整合素αvβ3在血管紧张素Ⅱ诱导人脐静脉内皮细胞衰老中的变化

目的观察整合素αvβ3在血管紧张素Ⅱ(AngⅡ)诱导的人脐静脉内皮细胞(HUVECs)衰老中的变化。方法体外培养HUVECs,采用CCK-8法检测细胞存活率,用AngⅡ(终浓度10-6mol/L)干预,分为实验对照组、AngⅡ诱导组。以衰老相关β-半乳糖苷酶活性和细胞增殖能力两种衰老标志物为主要观察指标。其中衰老相关β-半乳糖苷酶活性采用免疫化学染色方法,流式细胞术检测细胞周期来反应细胞的增殖能力;利用Western印迹法分析AngⅡ诱导HUVECs 0、12、24、36、48 h的整合素αvβ3表达的时间效应关系。结果与对照组相比,10-6 mol/L AngⅡ诱导组存活的细胞数为对照组的(77.15±6.83)%;(81.80±0.92)%的细胞呈现β-半乳糖苷酶阳性染色,流式细胞仪检测细胞周期停滞于G0～G1,证实细胞衰老;AngⅡ呈时间依赖性上调整合素αvβ3表达。结论 AngⅡ可以诱导HUVECs衰老,其机制可能与上调衰老细胞整合素αvβ3表达有关。     

TM4SF1对人脐静脉内皮细胞管腔形成的影响

目的研究TM4SF1对内皮细胞体外管腔形成的影响。方法应用qRT-PCR方法检测人脐静脉内皮细胞（HUVECs）中TM4SF1的mRNA表达量;应用siRNA方法瞬时转染HUVECs,采用qRT-PCR方法检测转染48h与72h后TM4SF1的沉默效果,选择最佳的转染时间,应用In Vitro Angiogenesis Assay Kit观察siControl与siTM4SF1转染后HUVECs体外管腔形成情况。结果qRT-PCR实验结果显示,HUVECs中表达TM4SF1的RQ值为（0．71±0．11）,已知高表达TM4SF1胰腺癌细胞株MPanc96表达TM4SF1的RQ值为（0．56±0．13）,两种细胞中TM4SFl表达量差异无统计学意义（P〉0．05）;siRNA瞬时转染HUVE Cs48h与72E后,TM4SF1表达分别下降了47．06％与93．14％,HUVECs瞬时转染72h后进行体外管腔形成实验,通过对管腔形成情况进行评级,siControl转染后HUVECs管腔形成情况为5级（多个闭合管状结构相连形成筛状）,siTM4SF1转染后为2级（细胞排列形成夹角）。结论TM4SF1在HUVECs中高表达,沉默TM4SF1后可以明显抑制HUVECs的体外管腔形成,提示TM4SF1与肿瘤血管形成相关。     

Taspine downregulates VEGF expression and inhibits proliferation of vascular endothelial cells through PI3 kinase and MAP kinase signaling pathways

Taspine is an active component isolated from Radix et Rhizoma Leonticis with inhibiting tumor angiogenic properties. The molecular mechanism(s) of taspine on tumor angiogenic inhibition have not been well documented. The aim of this study was to elucidate in detail the effects of taspine on genetic expressions of VEGF in human umbilical vein endothelial cells, and on VEGFR2-mediated intracellular signaling of human umbilical vein endothelial cells. The genetic expression of vascular endothelial growth factor (VEGF) in the human umbilical vein endothelial cells (HUVECs) treated with taspine in vitro was measured by the ELISA and RT-PCR methods. The effects of taspine on cell proliferation of HUVECs and HUVECs induced by VEGF165 were considered by using MTT assay. And also, a western blot was used to detect Akt and Erk1/2 expressions and their phosphorylation levels in HUVECs treated with taspine. Our results show that VEGF protein and mRNA expressions in the cells treated with taspine were significantly decreased. Taspine also significantly inhibited cell proliferation of HUVECs induced by VEGF165. HUVECs treated with taspine showed decreased Akt and Erk1/2 activities.     

Protective effects of timosaponin B-II on high glucose-induced apoptosis in human umbilical vein endothelial cells

This study was designed to investigate the action of timosaponin B-II, a main bioactive compound in Anemarrhena asphodeloides Bunge, on the prevention from high glucose-induced cytotoxicity and apoptosis in human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved. The results showed that compared with the normal control group, exposure of HUVECs to high glucose media for 72 h resulted in a significant increase in lactates dehydrogenise release, reactive oxygen species production, Caspase-3 activity and the percentage of apoptotic cells (p < 0.01). However, pretreatment with timosaponin B-II significantly increased the viability of HUVECs and decreased lactates dehydrogenise release, Caspase-3 activity and the apoptosis rate in a concentration-dependent manner (p < 0.05). In addition, timosaponin B-II notably decreased the amount of reactive oxygen species and malondialdehyde, as well as promoted glutathione peroxidase activity, endothelial nitric oxide synthase activity and nitric oxide release (p < 0.05). These results suggest that timosaponin B-II has the antiapoptotic effect in endothelial cells through inhibition of high glucose-induced oxidative stress and has the potential for preventing diabetic cardiovascular complications.     

等鞭金藻多肽IEC对脂多糖诱导血管内皮炎症的保护作用研究

目的 探究氨基酸序列为Ile-Ile-Ala-Val-Glu-Ala-Gly-Cys的等鞭金藻多肽IEC对LPS诱导人脐静脉内皮细胞（HUVEC）炎症反应的保护作用。方法 用噻唑蓝（MTT）法确定细胞活力,DCFH-DA探针法和DAF-FM DA探针法分别测定活性氧（ROS）和NO的表达情况,蛋白免疫印迹法检测细胞间Toll样受体（TLR4）、一氧化氮合酶（NOS-2）和环氧合酶（COX-2）蛋白的表达水平,酶联免疫吸附法测定白细胞介素-6 （IL-6）和肿瘤坏死因子（TNF-α）的释放量以及分子对接分析IEC与COX-2、TLR-4蛋白的相互作用。结果 MTT法证明IEC对HUVEC细胞无明显毒性作用(P>0.05);与对照组相比,随着实验组多肽浓度增加,可以明显降低ROS和NO的释放(P<0.05),炎症相关细胞因子TLR-4、COX-2、NOS-2和IL-6、TNF-α的表达也明显逐渐减少(P<0.000 1);分子对接结果表明,IEC与COX-2、TLR4能形成稳定的化学键,说明存在相互作用的可能性。结论 IEC具有良好的抑制ROS和NO生成效果,并可明显抑制TLR-4/NOS-2/COX-2信号通路的激活以及抑制炎症因子的表达,是潜在的炎症抑制剂。     

蛋白激酶C-胞外信号调节激酶1/2信号通路在尼古丁诱导的血管内皮细胞表达纤维溶解酶原激活物抑制物-1中的作用

目的 探讨蛋白激酶C(PKC)-胞外信号调节激酶(ERK)1/2信号通路在尼古丁诱导人脐静脉内皮细胞(HUVECs)表达纤维溶解酶原激活物抑制物-1(PAI-1)中的作用.方法 体外培养HUVECs,采用不同实验条件尼古丁进行干预,ELISA法测定细胞上清液中PAI-1的浓度,观察尼古丁作用的最佳浓度和时间.进一步分别用PKC的抑制剂星型胞菌素staurosporine (STS)和ERK的抑制剂PD98059干预HUVECs,观察PKC或ERK被阻断后对尼古丁诱导的HUVECs 表达PAI-1的影响,ELISA测定各组细胞上清液中PAI-1蛋白的表达水平,RT-PCR检测各组细胞PAI-1 mRNA的表达.结果 100 μmol/L尼古丁组PAI-1蛋白水平[(22.6 ± 1.1)μg/L]明显高于对照组[(14.2± 2.8)μg/L,q=5.64,P＜0.05];以100 μmol/L 尼古丁分别与HUVECs孵育0、4、6、8、12及24 h,各组PAI-1蛋白表达呈时间依赖性升高,并在12 h达到高峰(F=32.063,P＜0.05);尼古丁组PAI-1 mRNA及蛋白含量[(1.32±0.20),(21.08±0.83)μg/L]明显高于对照组[(0.73±0.10),(13.39± 0.93)μg/L,q=8.43、11.97,均P＜0.05];尼古丁+STS组PAI-1 mRNA及蛋白含量[(1.07±0.10),(16.19±2.15)μg/L]较尼古丁组降低(q=5.61、7.61,均P＜0.05),但仍高于对照组(q=7.84、4.36,均P＜0.05);尼古丁+ PD98059组PAI-1 mRNA及蛋白表达[(1.12±0.11),(17.52±1.72)μg/L]低于尼古丁组(q=4.68、5.54,均P＜0.05),仍高于对照组(q=8.77、6.43,均P＜0.05).结论 PKC-ERK1/2信号通路在尼古丁诱导的血管内皮细胞PAI-1表达上调中发挥一定作用.

Abstract：

Objective To explore the role of protein kinase C (PKC)- extracellular signal-regulated kinase (ERK)1/2 signal pathway in the process of plasminogen activator inhibitor-1(PAI-1) protein and mRNA expression in cultured human umbilical vein endothelial cells(HUVECs) induced by nicotine. Methods HUVECs were cultured to examine the effect of nicotine on the expression of secreting PAI-1 in HUVECs on different experimental conditions. The expression of PAI-1 protein was measured by ELISA. PKC inhibitor staurosporine (STS)and ERK1/2 inhibitor PD98059 were used to detect PKC or ERK1/2 function on the expression of PAI-1 in HUVECs induced by nicotine. The PAI-1 mRNA expression was determined by RT-PCR. Results The expression level of PAI-1 protein in 100 μmol/L nicotine treated group [(22.6±1.1) μg/L] increased significantly compared to the control group [(14.2±2.8) μg/L; q=5.64,P<0.05]. After stimulation with 100 μmol/L nicotine for 0,4,6,8,12 and 24 h, the levels of PAI-1 protein increased over time and reached the peak at 12 h (F=32.063,P<0.05).The PAI-1 mRNA and protein expression in nicotine treated group [(1.32±0.20), (21.08 ± 0.83) μg/L] increased significantly compared to the control group [(0.73±0.10), (13.39±0.93) μg/L; q=8.43,11.97,all P<0.05].Compared with nicotine treated group , the PAI-1 mRNA and protein expression in nicotine and STS treated group [(1.07±0.10),(16.19±2.15) μg/L] decreased significantly(q=5.61,7.61, all P<0.05), but still higher than the control group (q=7.84,4.36, all P<0.05). In nicotine and PD98059 treated group, the PAI-1 mRNA and protein expression [(1.12±0.11),(17.52±1.72) μg/L] decreased significantly compared to the nicotine treated group(q= 4.68,5.54, all P<0.05), still higher than the control group (q=8.77,6.43, all P<0.05). Conclusion PKC-ERK1/2 signal pathway may play a partial role in the up-regulation of PAI-1 induced by nicotine in HUVECs.     

Arsenic trioxide induces procoagulant activity through phosphatidylserine exposure and microparticle generation in endothelial cells

Background

Coagulopathy is a major cause of early death when arsenic trioxide (As₂O₃) therapy fails. In addition to the procoagulant properties of blast cells, the cytotoxic therapy may contribute to the coagulation disorders. The aim of the present study was to evaluate the possible impact of As₂O₃ on membrane alterations, including phosphatidylserine (PS) exposure and microparticle generation, and the consequent procoagulant properties of endothelial cells.

Methods

Procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVECs) was assessed by measuring clotting time and through purified coagulation complex assays. PS exposure on HUVEC membrane was observed by confocal microscopy and quantified with flow cytometry. In addition, counts and PCA of endothelial microparticles were determined by flow cytometry and plasma coagulation assay.

Results

As₂O₃ increased the ability of HUVECs to accelerate coagulation process and promote formation of coagulation complexes. Procoagulant activity corresponded to PS exposed on HUVECs. In coincidence with the PS externalization, As₂O₃ increased the production of PS-bearing microparticles, which then accelerated fibrin strand formation significantly. By blocking PS, lactadherin was able to inhibit over 90% of the intrinsic tenase/prothrombinase activity of As₂O₃-treated HUVECs, and restored coagulation times of As₂O₃-treated cells and microparticles to control levels.

Conclusions

As₂O₃ increases PCA of HUVECs through PS exposure and PS-bearing microparticle generation, which might cause thrombosis and act as a contributing factor in As₂O₃ therapy-related coagulopathy.     

pSilencer1.0-U6-Ang2/Tie2-siRNA重组质粒的构建及其在体外人脐静脉内皮细胞中抑制Ang2.Tie2基因的表达

目的 探讨应用RNA干扰(RNAi)技术抑制Ang2、Tie2基因的表达情况,为进一步研究其在体外抑制血管生成以及抑制肿瘤血管生成的动物实验研究奠定基础.方法 构建pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒各2条并予以鉴定.用pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒转染人脐静脉内皮细胞(HUVECs),采用免疫细胞化学染色和RT-PCR方法检测各组HUVECs中Ang2及Tie2的蛋白及mRNA的表达状况.结果 成功构建pSilencer 1.0-U6-Ang2/Tie2-siRNA重组质粒各2条,用其转染HUVECs后,免疫细胞化学染色和RT-PCR检测结果显示,Ang2、Tie2在蛋白和mRNA的表达水平均受到明显抑制(P＜0.05),并且siRNA的2条重组质粒之间均差异无统计学意义(P＞0.05).结论 pSilencer 1.0-u6-Ang2/Tie2-siRNA在体外能够抑制HUVECs的Ang2和Tie2的蛋白及mRNA的表达.     

Innate response of human endothelial cells infected with mycobacteria

Endothelial cells are susceptible to infection by several pathogens, but little is known about mycobacterial infection. We analyzed some features of mycobacteria-endothelial cell interactions and the innate response to the infection. Intracellular growth in human umbilical vein endothelial cells (HUVECs) of three Mycobacterium species: M. tuberculosis (MTB), M. abscessus (MAB) and M. smegmatis (MSM) was analyzed. M. smegmatis was eliminated; M. abscessus had an accelerate intracellular replication and M. tuberculosis did not replicate or was eliminated. M. abscessus infection induced profound cytoskeleton rearrangements, with M. tuberculosis infection changes were less marked, and with MSM were slight. Nitric oxide (NO) production was induced differentially: M. abscessus induced the highest levels followed by M. tuberculosis and M. smegmatis; the contrary was true for reactive oxygen species (ROS) production. Only M. tuberculosis infection caused beta-1 defensin over-expression. As a whole, our results describe some aspects of the innate response of HUVEC infected by mycobacteria with different virulence and suggest that a strong cytoskeleton mobilization triggers a high NO production in these cells.