制率,苏木精-伊红（HE）染色光镜下观察细胞形态变化,用免疫组化法检测细胞内血管内皮生长因子（VEGF）阳性表达的差异,流式细胞术分析细胞凋亡。结果r：HES、PYM作用后的吸光度值（OD值）均随浓度增大而减小,VEGF免疫组化染色细胞灰度值均增大,流式细胞仪测得两药均会引起细胞凋亡,联合使用表现出协同效应。光镜下可见：rHES作用后细胞形态明显改变,可看到大量凋亡细胞,PYM作用后大量细胞悬浮,贴壁细胞数量明显减少。结论r：HES、PYM均能促进HUVEC细胞凋亡,联合使用有协同作用。     

Angiogenesis in renal cell carcinoma: Evaluation of microvessel density, vascular endothelial growth factor and matrix metalloproteinases

BACKGROUND: Angiogenesis, the growth of new blood vessels, has a critical role in tumor growth and metastasis. The purpose of this study was to investigate the involvement of angiogenesis and angiogenic factors in the pathogenesis of renal cell carcinoma (RCC). METHODS: Formalin-fixed and paraffin-embedded tissue blocks from 70 patients with RCC were studied. The situations of tumor angiogenesis were evaluated by assessing microvessel density (MVD) through CD31 immunostaining. The expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) was detected immunohistochemically. RESULTS: The value of MVD ranged from 12.0 to 93.0 with a median of 39.91 in RCC. Of the 70 RCCs, the expression of VEGF was detected in 52 (74.3%), MMP-2 in 29 (41.4%) and MMP-9 in 19 (27.1%) cases. Statistical analysis revealed significant associations of the tumor stage with MVD, and the expression of VEGF and MMP-2 in RCC. Additionally, MVD was closely related to the expression of VEGF but was not related to the expression of MMP-2 and MMP-9 in RCC. CONCLUSION: The degree of angiogenesis may be closely related to the tumor progression of RCC. The expression of VEGF may be responsible for angiogenesis in RCC, and both VEGF and MMP-2 expression may function as tumor associated angiogenic factors in RCC.     

神经生长因子对人血管内皮细胞增殖作用的研究

目的:研究神经生长因子(Nerve growth factor,NGF)对人血管内皮细胞(Human vascular endothelial cell,HEVC)增殖作用的影响。方法:将NGF作用于体外培养的HEVC304,利用酶联免疫反应检测其增殖率,利用免疫组织化学的方法检测对增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)的表达和标记指数。结果:NGF作用组较对照组其细胞增殖率明显增加(P<0.001),NGF中和抗体组其细胞增殖率下降明显(P<0.05)。NGF作用组较对照组能明显增加HEVC对PCNA的表达和PCNA标记指数(P<0.05),NGF中和抗体组其PCNA表达和PCNA标记指数明显减少(P<0.05)。结论:NGF能明显促进HEVC的增殖。     

Synergistic effect of estrogen and VEGF on the proliferation of hemangioma vascular endothelial cells

Purpose

The aim of this study was to observe the influences of estradiol (E₂), vascular endothelial growth factor (VEGF), and 4OH-tamoxifen (TAM) on the proliferation of hemangioma vascular endothelial cells (HVECs).

Methods

Two strawberry hemangiomas with positive estrogen receptor staining from 2 infants (cases 1 & 2) were used for HVECs culture. The HVECs of passage 3 were cultured in estrogen-free improved minimum essential medium and divided into 5 groups based on different treatments: group 1, no treatment; group 2, treated with E₂; group 3, treated with VEGF; group 4, treated with both E₂ and VEGF; group 5, treated with E₂, VEGF, and TAM. Cell count (CC) and DNA proliferation index (PI) were determined on culture days 0, 3, 6, and 9.

Results

On day 9 in case 1, CC and PI were the following: in group 1, 1.15 ± 0.18 × 10⁵ mL and 19.96% ± 1.45%, respectively, presenting no statistically significant changes; in group 2, 1.38 and 1.61 times those of group 1, respectively (P < .01); in group 3, 2.10 and 1.61 times those of group 2, respectively (P < .01); in group 4, 1.62 and 1.40 times as high as with group 3, respectively (P < .01); in group 5, down to the levels of group 1. The results in case 2 were similar to those in case 1.

Conclusions

In vitro, the promoting effect of VEGF on HVECs proliferation is stronger than that of estrogen. Estrogen and VEGF enhance this proliferation in a synergistic fashion, which can be inhibited by tamoxifen.     

肾移植受者血清抗血管内皮细胞特异性抗体的提取和纯化

目的  探讨肾移植术后出现排斥反应的受者血清中抗血管内皮细胞特异性抗体的提取和纯化方法。方法  首先分离培养人脐静脉血管内皮细胞(HUVEC)。收集肾移植术后肾功能不良受者的血清样本, 应用流式细胞术进行抗血管内皮细胞特异性抗体的筛选; 用Luminex抗体检测平台检测血清中抗人类白细胞抗原(HLA)和主要组织相容性复合体Ⅰ类相关链A (MICA)的抗体。在确定血清标本中存在有抗血管内皮细胞特异性抗体后, 用HUVEC将其吸附。洗涤细胞后再将吸附的抗体从细胞膜上洗脱下来, 再用Protein-A/G磁珠再次纯化和浓缩洗脱液中的抗体IgG。采用流式细胞术检测洗脱液中抗体活性, 用SDS-聚丙烯酰胺凝胶(SDS-PAGE)凝胶和免疫印迹法鉴定纯化的抗血管内皮细胞特异性抗体(IgG)。结果  在386例肾移植受者的血清中, 选取血清肌酐(Scr)>400 μmoI/L、抗HLA抗体阴性、抗MICA抗体阴性和荧光反应强度(MFI)>16的5例受者的血清样本, 纯化后的抗血管内皮细胞特异性抗体IgG在SDS-PAGE凝胶显示免疫球蛋白重链(纯度>95%)。流式细胞术结果显示纯化的抗体具有重新结合血管内皮细胞表面抗原的特性。结论  采用人脐静脉血管内皮细胞吸附肾移植受者血清中抗血管内皮细胞特异性抗体的提纯方法可取得较好效果。