硫氧还蛋白对缺氧/复氧人脐静脉内皮细胞的保护作用

目的 探讨硫氧还蛋白(thioredoxin,TRX)对缺氧/复氧损伤内皮细胞(ECV)的保护作用.方法 对体外培养的内皮细胞株ECV304(缺氧/复氧组)及TRX修饰的ECV304(TRX组)进行缺氧/复氧实验,另一组ECV304细胞不作缺氧/复氧作为正常对照组,检测复氧不同时间点细胞培养上清液中丙二醛(MDA)、内皮素-1(ET-1)及一氧化氮(NO)的含量变化.结果 正常对照(ECV304组),细胞内的MDA、ET-1及NO含量在24 h内变化不大；转染空载体的ECV304细胞在缺氧3h时,MDA、ET-1含量显著增加,复氧12 h时,MDA、ET-1含量达到最高,然后缓慢下降；ECV304/Trx组与转染空载体组相比,在3h及12时,MDA含量极显著下降(P＜0.01),而在24 h MDA水平显著下降(P＜0.05).ET-1水平在复氧的各时间点均比有下降,在复氧24 h下降最明显(P＜0.01)；转染空载体的ECV304细胞在缺氧3h时,NO含量显著下降,复氧12 h时,NO含量下降到最低,然后缓慢上升；ECV304/Trx组与转染空载体组相比,在3h及12时,NO含量极显著上升(P＜0.01),在12 h NO水平最高.ECV304/TRX组与正常对照组相比,在12 h时,有显著差异.结论 TRX能抑制缺氧/复氧对ECV的损伤,对缺氧/复氧环境下的ECV起保护作用.     

脑通汤含药血清对缺氧/复氧大鼠海马神经元抗氧化应激相关指标的影响

目的探讨脑通汤通过海马神经元抗氧化应激治疗血管性痴呆(VD)的机制。方法培养大鼠原代海马神经元及鉴定,取培养9 d的神经元,随机分为正常细胞组、缺氧/复氧模型组、正常血清组、脑通汤大、中、小剂量含药血清组。除正常细胞组以外,各组均置入(95%N2+5%CO2)混合气体的三气培养箱缺氧24 h再复氧2 h造模。采用MTT法检测缺氧24 h和复氧2 h时间点海马神经元活性并计算细胞存活率;实时荧光定量PCR法检测海马神经元核因子-E2-相关因子(Nrf)2、血红素氧合酶亚型(HO)-1、醌氧化还原酶(NQO)1 mRNA的表达。结果 MTT显示:缺氧24 h:模型组的海马神经元活性及存活率较正常细胞组明显下降(P0.05),与正常血清组及脑通汤不同剂量含药血清组比较无明显差异(P0.05)。复氧2 h:脑通汤不同剂量含药血清组均较模型组明显升高(P0.05),正常血清组较模型组无明显差异(P0.05)。实时荧光定量PCR结果显示:脑通汤含药血清能够促进缺氧/复氧海马神经元抗氧化应激相关基因Nrf2、HO-1、NQO1的表达(P0.05),并且脑通汤含药血清大剂量组与中剂量组的上述3个基因水平明显高于缺氧/复氧模型组(P0.01)。结论脑通汤可通过上调抗氧化相关基因Nrf2、HO-1、NQO1的表达,减轻海马神经元缺氧/复氧损伤的氧化应激反应,从而保护大鼠海马神经元缺氧/复氧损伤。     

心肌细胞缺氧复氧损伤时Mipu1启动子活性、Mipu1 mRNA表达变化及意义

目的探讨心肌细胞缺氧复氧损伤时心肌缺血预适应表达上调蛋白1(Mipu1)启动子活性的变化及其作用。方法按本课题组的常规方法复制心肌细胞缺氧复氧模型,先予缺氧(95%N2-5%CO2,无血清低糖DMEM)处理6 h,再分别复氧(95%空气-5%CO2,10%FBS-高糖DMEM)6、12、24 h。采用MTT法检测心肌细胞活性,比色法测定心肌细胞LDH活性,双荧光素酶报告基因技术检测启动子PGL3-Mipu1活性,实时定量PCR法检测Mipu1mRNA表达。结果缺氧6 h复氧(6、12、24 h)后心肌细胞活性减低,LDH释放增加,启动子PGL3-Mipu1活性增加,Mipu1 mRNA表达上调,且在缺氧6 h复氧12 h时变化最明显。结论 Mipu1可能参与了心肌细胞的缺氧复氧损伤,其可能通过调控凋亡相关基因发挥作用。     

长期缺氧对肺腺癌SPCA1细胞谷胱甘肽转移酶表达及耐药性的影响

目的 探讨长期缺氧对SPCA1细胞谷胱甘肽转移酶（GST-π）表达的影响及耐药性的改变。方法 SPCA1细胞常氧（20％O2）和缺氧（0．5％O2）条件下作用16h后，提取RNA和蛋白，用定量RT-PCR的方法检测缺氧诱导因子1α（HIF-1α）和GST-π mRNA的表达情况，用Western blotting法检测HIF-1α蛋白；制备细胞悬液，用流式细胞仪测定GST-π表达；通过克隆形成实验分析SPCA1细胞对阿霉素和丝裂霉素的药物敏感性。通过 RNA干扰技术下调HIF-1α表达后，分为对照组和干扰组，观察GST-π的表达。结果 设常氧组GST-π mRNA表达率为1，缺氧组为12．2。常氧组GST-π蛋白阳性表达率为（35．78±1．25）％，缺氧组为（72．13±2．11）％。与常氧组比较缺氧组HIF-1α表达明显增加。计算1％克隆存活的药物浓度，阿霉素处理细胞，常氧组浓度为（0．29±0．05）μg／ml；缺氧组浓度为（0．48±0．07）μg／ml；丝裂霉素处理细胞，常氧组浓度为（0．71±0．10）μg／ml；缺氧组浓度为（0．79±0．12）μg／ml。干扰HIF-1α后，HIF-π mRNA与对照组比较下降了70％，而GST-π mRNA稍有下降。GST-π蛋白表达常氧条件下对照组为（32．14±1．23）％，干扰组为（30．88±1．65）％；缺氧条件下对照组为（64．78±2．45）％，干扰组为（67．42±2．21）％。结论 长期缺氧可诱导SPCA1细胞HIF-1α和GST-π表达增加，对阿霉素耐药性增加，对丝裂霉素没有影响，HIF-1α和GST-π并没有直接的相关性。     

黄芪注射液与缺氧缺糖/复氧复糖大鼠海马神经元caspase-3表达的关系

目的探讨黄芪注射液与缺氧缺糖/复氧复糖大鼠海马神经元凋亡相关基因caspase-3表达的关系。方法取原代培养8 d的大鼠海马神经元,随机分为正常对照组、缺氧缺糖/复氧复糖组、黄芪注射液溶剂对照组和黄芪注射液组。除正常对照组外均进行缺氧缺糖0.5h再复氧复糖。各组于复氧复糖后0、0.5、2、6、24、48、72 h和120 h采用Western印迹检测caspase-3蛋白的表达,采用RT-PCR方法检测caspase-3 mRNA的表达。结果除0 h和0.5 h外,各时间点缺氧缺糖/复氧复糖组海马神经元caspase-3蛋白的平均灰度值均较正常对照组明显增加(P0.05),复氧复糖24h平均灰度值最高;与缺氧缺糖/复氧复糖组相比,黄芪注射液溶剂对照组各时间点caspase-3蛋白的平均灰度值无明显变化(P0.05),而黄芪注射液组除0h和0.5h外,在各个时间点caspase-3蛋白的平均灰度值明显降低(P0.05)。RT-PCR结果显示:除0h和0.5h外,各时间点缺氧缺糖/复氧复糖组海马神经元caspase-3 mRNA的平均光密度值均较正常对照组明显增加(P0.05),24h平均光密度值最高;与缺氧缺糖/复氧复糖组相比,黄芪注射液溶剂对照组各时间点caspase-3 mRNA的平均光密度值无明显变化(P0.05),而黄芪注射液组在除0 h和0.5 h外的各个时间点caspase-3 mRNA的平均光密度值均明显降低(P0.05)。结论黄芪注射液抑制缺氧缺糖/复氧复糖大鼠海马神经元的凋亡作用,是通过抑制海马神经元凋亡相关基因caspase-3的表达来实现的。     

缺氧后处理对缺氧复氧心肌线粒体活性氧及细胞凋亡的影响

目的 观察缺氧后处理对缺氧复氧心肌线粒体活性氧及细胞膜和线粒体Bcl-2和Bax蛋白表达的影响,探讨其调控心肌细胞凋亡的机制.方法 构建大鼠乳鼠心肌细胞缺氧复氧损伤模型,将细胞分为对照组、缺氧/复氧纽(缺氧3h后复氧6h)、缺氧后处理组(缺氧3h后行复氧5min、缺氧5 min,反复3次,再复氧6 h).应用荧光酶标仪测定线粒体活性氧量,流式细胞仪检测心肌细胞凋亡,Western blot检测细胞膜和线粒体Bcl-2和Bax蛋白的表达.结果 缺氧/复氧组和缺氧后处理组心肌细胞线粒体活性氧量较对照组显著升高(P＜0.01).缺氧后处理组心肌细胞线粒体平均荧光强度为30.74±1.88a.u./μg,显著低于缺氧/复氧组(63.17±2.75a.u./μg,P＜0.01),仍高于对照组(14.41±2.15a.u./μg).缺氧/复氧组和缺氧后处理组心肌细胞凋亡率较对照组显著升高(45.86％±3.29％和26.99％±3.35％比5.72％±1.63％,P＜0.01),缺氧后处理组低于缺氧/复氧组(P＜0.01).细胞膜和线粒体Bcl-2蛋白在缺氧后处理组显著上调,在缺氧/复氧组显著下调;Bax蛋白在缺氧后处理组显著下调,在缺氧/复氧组显著上调.结论 缺氧后处理抑制线粒体活性氧爆发,减轻缺氧/复氧诱导的心肌细胞凋亡,其抗凋亡机制可能与线粒体和细胞膜Bcl-2蛋白表达上调及Bax蛋白表达下调有关.     

灯盏花素对缺氧/复氧大鼠心肌细胞凋亡及Stat1,3 mRNA表达的影响

目的观察灯盏花素对缺氧复氧大鼠心肌细胞凋亡及其凋亡相关基因信号转导及转录激活因子Stat1,3 mRNA表达的影响。方法用原代培养的乳鼠心肌细胞建立缺氧复氧损伤模型,随机分为4组,正常对照组,模型组、灯盏花素(25、50 mg/L)组,其中模型组、灯盏花素组进行缺氧2 h再复氧4 h。采用AnnexinⅤ-PI双染,流式细胞仪检测心肌细胞的凋亡情况;RT-PCR检测大鼠心肌细胞的Stat1,3 mRNA表达变化。结果缺氧/复氧损伤后,与正常对照组比较,大鼠心肌细胞凋亡及Stat1 mRNA表达均增加(P<0.01),Stat3 mRNA表达降低(P<0.01);灯盏花素组处理后,大鼠心肌细胞凋亡及Stat1 mRNA表达均减少(P<0.05),Stat3 mRNA表达升高(P<0.05,P<0.01)。结论灯盏花素上调凋亡抑制基因Stat3mRNA表达,下调凋亡促进基因Stat1 mRNA表达,从而抑制缺氧/复氧大鼠心肌细胞的凋亡。     

亚低温对缺氧/复氧内皮细胞胞间黏附分子-1表达和黏附率的影响

目的 观察亚低温对缺氧/复氧内皮细胞胞间黏附分子-1(ICAM-1)表达和黏附率的影响.方法 以ECV304内皮细胞为研究对象,利用简易缺氧室,以缺氧模拟体内缺血性损伤,建立缺氧/复氧内皮细胞模型.将细胞分为33℃及37℃缺氧/复氧组.各组又分为缺氧6,12,18 h后再复氧6 h 3个亚组.应用Western印迹检测两组各亚组ICAM-1的表达并应用细胞计数法检测各亚组细胞和中性粒细胞(PMN)的黏附率.结果 ①在缺氧6,12,18 h亚组,37℃组内皮细胞表达ICAM-1的积分光密度值明显高于33℃组[(0.185±0.026) vs (0.120±0.064)、(0.329±0.057) vs (0.218±0.027)、(0.443±0.068) vs (0.326±0.019)](P均＜0.05).②在缺氧6,12,18 h亚组,33℃组内皮细胞和中性粒细胞的黏附率明显高于37℃组[(29.41±1.77)% vs (22.30±3.25)%、(43.66±2.57)% vs (34.51±5.06)%、(62.92±4.82)% vs (49.68±4.34)%](P＜0.05).结论 亚低温能有效抑制缺氧/复氧内皮细胞与中性粒细胞的黏附,其机制可能与降低缺氧/复氧内皮细胞ICAM-1的表达有关.     

心肌细胞系H9c2缺氧上清诱导人脐静脉内皮细胞ICAM-2的表达

目的探讨心肌细胞H9c2的缺氧培养上清对人脐静脉内皮细胞（HUVEC）细胞间黏附分子-2（ICAM-2）表达的影响及其分子机制。方法大鼠心肌细胞系H9c2在氧浓度为1%的条件下缺氧培养24 h后收集上清,用于HUVEC培养。收集不同时间点HUVEC细胞,提取总RNA和总蛋白,RT-PCR方法检测ICAM-2 mRNA表达,Western杂交方法检测ICAM-2蛋白表达。适当浓度细胞因子孵育HUVEC 6 h,收集细胞提取总蛋白,Western杂交方法检测ICAM-2蛋白表达。ELISA方法测定H9c2缺氧培养上清和常氧培养上清的PDGF-BB和PDGF-AB浓度。结果在mRNA水平,HUVEC在H9c2缺氧培养上清孵育1 h后ICAM-2的表达明显升高,3 h后达到最高;同时在HUVEC表达ICAM-2蛋白在3 h、6 h明显增高。细胞因子刺激实验表明PDGF-BB和PDGF-AB可以使HUVEC表达ICAM-2显著增加。在缺氧上清中PDGF-BB有显著性增加（P〈0.05）,而缺氧上清中的PDGF-AB却有显著减少（P〈0.01）。结论心肌细胞系H9c2的缺氧培养上清可以诱导内皮细胞HUVEC表达ICAM-2,H9c2在缺氧条件下产生的因子PDGF-BB可能是其中起作用的物质。     

奇智方对人脐静脉内皮细胞ICAM-1和ICAM-1 mRNA表达的影响

目的采用人脐静脉内皮细胞（HUVEC-304）进行细胞间黏附分子-1（ICAM-1）及其转录水平（ICAM-1mRNA）表达的离体实验研究,观察奇智方对血管内皮功能的作用。方法应用ELISA方法检测ICAM-1;用原位杂交的方法检测ICAM-1 mRNA。结果中药治疗组和阳性对照组与生理盐水组比较,ICAM-1蛋白分子表达均有统计学意义（P〈0．01）;中药治疗组和阳性对照组均能明显使缺氧／复氧后细胞ICAM-1 mRNA下调,与缺氧组比较有统计学意义（P〈0．01）。结论奇智方抑制缺氧／复氧后血管内皮细胞表达ICAM-1和ICAM-1 mRNA,从而保护血管内皮细胞的屏障完整和功能正常,可能是其免疫抑制机理的分子基础之一。     

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury.METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (ARC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia-preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group ARC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively.RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.     

血府逐瘀口服液对大鼠缺氧-再给氧损伤心脏微血管内皮细胞黏附分子表达的影响

目的 观察血府逐瘀口服液对心脏微血管内皮细胞缺氧／复氧损伤中黏附分子表达的影响。方法 通过心脏微血管内皮细胞体外培养技术，建立缺氧／复氧损伤模型，模拟心肌缺血再灌注损伤。用免疫细胞化学法和图像定量分析系统，观察心脏微血管内皮细胞的细胞间黏附分子-1（ICAM-1）和血管细胞黏附分子-1（VCAM-1）的表达变化。结果大鼠心脏微血管内皮细胞缺氧4h后ICAM-1和VCAM-1的蛋白表达较对照组升高，但无统计学意义（P〉0．05），再给氧6h、12h后ICAM-1和VCAM一1的表达较对照组明显增高（P〈0．01）。血府逐瘀口服液能显著降低ICAM-1和VCAM-1的蛋白表达，这种作用随着剂量的增加而增强。结论 血府逐瘀口服液可通过降低心脏微血管内皮细胞的ICAM-1和VCAM-1表达，减少白细胞浸润，从而减轻缺血再灌注损伤中的炎性反应对心功能造成损害。     

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury. METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia- preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively. RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.     

细胞间粘附分子-1表达水平对老年大鼠心肌缺血再灌注损伤的影响

目的　探讨细胞间粘附分子 1(ICAM 1)mRNA及蛋白表达水平对老年大鼠心肌缺血再灌注损伤 (IRI)的影响。方法　大鼠 72只 ,制作心肌IRI模型 ,设青、老年组 ;各组分设缺血 1h、再灌注 3、6、12和 2 4h时相点 ,用原位杂交和免疫组织化学测定ICAM 1mRNA及蛋白表达水平 ,用酶法测定中性粒细胞 (PMNs)浸润数 ,红四唑 (TTC)染色法测梗死范围。结果　心肌IRI时 ,青、老年组ICAM 1表达均明显增高 ,其mRNA表达至 6h达高峰 ,而蛋白质表达、PMNs浸润和梗死范围改变于 12～ 2 4h达高峰 ,后三者间呈显著正相关。青年组上述指标于再灌注时虽也明显增高 ,但比老年组明显减轻。结论　心肌缺血再灌注时 ,ICAM 1参与介导了PMNs对心肌组织的直接损伤和IRI的发生、发展 ;老年大鼠IRI时ICAM 1表达水平的增高可能是导致其损伤较青年组严重的重要因素之一     

C1酯酶抑制剂对大鼠急性心肌缺血再灌注损伤的保护作用

目的探讨C1酯酶抑制剂对大鼠心肌缺血再灌注损伤的保护作用及其可能机制。方法将48只Wistar大鼠随机分为:假手术组、模型组、治疗组、预适应组,每组12只。建立急性心肌缺血再灌注损伤模型,模型组、治疗组、预适应组大鼠结扎左前冠状动脉30 min后再灌注2 h,假手术组不结扎。检测血清肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)活性;检测各组大鼠心肌梗死范围;免疫组织化学法检测组织细胞间黏附分子1(ICAM 1)的表达及RT-PCR检测ICAM-1 mRNA的表达。结果与假手术组比较,模型组大鼠左心室收缩压、左心室压力最大上升速率及最大下降速率(±dp/dt_(max))明显降低,左心室舒张末压、CK、CK-MB、ICAM-1、ICAM-1 mRNA明显升高(P<0.01);与模型组比较,治疗组和预适应组大鼠左心室收缩压、±dp/dt_(max)明显升高(P<0.01),左心室舒张末压、CK、CK-MB、ICAM-1、ICAM-1 mRNA明显降低(P<0.05,P<0.01),心肌梗死面积明显缩小(P<0.01)。结论 C1酯酶抑制剂可能通过阻断ICAM-1的表达,从而限制中性粒细胞聚集起到心肌保护作用。     

Effect of hypoxia and hypoxia/reoxygenation on proteoglycan metabolism by vascular smooth muscle cells

Hypoxia and hypoxia/reoxygenation are known to affect vascular smooth muscle cell physiology. In this study, we first investigated proteoglycan synthesis by human aortic smooth muscle cells exposed to normoxia, hypoxia, or hypoxia/reoxygenation. We then compared the newly synthesized proteoglycans from normoxic and hypoxic-reoxygenation cultures for their ability to bind low density lipoprotein (LDL). Confluent smooth muscle cells under normoxia, hypoxia, or hypoxia/reoxygenation were pulsed with [35S]sulfate, and secreted and cell-associated proteoglycans were analyzed. Secreted proteoglycans in cultures exposed to hypoxia (4 h)/reoxygenation (19 h) increased 28% over those of cells continuously exposed to normoxia. Cell-associated proteoglycans did not differ significantly between the two groups. In contrast, hypoxia (4 h) followed by a 30-min reoxygenation produced a 37% decrease in newly synthesized proteoglycans. Hypoxia alone also resulted in a 24% decrease in secreted proteoglycans and a 20% decrease in cell-associated proteoglycans. Proteoglycans newly synthesized by smooth muscle cells exposed to normoxia and hypoxia/reoxygenation did not differ in their charge densities and molecular size but did differ in glycosaminoglycan composition. Exposure of smooth muscle cells to hypoxia/reoxygenation produced a 60% increase in a proteoglycan subfraction that bound LDL with very high affinity. The incorporation of [3H]leucine into total cellular protein decreased significantly following exposure of smooth muscle cells to hypoxia as well as hypoxia/reoxygenation. These results indicate that hypoxia and hypoxia/reoxygenation cause major alterations in proteoglycan metabolism by vascular smooth muscle cells.     

Effect of hypoxia/reoxygenation on CD73 (ecto-5'-nucleotidase) in mouse microvessel endothelial cell lines

Cerebral ischemia and post-ischemic reperfusion commonly result in significant brain damage. Brain microvessel endothelial cells, the key target cells and regulating sites, can secrete adenosine which plays an important neuroprotective role in the ischemic brain. A primary determinant of localized production of adenosine at tissue interfaces is ecto-5'-nucleotidase (CD73). In our experiments, we used bEnd.3 cells, immortalized mouse brain microvessel endothelial cell lines, as the target cells to study the effect of hypoxia and posthypoxic reoxygenation on CD73 in brain microvessel endothelial cells. CD73 activity in bEnd.3 cells exposed to hypoxia significantly increased in time-dependent way. The upregulation of CD73 mRNA and protein expression induced by hypoxia in bEnd.3 cells were detected by RT-PCR and Western blot. However, for reoxygenation studies, CD73 activity, mRNA and protein expression decreased at the initial stages, but increased at prolonged reoxygenation. Our results suggest that hypoxia can induce upregulation of CD73 expression in brain microvessel endothelial cells, which can be reversed by reoxygenation of short duration. But CD73 expression increased gradually with the duration of reoxygenation. Then, we infer that CD73 in brain microvessel endothelial cells plays a very important role through forming adenosine during brain ischemia and reperfusion.     

低氧致大鼠肺动脉平滑肌细胞中PTEN/Akt1表达的变化与细胞增殖的关系

目的 通过观察低氧致大鼠肺动脉平滑肌细胞(PASMC)与细胞张力蛋白同源在10号染色体有缺失的磷酸酶(PTEN)和丝氨酸/苏氨酸蛋白激酶1(Akt1)mRNA及其蛋白表达水平的变化与低氧PASMC增殖的关系,探讨PTEN/Akt1信号途径在低氧肺血管重建中的可能调控作用.方法 用组织块法培养PASMC.采用半定量逆转录-PCR技术检测常氧组、低氧2、8、12、24 h组PTEN、Akt1基因mRNA的表达水平,采用Western blot技术检测相应的蛋白表达水平.采用噻唑蓝比色法和氚-胸腺嘧啶脱氧核苷(3H-TdR)掺入法检测PASMC的增殖改变.数据用x±s表示,采用Excel 2003软件进行t检验,P<0.05为差异有统计学意义.结果低氧刺激PASMC不断增殖,3H-TdR法检测的吸光度值低氧12 h组(0.70±0.10)比常氧组(0.37±0.06)明显增高(t=14.29,P<0.01);噻唑蓝法检测的吸光度值低氧24 h组(11 208±679)比常氧组(8374±545)明显增高(t=19.56,P<0.01).各组均检测出PTEN、Akt1基因mRNA及蛋白表达的变化.常氧组Akt1的mRNA、总蛋白、磷酸化蛋白的吸光度值分别为0.76±0.09、25±6和48±8,随着低氧培养时间延长,吸光度值逐渐升高,低氧8 h组达到高峰,分别为1.05±0.09、41±7和79±14,与常氧组比较,差异均有统计学意义(t值为8.31～168.00,P<0.05和P<0.01),随后开始下降,低氧24 h组恢复至常氧组水平.常氧组PTEN的mRNA、磷酸化蛋白的吸光度值分别为0.25±0.06和98±8,并随着低氧培养时间延长而逐渐升高,低氧24 h组达到高峰,分别为0.38±0.05和232±12,与常氧组比较,差异均有统计学意义(t值分别为22.04和50.46,均P<0.01).结论 PTEN/Akt1的转录和激活与低氧肺血管重建的PASMC增殖密切相关.     

Gax基因双向调节低氧性肺动脉内皮细胞增殖及影响HIF-1α基因表达的研究

目的观察Gax基因对低氧性肺动脉内皮细胞（PAECs）增殖和低氧诱导因子-1α（HIF-1α）基因表达的影响,为进一步研究Gax基因调节低氧性肺动脉高压（HPH）的作用与机制奠定基础。方法取大鼠肺动脉,用酶消化法获取PAECs并进行原代培养;PAECs分4组：未转染常氧对照组（常氧组）、未转染低氧处理组（低氧组）、Ad—SGal转染再行低氧处理组（Ad—SGal＋低氧组）、Ad—Gax转染再行低氧处理组（Ad—Gax＋低氧组）。分别在常氧（21％O2）和低氧（2．5％O2）1h、3h、6h和12h各时相点,采用3H-胸腺嘧啶核苷（3H-TdR）掺入法检测PAECs增殖;使用RT—PCR和Weatern blot方法分别检测PAECs中HIF-1α mRNA和蛋白表达水平。结果①PAECs的3H-TdR掺入量：与常氧组同时相点比较,低氧组和Ad—pGaJ＋低氧组均显著升高（P均〈0．01）,在低氧6h达最大值;Ad．Gax＋低氧组与常氧组同时相点比较均显著升高（P均〈0．01）,但与低氧组比较却均明显降低（P〈0．01、P〈0．05）,到低氧6h降幅最大;②在低氧处理6h,与常氧组比较,低氧组和Ad—BGal＋低氧组HIF-1α mRNA和蛋白表达均明显上调;与低氧组或Ad—BGal＋低氧组比较,Ad—Gax＋低氧组HIF-1α mRNA和蛋白表达皆显著下调,差异有统计学意义（P〈0．05、P〈0．01）。低氧早期内皮细胞异常增殖加速,而此时增强Gax基因的表达可抑制细胞的异常增殖;随着低氧时间的不断延长,细胞增殖受到抑制,而此时增强内皮细胞中Gax基因表达却又促进细胞增殖,以此来维持细胞的数量。结论Gax基因对维持内皮细胞数量的稳态具有双向调节作用。增强Gax基因的表达能下调低氧诱导的HIF-1α mRNA和蛋白表达,这可能与Gax基因抑制低氧性内皮细胞异常增殖的机制相关。     

Adenosine prevents neutrophil adhesion to human endothelial cells after hypoxia/reoxygenation

BACKGROUND: Neutrophil adhesion to vascular endothelium has been implicated in the pathogenesis of myocardial injury after ischaemia/reperfusion (IR) and the "no-reflow" phenomenon. Adenosine and sodium-nitroprusside (SNP) have been used clinically to ameliorate this injury. We set out to establish a human cellular model for the study of IR and to evaluate the effects of adenosine and SNP on neutrophil adhesion in vitro. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) were exposed to hypoxia (5% CO2, 95% N2) or normoxia (room air, 5% CO2) for 2 h, followed by reoxygenation for 30 min (IR condition). Human neutrophils were then added together with adenosine (50 microM), SNP (10 microM) or no additive (control). After incubation for 1 h, neutrophil adhesion to endothelial cells was quantified via automated cell counts. The experiment was repeated with the adenosine treatment alone, with and without the addition of the adenosine A2A receptor blocker ZM-241385. RESULTS: Compared with baseline neutrophil adhesion after normoxia, hypoxia followed by reoxygenation increased adhesion to 189+/-43% (p=0.01), but this effect was prevented by the addition of adenosine (109+/-17%, p=NS compared to control conditions). SNP did not affect the increased adhesion caused by hypoxia (166+/-25%, p=NS). The addition of ZM-241385 did not inhibit the effect of adenosine on neutrophil adhesion after hypoxia/reoxygenation. CONCLUSIONS: Exposure of human endothelial cells to hypoxia/reoxygenation causes increased neutrophil adhesion. This effect is prevented by adenosine, but not mediated by the A2A receptor. SNP does not prevent neutrophil adhesion after IR in vitro.