apelin对脂多糖诱导的大鼠肺微血管内皮细胞凋亡和骨架改变的影响及机制

目的探讨apelin对脂多糖(LPS)诱导的大鼠肺微血管内皮细胞(PMVEC)凋亡、骨架改变的影响及作用机制。方法采用体外组织贴块法培养大鼠PMVEC,激光共聚焦显微镜观察LPS 10 mg·L~(-1)分别处理0,3,6,12,24和48 h PMVEC骨架结构的改变;另取PMVEC加入apelin 1和10 nmol·L~(-1)或p38抑制剂SB203580 10μmol·L~(-1)预处理,2 h后加入LPS 10 mg·L~(-1)作用24 h后,AnnexinⅤ/PI染色法检测大鼠PMVEC凋亡,Western蛋白印迹法检测凋亡相关蛋白BAX和BCL-2的水平;同时还采用Western蛋白印迹法检测LPS 10 mg·L~(-1)作用0,5,15,30,60,120和240 min,或apelin 1和10 nmol·L~(-1)预处理2 h后加入LPS 10 mg·L~(-1)作用30 min后p38丝裂原激活蛋白激酶(MAPK)磷酸化水平的变化。结果激光共聚焦显微镜观察结果显示,LPS明显诱导了大鼠PMVEC骨架重排,apelin 1和10 nmol·L~(-1)预处理明显干预了LPS诱导的PMVEC应力纤维的形成和细胞骨架形态的改变。AnnexinⅤ/PI染色结果表明,apelin 1和10 nmol·L~(-1)预处理明显抑制了LPS诱导的大鼠PMVEC凋亡,早期凋亡率由(43.8±4.6)%分别降至(33.7±6.9)%和(11.2±3.0)%(P<0.05),晚期凋亡率由(54.3±3.4)%分别降至(29.5±4.6)%和(9.0±1.6)%(P<0.05),并不同程度地逆转了BAX和BCL-2的表达失衡情况(P<0.05)。Western蛋白印迹结果显示,LPS作用5 min即明显诱导大鼠PMVEC中p38 MAPK磷酸化水平增高(P<0.05),且在30 min时磷酸化水平达到最高(P<0.01),apelin预处理明显抑制了LPS作用30 min诱导的p38 MAPK磷酸化水平的增高(P<0.01)。p38抑制剂SB203580预处理明显抑制了LPS诱导的大鼠PMVEC凋亡,早期(36.7±3.8%)和晚期凋亡率(38.3±7.5%)分别降至(19.7±4.7)%和(15.7±3.6)%(P<0.01)。结论 apelin明显干预了LPS诱导的大鼠PMVEC骨架蛋白重排以及凋亡损伤,这一保护作用与其抑制p38 MAPK信号通路有关。     

米诺环素对脂多糖诱导的肺微血管内皮细胞炎症损伤的抑制作用

目的探讨米诺环素对急性肺损伤肺微血管内皮细胞炎症损伤的抑制作用。方法以脂多糖(LPS)10 mg·L^-1与肺微血管内皮细胞作用24 h,构建炎症损伤的细胞模型;米诺环素10和25μmol·L^-1共孵育24 h,用CCK-8法检测细胞存活率,ELISA检测培养液中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-8和细胞间黏附分子1(ICAM-1)水平,流式细胞术分析细胞活性氧(ROS)水平,Western印迹法检测细胞聚腺苷二磷酸核糖聚合酶1(PARP-1)和NF-κB表达。结果米诺环素可保护LPS诱导的肺微血管内皮细胞炎症损伤,LPS组细胞存活率为82.4%,米诺环素10和25μmol·L^-1组细胞存活率可达90.6%和96.9%(P<0.01)。米诺环素可降低LPS诱导的肺微血管内皮细胞炎症因子的表达,与LPS组相比,米诺环素组细胞TNF-α,IL-6,IL-8和ICAM-1的表达显著降低(P<0.01)。米诺环素还显著抑制LPS诱导的细胞氧化应激,LPS组细胞ROS相对水平为3.19,米诺环...     

虎杖苷对低氧所致大鼠肺微血管内皮细胞损伤的保护作用

目的探讨虎杖苷对低氧所致大鼠肺微血管内皮细胞(PMVECs)损伤的保护作用及其可能的机制。方法采用组织块法培养PMVECs,免疫荧光检测特异性标志物Ⅷ因子相关抗原的表达,结合形态学鉴定细胞;不同浓度的虎杖苷(30,60和120μmol.L-1)干预后,分别在常氧与低氧环境下培养24,48和72 h,MTT法观察细胞生长情况,检测LDH活性,ELISA法测定细胞上清液中血管内皮细胞生长因子(VEGF)的含量,免疫组化法检测VEGF的表达。结果培养的PMVECs具有鹅卵石形态,Ⅷ因子相关抗原呈阳性表达,鉴定为PMVECs;虎杖苷对低氧下PMVECs活性的影响作用明显,并能显著降低低氧所致的细胞培养液中LDH活性升高、VEGF的含量升高及表达增加。结论虎杖苷在低氧环境下对PMVECs有明显的保护作用,其机制可能与虎杖苷对抗低氧致LDH活性升高、VEGF表达增加有关。     

cAMP反应元件结合蛋白在LPS致肺微血管内皮细胞损伤过程中的作用

目的探讨cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)在脂多糖(lipopolysaccharide,LPS)致大鼠肺微血管内皮细胞(rat pulmonary microvascular endothelial cell,RPMVEC)炎症损伤过程中的作用。方法体外分离、培养RPMVEC基础上,Western blot法检测CREB磷酸化CREB水平,伊文思蓝-白蛋白法检测RPMVEC通透性。结果 LPS刺激RPMVEC后,CREB中Ser133位点磷酸化水平呈时间依赖性地增加,30 min时达到峰值,120 min仍未降至正常水平,加入10μmol·L-1V5681(PKA通路特异性抑制剂)共孵育后,CREB磷酸化几乎被完全抑制,RPMVEC单层通透性明显增加。结论 LPS能诱导RPMVEC中CREB快速磷酸化,PKA通路介导上述过程,CREB在LPS致RPMVEC炎症损伤过程中可能起到保护作用。     

增殖分子在甲状腺组织中的表达及意义

目的探讨增殖分子在甲状腺良恶性组织中表达的意义,以及在分化性腺癌中与AMES分级的相关性。方法应用免疫组织化学方法,检测术前未行特殊治疗的48例甲状腺癌和15例良性病变中P53突变蛋白及增殖细胞核抗原(PCNA)的表达。结果P53突变蛋白在良性病变中无阳性表达,恶性肿瘤中为16/48(33.3%),分化性腺癌中为12/43(27.9%),未分化癌中为4/5(80.0%)。良恶性病变中,其表达差异有显著意义(P<0.05)。但与年龄、性别、肿瘤直径、AMES分级无明显相关。PCNA表达在恶性肿瘤中明显升高。分化性腺癌中,其表达与年龄、肿瘤直径、AMES分级显著相关(P<0.01),与性别无关。随着肿瘤分化程度的降低,P53突变蛋白与PCNA表达逐渐升高,两者间呈现正性相关。结论P53突变蛋白与PCNA协同表达于甲状腺恶性肿瘤,联合测定有助于判断肿瘤恶性度及预后。AMES分级能有效地判断分化性腺癌的恶性程度。     

白藜芦醇激活Akt/eNOS信号缓解LPS诱导血管内皮细胞功能损伤的作用探讨

探究白藜芦醇调控Akt/eNOS信号影响脂多糖(LPS)诱导的血管内皮细胞(VE)增殖、迁移和侵袭功能损伤。在VE细胞中添加不同浓度LPS(0,10,50,100,200,400,800和1 000μg/mL)和白藜芦醇(0,1,5,10,50,100,200和500μg/mL),确定LPS和白藜芦醇有效作用浓度,将VE细胞分为：Control组、LPS组(200μg/mL)和LPS+resveratrol组(200μg/mL LPS+50μg/mL白藜芦醇)。通过MTT法、划痕实验和Transwell小室实验评估白藜芦醇对LPS诱导的VE细胞增殖、迁移和侵袭能力的影响。Western blot检测VE细胞中p-Akt和p-eNOS表达。VE细胞活力随LPS和白藜芦醇处理浓度的增加而降低,在VE细胞中利用200μg/mL的LPS建立血管内皮细胞损伤模型,同时用50μg/mL的白藜芦醇处理。LPS组VE细胞的增殖(P<0.01)、迁移(P<0.01)和侵袭能力(P<0.01)均低于Control组。LPS+resveratrol组VE细胞的增殖(P<0.01)、迁...     

泮托拉唑对急性肺损伤模型大鼠和人肺微血管内皮细胞损伤的作用及作用机制

目的 探讨泮托拉唑对急性肺损伤(ALI)模型大鼠和人肺微血管内皮细胞(HPMECs)损伤的作用及作用机制.方法 将48只SD大鼠随机分为正常对照组,脂多糖组,阳性对照组,泮托拉唑低、高剂量组和泮托拉唑高剂量+氯喹组,各8只.除正常对照组外,其余各组大鼠均腹腔注射5 mg/kg脂多糖复制ALI模型;阳性对照组大鼠腹腔注射...     

血小板内皮细胞粘附分子-1在大鼠肺缺血-再灌注损伤中的作用研究

目的 探讨血小板内皮细胞粘附分子-1（PECAM-1/ CD31）在大鼠肺缺血 再灌注损伤过程中的作用。方法建立大鼠单肺原位热缺血-再灌注模型。将清洁SD大鼠随机分为对照组和实验组（缺血-再灌注组）,应用流式细胞术测定大鼠肺缺血60 min后再灌注损伤不同阶段（0,2,4,6,8 h）血液中中性粒细胞和淋巴细胞PECAM-1/ CD31的变化情况。并采用光镜和透射电镜观察组织、细胞形态及亚显微结构,确定凋亡发生情况。结果肺缺血 再灌注后,血液中中性粒细胞和淋巴细胞PECAM-1/ CD31均开始升高,2 h达到高峰（P＜0.01＝,然后逐渐下降,至6和8 h基本恢复到缺血 再灌注前水平。肺缺血 再灌注后,肺组织透射电镜观察可见肺泡Ⅱ型上皮细胞增多,并且不同程度出现形态学改变,细胞体积缩小变圆,细胞核呈多边形,核被膜外折或内陷,核固缩并边集在核膜下呈月牙状或腰带状,胞浆浓缩,胞浆内嗜锇性板层体减少,排空增多,细胞膜上微绒毛减少或消失,并可见凋亡小体,提示细胞凋亡发生。此种亚显微结构的改变在再灌注2 h最为明显,与PECAM-1的变化相似。结论血小板内皮细胞粘附分子-1参与并介导了肺缺血 再灌注损伤的病理过程,可作为肺缺血-再灌注损伤的标志分子。     

灯盏花素对大鼠脑微血管内皮细胞OGD损伤的保护作用及机制研究

<正>灯盏花素(breviscapine,Bre)是从短亭飞蓬中提取的黄酮类化合物,具有保护缺血性脑血管疾病、调节血管内皮功能、减轻炎性反应等作用[1]。本实验通过分离培养大鼠脑微血管内皮细胞(BMECs),建立氧糖剥夺(OGD)BMECs模型,研究Bre对BMECs损伤是否具有保护作用,并探讨其可能的机制。相关研究尚未见报道。1材料与方法     

灯盏花素对血小板活化因子致肺微血管内皮细胞损伤的影响

目的　探讨血小板活化因子 (PAF)诱导肺微血管内皮细胞损伤的机制及灯盏花素的干预作用。方法　观察PAF对体外培养大鼠肺微血管内皮细胞 (RPMVEC)的形态、单层通透性、F 肌动蛋白 (F actin)的影响和灯盏花素干预后的变化 ,F actin用流式细胞仪测定。结果　PAF浓度大于 0 1mg·L-1作用 4 8h内观察到细胞脱落和破裂。 10mg·L-1PAF 12 0min内可使RPMVEC单层通透性增高、F actin解聚 ,灯盏花素可抑制上述变化。结论　①PAF引起RPMVEC脱落和破裂呈时间和剂量依赖性。②PAF引起内皮单层通透性的增高的机制与F actin解聚密切相关。③灯盏花素可抑制PAF引起RPMVEC单层通透性的增高和F actin下降 ,对PAF引起的RPMVEC损伤有一定保护作用。     

葛根素对缺氧复氧心脏微血管内皮细胞的保护作用

目的:探讨葛根素在心脏微血管内皮细胞(cardiac microvascular endothelial cell,CMEC)缺氧复氧损伤中的作用。方法:将CMEC随机分为空白组、模型组、葛根素低、中、高剂量(25,50,100 mg·L^-1)共3组。四甲基偶氮唑(methylthiazolyl tetrazolium,MTT)法检测细胞活力,流式细胞仪检测细胞凋亡,比色法检测乳酸脱氢酶(lactate dehydrogenase,LDH)活性、丙二醛(malondialdehyde,MDA)含量及超氧化物歧化酶(superoxide dismutase,SOD)活性,Western blot法检测Caspase-3和Bcl-2蛋白表达。结果:葛根素预处理后,缺氧复氧CMEC细胞活力增强,细胞凋亡率、LDH活性及MDA含量降低,SOD活性增强,Caspase-3蛋白表达减少,Bcl-2蛋白表达增加。结论:葛根素可拮抗缺氧复氧所致的CMEC损伤,可能与其抗氧化及抗凋亡活性相关。     

过氧化氢诱导肺微血管内皮损伤的实验研究

目的　探讨H2 O2 诱导培养肺微血管内皮细胞损伤的机制。方法　观察H2 O2 对大鼠肺微血管内皮细胞 (RP MVEC)的形态、单层通透性、F 肌动蛋白和 β AR的影响。结果　H2 O2 浓度大于 1mmol·L-1作用 48h内观察到细胞脱落和破裂。 10mmol·L-1H2 O2 90min内可使RPMVEC单层通透性增高、F 肌动蛋白发生明显解聚和 β AR显著下调 ;FOR、山莨菪碱和CTX可抑制上述变化。结论　H2 O2引起的RPMVEC脱落与破裂呈浓度和时间依赖性。H2 O2引起RPMVEC单层通透性增高的机制与F 肌动蛋白解聚密切相关 ;β AR参与内皮通透性调节。FOR、山莨菪碱和CTX对H2 O2 引起RPMVEC单层通透性增高有一定的保护作用。     

Paraquat-induced membrane dysfunction in pulmonary microvascular endothelial cells

Membrane dysfunction monitored by lactate dehydrogenase release from cultured pulmonary microvascular endothelial cells of pigs, which were exposed to paraquat at different concentrations (0.1-2 mM), was examined. Paraquat caused a time-dependent increase in lactate dehydrogenase release. Lactate dehydrogenase releases after 72 hr, 32, 58, and 84% by 0.1, 0.5, and 2 mM paraquat, respectively, were well correlated with cell viability measured by cell adherence. In contrast, reductions of two tetrazolium compounds were depleted profoundly by 72 hr after exposure to 0.5 mM paraquat, suggesting depletion of intracellular reductive substances. Extracellular hydrogen peroxide began to significantly increase 56 hr or 32 hr after exposure to 0.5 mM or 1.5 mM paraquat, respectively, preceding the initial increase of lactate dehydrogenase release (64 hr by 0.5 mM or 48 hr by 1.5 mM). Lactate dehydrogenase release 72 hr after exposure to 0.5 mM paraquat was prevented strongly by catalase (1000 units/ml), but weakly by superoxide dismutase (1000 units/ml). These enzymes failed to restore the reduced acid phosphatase activity. Also, 0.1 mM desferal or alpha,alpha'-dipyridyl protected lactate dehydrogenase release. Similarly, 1 mM thiourea or dimethylthiourea, and 0.5 mM alpha-tocopherol or trolox, were effective, but diethylenetriaminepentaacetic acid (0.1 mM) and probucol (5 or 10 microM) were ineffective. Exposure of 0.5 or 1.5 mM paraquat suppressed levels of lipid peroxidation. These results indicate that membrane dysfunction by paraquat is ascribed to an iron-catalyzed reaction of extracellularly increased hydrogen peroxide. A deleterious species for the membrane dysfunction is discussed.     

利多卡因调控Apelin/APJ系统对脓毒症相关性脑病大鼠的脑保护作用

目的 探究利多卡因对脓毒症相关性脑病模型大鼠的脑保护作用及机制。方法 将40只大鼠按随机数字表法分为对照组、模型组、利多卡因组、利多卡因＋血管紧张素受体AT1相关的受体蛋白拮抗剂Apelin13（F13A）组,每组10只。除对照组外,其余3组大鼠均构建脓毒症相关性脑病模型,利多卡因组和利多卡因＋F13A组造模后即刻给予利多卡因10 mg/kg负荷剂量,随后尾iv利多卡因10 mg/kg并持续3 h,利多卡因＋F13A组同时ip APJ拮抗剂F13A 100 μg/kg。24 h后,Morris水迷宫实验检测各组大鼠认知功能,酶联免疫吸附（ELISA）法检测各组大鼠血清白细胞介素（IL）-6、IL-10、肿瘤坏死因子-α（TNF-α）水平,苏木精-伊红（HE）染色观察各组大鼠脑组织病理变化,TdT介导的dUTP缺口末端标记法（TUNEL）和神经元特异核蛋白（NeuN）双免疫荧光染色检测各组大鼠脑组织皮质内神经元凋亡,免疫荧光双染法观察各组大鼠脑组织皮质内Apelin与APJ表达,实时荧光定量逆转录聚合酶链反应（RT-qRCR）检测各组大鼠脑组织Apelin和APJ的mRNA表达,蛋白质免疫印迹（Western blotting）法检测各组大鼠脑组织Apelin和APJ的蛋白表达。结果 与模型组比较,利多卡因组大鼠逃避潜伏期缩短,通过目标象限次数增加（P＜0.05）,血清IL-6、TNF-α水平降低而IL-10水平显著升高（P＜0.05）,海马区神经元损伤明显减轻,形态和分布均趋于正常,脑组织皮质内凋亡神经元数目减少（P＜0.05）;Apelin与APJ荧光染色表达均明显增强,脑组织内Apelin、APJ的mRNA与蛋白相对表达量均显著上调（P＜0.05）;与利多卡因组比较,利多卡因＋F13A组大鼠逃避潜伏期延长,通过目标象限次数减少（P＜0.05）,血清IL-6、TNF-α水平升高,血清IL-10水平显著降低（P＜0.05）,海马区神经元损伤加重,有大量细胞肿胀与细胞核固缩,脑组织皮质内凋亡神经元数目增加（P＜0.05）。同时,Apelin与APJ荧光染色表达均明显减弱,脑组织内Apelin、APJ的mRNA与蛋白相对表达量均显著下调（P＜0.05）。结论 利多卡因能够改善脓毒症相关性脑病模型大鼠脑损伤,抑制炎症反应,并减少神经元凋亡,其机制可能与激活Apelin/APJ系统有关。     

碘化N-正丁基氟哌啶醇对大鼠心脏微血管内皮细胞缺氧复氧损伤保护机制的研究

目的观察碘化N-正丁基氟哌啶醇(N-n-butyl haloperidol iodide,F2)对培养的内皮细胞缺氧/复氧(anoxia/reoxygenation,A/R)损伤及早期生长反应基因-1(early growth response gene 1,Egr-1)蛋白表达的影响。方法应用新生SD大鼠进行心脏微血管内皮细胞(cardiac microvascular endothelial cells,CMECs)原代培养,取3⁴代的CMECs建立A/R模型。通过测定细胞培养上清液中乳酸脱氢酶(LDH)及CMECs中超氧化物歧化酶(SOD)、丙二醛(MDA)含量,观察CMECs损伤程度;应用ELISA法测定细胞培养上清液肿瘤坏死因子-α(TNF-α)的含量,观察CMECs炎症反应水平;Western blot法检测培养CMECs中Egr-1的蛋白表达水平。结果 A/R造成CMECs内MDA升高,SOD下降,上清液中LDH、TNF-α含量升高,A/R刺激下细胞Egr-1的蛋白表达水平明显升高;A/R刺激前给予F2可减轻CMECs的损伤及炎症反应程度,抑制Egr-1蛋白的表达。结论 F2对心脏微血管内皮细胞A/R损伤具有保护作用,该作用可能与其抑制Egr-1的表达有关。     

大鼠心肌微血管内皮细胞的体外培养

目的 探讨大鼠心肌微血管内皮细胞离体培养方法.方法 选取1～2周龄的SD大鼠,无菌条件下快速取出心脏,彻底去除大血管、心房、右心室和心内外膜.用植块法培养心肌微血管内皮细胞.倒置显微镜下形态学观察结合免疫细胞化学方法检测第Ⅷ因子相关抗原和CD34对培养细胞进行鉴定.结果 细胞呈单层贴壁生长,融合后呈"铺路石样";第Ⅷ因子相关抗原和CD34免疫细胞化学鉴定阳性.结论 成功培养出纯度较高的大鼠心肌微血管内皮细胞,方法简便,可用于心血管疾病的研究.     

Metabolism of nicotine in rat lung microvascular endothelial cells

The aim of this study was to examine whether cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas-blood barrier, have the ability to metabolize nicotine. Nicotine was biotransformed to cotinine and nicotine N'-oxide by cytochrome 450 (CYP) and flavin-containing monooxyganase (FMO), respectively, in rat LMECs. The intrinsic clearance (Vmax1/Km1) for the cotinine formation was about 20 times as high as that for the trans-nicotine N'-oxide formation in the low-Km phase, indicating that oxidation by CYP was much higher than that by FMO. On the other hand, as shown in Eadie-Hofstee plots, the formation of cis-nicotine N'-oxide was monophasic, whereas the plot for the trans-nicotine N'-oxide formation was clearly biphasic. These results suggest that nicotine N'-oxide was stereoselectively metabolized to cis and trans forms. However, in the high-Km phase there was no significant difference in N'-oxidation between the cis and trans forms. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to cotinine of nicotine in rat LMECs using the respective enzyme inhibitors (tranylcypromine and troleandomycine). On the other hand, methimazole (5 microM) caused 73 and 45% decreases in the formation of N'-oxides of cis- and trans- enantiomers, respectively, demonstrating the presence of FMO in rat LMECs. These results suggest that rat LMEC enzymes can convert substrates of exogenous origin such as nicotine for detoxication, indicating LMECs are an important barrier for metabolic products, besides hepatic cells.     

Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells

Regulation of the rolipram-sensitive cAMP-specific phosphodiesterase 4 (PDE4) gene family was studied in rat pulmonary microvascular endothelial cells (RPMVECs). Total PDE4 hydrolysis was increased within 10 min after addition of forskolin (10 microM), reached a maximum at 20-40 min, and then gradually declined in the cells. A similar activation of PDE4 activity was observed using a protein kinase A (PKA) activator, N(6)-monobutyryl cAMP. Both the forskolin and the N(6)-monobutyryl cAMP activated PDE4 activities were blocked by the PKA-specific inhibitor, H89. This forskolin-stimulated and PKA-mediated short-term activation of PDE4 activity was further confirmed by in vitro phosphorylation of 87kDa PDE4A6 and 83kDa PDE4B3 polypeptides using exogenous PKA Calpha. Increased immunoreactivity of phosphorylated PDE4A6 in situ was detected in Western blots by a PDE4A-phospho antibody specific to the putative PKA phosphorylation sites. Following long-term treatment of RPMVECs with rolipram and forskolin medium (RFM) for more than 60 days, PDE4 activity reached ten-fold higher values than control RPMVECS with twenty-fold increases detected in intracellular cAMP content. The RFM cells showed increased immunoreactivities of the constitutive 4A6 and 4B3 isoforms plus two novel splice variants at 101kDa (4B1) and 71kDa (4B2). Treatment with H89 did not inhibit the PDE4 elevation in RFM cells. In addition to the increased levels of PDE4 in RFM cells, immunofluorescence showed a translocation of PDE4A and 4B to a nuclear region, which was normally not observed in RPMVECs. The PDE4 activity in RFM cells decayed rapidly with an even faster decline of intracellular cAMP content when forskolin/rolipram were removed from the medium. These results suggest that both the activation (short-term) and induction (long-term) of PDE4A/4B isoforms in RPMVECs are closely modulated by the intracellular cAMP content via both post-translational and synthetic mechanisms.