流体切应力和Ox—LDL地内皮细胞表面ICAM—1表达的影响

目的研究动脉粥样硬化(AS)的危险因素高脂血症、流体切应力及二者共同作用对血管内皮细胞细胞间粘附分子-1(ICAM-1)表达的影响。方法以人脐静脉内皮细胞(HUVECs)为研究对象,应用免疫组化ACB方法,观测Ox-LDL、流体切应力及二者共同作用对HUVECs的粘附分子ICAM-1表达的影响。结果与对照组相比,(1)Ox-LDL显著增加HUVECs表面ICAM-1表达;(2)流体切应力使HUVECs表面ICAM-1表达增加,但ICAM-1的表达随时间依赖性增加的趋势不明显;(3)共同作用后,HUVECsICAM-1的表达较基础表达显著增加。结论Ox-LDL、流体切应力的改变及二者共同作用显著增加H-UVECs表面ICAM-1表达。     

瑞舒伐他汀对内皮细胞中组织因子途径抑制物表达的影响

目的探讨瑞舒伐他汀对组织因子途径抑制物(TF-PI)表达的调节作用及机制。方法培养人脐静脉内皮细胞(HUVECs),给予多种剂量的瑞舒伐他汀和(或)肿瘤坏死因子-α(TNF-α)、磷酸腺苷激活的蛋白激酶(AMPK)抑制剂与激活剂处理细胞,利用实时定量PCR、Western blot技术检测内皮细胞中TFPI表达的变化。结果 TNF-α下调内皮细胞中TFPI的的表达水平,而这一作用被瑞舒伐他汀所阻断。而且,瑞舒伐他汀呈时间及剂量依赖性上调TFPI的mRNA与蛋白水平。AMPK抑制剂———Compoud C则可逆转瑞舒伐他汀对TFPI表达的上调作用,而AMPK激动剂———Aicar则具有与瑞舒伐他汀相似的上调TFPI的作用。结论瑞舒伐他汀通过激活AMPK途径上调血管内皮细胞中TFPI的表达。     

Cell type-specific recycling of tetrahydrobiopterin by dihydrofolate reductase explains differential effects of 7,8-dihydrobiopterin on endothelial nitric oxide synthase uncoupling

(6R)-5,6,7,8-Tetrahydro-l-biopterin (BH4) availability regulates nitric oxide and superoxide formation by endothelial nitric oxide synthase (eNOS). At low BH4 or low BH4 to 7,8-dihydrobiopterin (BH2) ratios the enzyme becomes uncoupled and generates superoxide at the expense of NO. We studied the effects of exogenously added BH2 on intracellular BH4/BH2 ratios and eNOS activity in different types of endothelial cells. Incubation of porcine aortic endothelial cells with BH2 increased BH4/BH2 ratios from 8.4 (controls) and 0.5 (BH4-depleted cells) up to ∼20, demonstrating efficient reduction of BH2. Uncoupled eNOS activity observed in BH4-depleted cells was prevented by preincubation with BH2. Recycling of BH4 was much less efficient in human endothelial cells isolated from umbilical veins or derived from dermal microvessels (HMEC-1 cells), which exhibited eNOS uncoupling and low BH4/BH2 ratios under basal conditions and responded to exogenous BH2 with only moderate increases in BH4/BH2 ratios. The kinetics of dihydrofolate reductase-catalyzed BH4 recycling in endothelial cytosols showed that the apparent BH2 affinity of the enzyme was 50- to 300-fold higher in porcine than in human cell preparations. Thus, the differential regulation of eNOS uncoupling in different types of endothelial cells may be explained by striking differences in the apparent BH2 affinity of dihydrofolate reductase.     

汉防己甲素抑制血管生成的作用

背景与目的:血管生成在肿瘤从良性向恶性转变、癌细胞进入血液循环、转移灶发展和破裂中都起着重要作用.本研究探讨汉防己甲素对体外、体内血管生成的抑制作用及其可能的机制.方法:采用M1_r法观察汉防己甲素对人脐静脉血管内皮细胞(human umbilical vein endothelial cell HUVEC)和人肠癌LoVo细胞增殖的影响.通过Transwell小室趋化实验、体外小管形成实验观察汉防己甲素对HUVEC迁移、成血管能力的影响.建立裸鼠kIVo细胞皮下移植瘤模型.给予汉防己甲素灌胃,观察用药对肿瘤微血管密度的影响.结果:2-8 μg/mL的汉防己甲素作用48 h时对HUVEC的细胞增殖抑制率为24.6%-76.9%,对LoVo细胞增殖抑制率为11.6%～14.0%;体外小管形成实验发现.2～8 μg/mL的汉防己甲素作用24 h时HUVEC小管形成数目减少,且管腔不完整,与对照组比较差异有统计学意义(P值均＜0.001).经2-8 μg/mL的汉防己甲素处理12 h后HUVEC迁移数明显少于对照组(P值均＜O.001).在裸鼠皮下移植瘤体内实验中,80 mg/kg汉防己甲素作用于裸鼠LoVo细胞皮下移植瘤后其微血管密度与生理盐水对照组比较.差异具有统计学意义(p0.035).结论:汉防己甲素在体外能有效抑制血管生成,其机制可能与抑制HUVEC增生、迁移和小管形成,诱导HUVEC凋亡,抑制HUVEC DNA的合成有关.汉防己甲素在体内对裸鼠LoVo移植瘤具有抗血管生成作用.     

PM2.5中不同组分对人脐静脉内皮细胞的联合毒性作用研究

目的：探讨PM_2.5中各种组分单独以及联合暴露对于人脐静脉内皮细胞（HUVECs）的毒性作用,并探讨不同组分之间是否存在交互作用。方法：以永生化的HUVECs细胞为研究对象,分别给予0、5、10、20、40、80、160 μg/mL纳米炭黑颗粒（下称炭黑）,0、2.5、5、10、20、40、80、160 μg/mL尘土颗粒（下称尘土）,0、6.25、12.5、25、50、100、200、400 μmol/L醋酸铅,0、5、10、20、40、80、160 μmol/L亚砷酸钠和氯化镉,染毒24 h,采用CCK-8法测定不同受试物对细胞存活率的影响,并计算不同受试物对HUVECs细胞的半数抑制浓度（IC₅₀）。然后进行联合染毒实验,选用细胞存活率为80%时的剂量,炭黑（或尘土）分别与铅、砷、镉对HUVECs进行联合染毒24 h,用CCK-8测定细胞存活率;另外根据PM_2.5中各组分实际占比,按m_铅：m_{炭黑/尘土}=1：50,m_砷：m_{炭黑/尘土}=1：100,m_镉：m_{炭黑/尘土}=1：500的比例进行两两联合染毒,以及按m_铅：m_砷：m_镉：m_{炭黑/尘土}=10：5：1：500的比例进行四者联合染毒。染毒24 h后,用CCK-8法测定细胞存活率。结果：HUVEC细胞存活率随着炭黑、尘土、醋酸铅、亚砷酸钠、氯化镉染毒浓度的增加而下降（P<0.05）,以上各受试物对HUVECs细胞的IC₅₀分别为16 μg/mL、110 μg/mL、184 μmol/L、15 μmol/L、14 μmol/L。炭黑与醋酸铅、亚砷酸钠、氯化镉联合染毒时,细胞存活率比炭黑单独染毒时分别下降了17.8%、43.8%、41.2%;尘土与醋酸铅、亚砷酸钠、氯化镉联合染毒时,细胞存活率比尘土单独染毒时分别下降了11.6%、27.8%、28.3%。联合染毒时细胞存活率比单独染毒时明显下降（P<0.05）。炭黑与亚砷酸钠、炭黑与氯化镉之间存在交互作用（均为P<0.05）。结论：PM_2.5中不同组分均可以对血管内皮细胞产生毒性作用,单独暴露时炭黑的细胞毒性作用比尘土大;但与3种金属联合暴露时,尘土比炭黑的联合暴露细胞毒性更大。铅、砷、镉对细胞的毒性作用大小排序为氯化镉>亚砷酸钠>醋酸铅,且炭黑颗粒与亚砷酸钠、炭黑颗粒与氯化镉之间存在交互作用,表现为协同作用。     

天麻、法半夏对ox-LDL诱导的HUVECs损伤的影响

摘要 目的：探讨天麻、法半夏对氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVECs）损伤的影响。方法：体外培养HUVECs，随机分为对照组、模型组、天麻组（5 mg/mL）、法半夏组（10mg/mL），流式细胞仪检测HUVECs细胞凋亡率、细胞增殖指数，Western blot法检测血管内皮生长因子（VEGF）和血管内皮生长因子受体-2（VEGFR-2）的表达，实时荧光定量PCR法检测半胱氨酸蛋白酶-3（Caspase-3）和半胱氨酸蛋白酶-9（Caspase-9 ）RNA的表达。结果：与对照组比较，模型组的细胞凋亡率升高[（4.32±0.11）%比（0.34±0.01）%，P<0.05]，细胞增殖指数下降[(43.40±4.04)%比(63.76±2.71)%，P<0.05]，VEGF表达下调（0.09±0.03比0.85±0.06，P<0.05），VEGFR-2表达下调（0.13±0.03比0.81±0.05，P<0.05），Caspase-3 RNA表达上调（1.67±0.02比0.79±0.01，P<0.05 ），Caspase-9 RNA表达上调（2.29±0.02比0.71±0.01，P<0.05）；与模型组比较，天麻组、法半夏组的细胞凋亡率下降[（2.51±0.45)% 、(2.12±0.13)%比(4.32±0.11)%，P<0.05]，细胞增殖指数升高[（50.83±2.46)%、(51.41±2.86)%比(43.40±4.04)%，P<0.05]，VEGF表达上调（0.60±0.04、0.30±0.03比0.09±0.03，P<0.05），VEGFR-2表达上调（0.55±0.05、0.55±0.03比0.13±0.03，P<0.05），Caspase-3 RNA表达下调（1.33±0.03、1.16±0.02比1.67±0.02 ，P<0.05），Caspase-9 RNA表达下调（1.86±0.09、1.82±0.02比2.29±0.02，P<0.05）；与天麻组比较，法半夏组的细胞凋亡率下降[（2.12±0.13)%比(2.51±0.45)%，P<0.05]，细胞增殖指数差异无统计学意义[（51.41±2.86)%比(50.83±2.46)%，P>0.05）]，VEGF表达下调（0.30±0.03比0.60±0.04，P<0.05），VEGFR-2表达差异无统计学意义（0.55±0.03比0.55±0.05，P>0.05），Caspase-3 RNA表达下调（1.16±0.02比1.33±0.03，P<0.05），Caspase-9 RNA表达差异无统计学意义（1.82±0.02比1.86±0.09，P>0.05）。结论：天麻和法半夏均能抑制ox-LDL诱导的HUVECs损伤，其机制可能与上调VEGF和VEGFR-2的表达、下调Caspase-3 RNA和Caspase-9 RNA的表达有关。     

Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells

Salviae miltiorrhizae (SM), a clinical, commonly used herb, can activate blood circulation and resolve stasis. We have investigated the effects of salvianolic acid B (Sal B), a pure compound extracted from the dried SM roots, on fibrinolytic (tissue-type plasminogen activator and plasminogen activator inhibitor, t-PA and PAI) and anticoagulant (thrombomodulin,TM) properties of cultured human umbilical vein endothelial cells (HUVECs). When HUVECs were treated with Sal B, a dose- (0.0125–0.5 mg/ml) and a time-dependent decrease in PAI activity were observed. PAI type 1 (PAI-1) antigen and PAI-1 mRNA expression significantly decreased compared to control values in the conditioned media of HUVECs pretreated with Sal B for 12 h. Moreover, TM activity reached a maximum stimulation of 1.25-fold over control levels in the pretreatment of Sal B for 12 h and t-PA and TM specific mRNA expression also increased (1.7- and 1.8-fold, respectively). In conclusion, Sal B increased the fibrinolytic and anticoagulant potential of cultured HUVECs by up-regulating the expression of t-PA and TM and by down-regulating the expression of PAI-1. These data suggest that Sal B is clinically effective because of its ability to change the gene expression profile of endothelial cells thereby preventing vascular events.     

栀子苷抗H_2O_2诱导人脐静脉内皮细胞凋亡的实验研究

目的探讨栀子苷对氧化应激诱导人脐静脉内皮细胞(HUVEC)凋亡的抑制作用及可能机制。方法体外培养的HUVEC细胞用不同浓度的栀子苷药物预处理24h后,加入终浓度为500μmol·L-1H2O2氧化损伤12h。荧光染色法观察细胞内DNA损伤情况,流式细胞术检测细胞凋亡率和线粒体膜电位的变化,Western blot检测Bcl-2和Bax蛋白的表达,酶免法测定caspase-3蛋白酶的活性,Real-time PCR检测超氧化物歧化酶(Mn-SOD)和谷胱甘肽过氧化物酶(GPx)的mRNA表达。结果与模型组相比,不同浓度栀子苷(12.5、25、50mg·L-1)可以明显改善H2O2对细胞内的DNA氧化损伤,降低细胞凋亡率,下调Bax蛋白表达,逐步恢复Bcl-2/Bax表达比例和线粒体膜电位的改变,降低caspase-3蛋白酶活性,上调细胞内Mn-SOD和GPx的表达,但对Bcl-2的表达无影响。结论栀子苷可以抑制H2O2诱导的HUVEC细胞凋亡,其机制可能与调节线粒体应激途径和发挥抗氧化损伤功能有关。     

[Gly14]-humanin restores cathepsin D function via FPRL1 and promotes autophagic degradation of Ox-LDL in HUVECs

Background and aimAbnormal aggregation of oxidized low-density lipoprotein (Ox-LDL) in vascular endothelial cells (VECs) is one of the major pathological changes in atherosclerotic lesions. Our research aimed to assess the mechanism of humanin (HN) in promoting autophagic degradation of Ox-LDL in HUVECs.Methods and resultsFlow cytometry and lipid quantitation results showed that Ox-LDL caused lipid and cholesterol accumulation in HUVECs. Western blot results showed that Ox-LDL increased the expression of autophagy-related proteins P62 and LC3-II in a concentration-dependent manner. The cathepsin D activity assay showed that Ox-LDL inhibited the function of cathepsin D. HNG pretreatment reduced lipid and cholesterol aggregation in HUVECs induced by Ox-LDL, increased LC3-II protein level, decreased P62 protein content, and reversed Ox-LDL-induced cathepsin D functional impairment. Inhibition of the FPRL1 pathway by FPRL1 siRNA or the FPRL1-specific inhibitor Boc-MLF blocked all HNG's protective effects. These results indicate that HNG could restore cathepsin D activity and protein level in HUVECs to repair lysosomal functional damage induced by Ox-LDL, further repairing Ox-LDL-induced autophagic damage in HUVECs.ConclusionHNG restores the activity of Ox-LDL-induced damaged lysosomal enzyme cathepsin D through its membrane protein receptor FPRL1 to promote autophagic degradation of Ox-LDL in HUVECs.