【作者】 李锁北；

【导师】 徐军美；

【作者基本信息】 中南大学 ， 麻醉学， 2010， 博士

【摘要】 研究目的(1)建立人脐静脉内皮细胞(HUVEC)体外培养方法,探讨肿瘤坏死因子-a (TNF-a)对HUVEC表达细胞间粘附分子(ICAM-1)、血管细胞间粘附分子(VCAM-1)的影响,筛选出TNF-a诱导粘附分子表达的最适浓度和时间；研究七氟醚预处理对HUVEC表达ICAM-1、VCAM-1及中性粒细胞粘附的影响；(2)研究七氟醚预处理对TNF-a诱导的血管内皮细胞NF-κB信号转导通路激活的影响,为七氟醚预处理抗炎作用的机制提供理论和实验依据；(3)探讨eNOS-NO信号系统对七氟醚预处理在体外培养的血管内皮细胞抗炎效应的影响。研究方法实验分为三个部分：(1)原代细胞培养参照参考文献报道方法并加以改进,通过形态学观察和免疫细胞化学法对细胞进行鉴定；应用Western blot及实时荧光定量PCR方法,检测不同浓度TNF-a (0、1ng/ml、2.5ng/ml、10ng/ml、40ng/ml)处理内皮细胞4小时,以及TNF-a (10ng/ml)作用不同时间(0、2h、4h、12h)后ICAM-1、VCAM-1蛋白及mRNA的表达,确定TNF-a诱导血管内皮细胞粘附分子稳定表达的最佳作用浓度和时间；然后将体外培养的HUVEC分为空白对照组、1.5MAC七氟醚组、10ng/ml TNF-a组以及10ng/ml TNF-a+0.5MAC、1.5MAC或2.5MAC七氟醚预处理组,分别检测七氟醚对TNF-a诱导HUVEC表达粘附分子及中性粒细胞粘附的影响。(2)应用核浆分离技术和Western blot方法,观察10ng/mlTNF-a刺激对HUVEC的IκBα、p-IκBα、核浆＼胞浆内NF-κB/p65亚基蛋白水平的影响及其时间趋势；将体外培养的HUVEC分为空白对照组、1.5MAC七氟醚单独刺激组、lOng/ml TNF-a单独刺激组以及1.5MAC七氟醚预处理组,检测各组细胞IκB-α、p-IκB-α以及核浆＼胞浆内NF-κB/P65亚基蛋白在各自变化高峰时点的水平。(3)分别以0.0.5MAC、1.5MAC和2.5MAC七氟醚预处理体外培养的HUVEC,应用western blot法检测p-eNOS和eNOS的蛋白水平,Griess reagent法检测细胞培养上清液中总NO含量,DAF-FM DA荧光探针法检测细胞内的NO含量；将体外培养的HUVEC分为10ng/ml TNF-a单独刺激组、1.5MAC七氟醚+10ng/ml TNF-a刺激组、eNOS抑制剂L-NAME组,并检测各相应时点的细胞培养上清液中总NO含量、细胞内NO含量以及p-eNOS\eNOS、IκB-a\p-IκB-α、核浆\胞浆内NF-KB/P65蛋白水平以及ICAM-1\VCAM-1的蛋白、mRNA水平和中性粒细胞与HUVEC的粘附率,确定eNOS-NO信号系统是否介导了七氟醚预处理在血管内皮细胞的抗炎作用。研究结果(1)改良的HUVEC的体外培养方法是成功的,经相差显微镜观察以及Ⅷ因子染色证实培养的细胞(97.5%)为HUVEC,10ng／ml的TNF-a处理HUVEC后,VCAM-1、ICAM-1在4h明显升高,之后稳定表达,台盼蓝染色显示95%以上细胞存活良好,TNF-a诱导HUVEC稳定表达粘附分子的最佳作用浓度为10ng/ml,时间为4h。与TNF-a单独刺激血管内皮细胞比较,1.5、2.5MAC七氟醚预处理明显降低TNF-a诱导血管内皮细胞的ICAM-1和VCAM-1蛋白及mRNA表达水平,并降低了中性粒细胞粘附率(P<0.05)。(2)10 ng/ml TNF-a刺激内皮细胞后,IκBa发生降解,同时磷酸化水平上调,随之p65亚基向核内移位,IκBa降解及磷酸化水平上调的时间高峰为30分钟,p65亚基入核的高峰时间为60分钟(P<0.01)；在各检测指标变化的高峰时点,与空白对照组比较,1.5MAC七氟醚单独刺激组细胞的IKBa蛋白降解、p-IκBa蛋白水平以及NF-KB/p65蛋白亚基的入核没有显著变化,而1.5MAC七氟醚预处理组IκBa蛋白降解、p-IκBa蛋白水平以及NF-KB/p65蛋白亚基的入核较TNF-a单独刺激组的有明显抑制(P<0.05)。(3)与空白对照组比较,1.5MAC和2.5MAC七氟醚预处理组的p-eNOS的表达明显增加,且p-eNOS/eNOS比值有显著性增高(P<0.05),且细胞培养上清液中总NO含量显著增加(P<0.05),细胞内NO含量有明显增加；与七氟醚预处理组比较,L-NAME组的细胞内p-eNOS/eNOS、NO含量及细胞培养上清液中总NO含量明显下降(P<0.05),同时,细胞的IKBa蛋白降解、p-IκBa蛋白表达水平以及NF-KB/p65蛋白亚基的入核、ICAM-1\VCAM-1的蛋白及mRNA水平和中性粒细胞粘附率均有显著性增加(P<0.05)。研究结论(1)七氟醚预处理抑制TNF-a诱导的血管内皮细胞粘附分子表达及与中性粒细胞的粘附;(2)七氟醚预处理下调TNF-a诱导的内皮细胞NF-κB信号通路激活,这可能介导了七氟醚预处理在TNF-a诱导的血管内皮细胞炎症反应中的抗炎作用；(3)七氟醚预处理促进血管内皮细胞eNOS磷酸化和随后的NO生成,进而抑制TNF-a诱导的NF-κB信号通路的激活,减轻了血管内皮细胞炎症反应。更多还原

【Abstract】 Objective (1) To establish culturing technique of HUVECs in vitro. To investigate the effects of TNF-a on the expression of ICAM-1, VCAM-1 of HUVECs and then to decide the optimal action time course and concentration. To investigate the inhibitory effects of sevoflurane pretreatment with different concentration on the expression of ICAM-1, VCAM-1 in TNF-a-induced HUVECs and neutrophils adhesion to HUVECs induced by TNF-a. (2) To investigate the effects of sevoflurane pretreatment on the activity of NF-κB signal transduction pathway in TNF-a-induced HUVECs, so as to provide theoretical and experimental evidence for the mechanism of anti-inflammatory effects of sevoflurane pretreatment. (3) To explore the effects of eNOS-NO signal system on anti-inflammatory effects of sevoflurane pretreatment on vascular endothelial cells in vitro.Methods The study is divided into three parts:(1) HUVECs were cultured according to references with minor modifications. Endothelial cells were identified by both morphology and immunocytochemical staining response to factorⅧrelated antigen. We measured the expression of ICAM-1, VCAM-1 of HUVECs induced by TNF-a at different concentrations (0, 1ng/ml,2.5ng/ml, lOng/ml,40ng/ml) by different time course (0, 1h,2h,4h,12h) by methods of Western blot and realtime quantitative chain reaction polymerase (qRT-PCR). Cultured HUVECs in vitro were randomly divided into six groups:vehicle, 1.5MAC sevoflurane, lOng/ml TNF-a,0.5MAC sevoflurane+lOng/ml TNF-a,1.5MAC sevoflurane+10ng/ml TNF-a,2.5MAC sevoflurane +10ng/ml TNF-a, the expression of ICAM-1 and VCAM-1 of HUVECs were detected by methods of Western blot and qRT-PCR and the adhesion ratio of neutrophils to HUVECs was measured by methods of myeloperoxidase. (2) In vitro study was performed on cultured HUVECs subjected to lOng/ml TNF-a, the expression of IκBa, p-IκBa and NF-KB/p65 subunit in nucleus or endochylema at different time course were detected by Western blot; the cultured HUVECs were randomly exposed to one of the following treatments:vehicle,1.5MAC sevoflurane, lOng/ml TNF-a,1.5MAC sevoflurane+10ng/ml TNF-a, the protein levels of IκBα, p-IκBa and NF-κB/P65 subunit in nucleus or endochylema were measured at respective alterative peak time points. (3) HUVECs were randomly exposed to one of the following treatments:vehicle,0.5MAC sevoflurane,1.5MAC sevoflurane,2.5MAC sevoflurane,30 min aftter sevoflurane pretreatment, the protein levels of p-eNOS and eNOS were detected by Western blot, the total NO content in cell medium by methods of Griess reagent and the NO content in vascular endothelial cells by methods of DAF-FM DA fluorescent probe; HUVECs were randomly assigned to receive lOng/ml TNF-a for 4 hours (control),1.5MAC sevoflurane sequently combined with TNF-a in the presence or absence the eNOS inhibitor N-nitro-L-arginine methyl ester (L-NAME; 1mM) pretreatment, the NO content in vascular endothelial cells and cultured medium, the protein levels of p-eNOS\eNOS, IKBa\p-IκBa, NF-κB/p65 subunit in nucleus\endochylema, the protein and mRNA levels of ICAM-1\VCAM-1 in vascular endothelial cells and the adhesion ratio of neutrophils to HUVECs were determined at corresponding time points.Results (1) HUVECs presented cobblestone-shaped and a positive immunochemistry reaction (97.5%). The increased expression of ICAM-1, VCAM-1 protein and mRNA levels in TNF-a-induced HUVECs was in a dose-dependent and time-dependent manner, HUVECs subjected to 10ng/ml TNF-a for 4 hours had a stable expression of ICAM-1, VCAM-1 protein and mRNA levels.sevoflurane pretreatment with 1.5MAC and 2.5MAC concentration both siginificantly inhibited the the expression of ICAM-1/VCAM-1 protein and mRNA levels in HUVECs, and the adhesion ratio of PMNs to HUVECs was also down-regulated (P<0.05). (2) Phosphorylation and degradation of IκBa, subsequent nucleus-bound translocation of p65 subunit of NF-κB were activated by lOng/ml TNF-a, the alterative peak time was respectively 30 minutes,60 minutes after TNF-a stimulti (P＜0.01).1.5 MAC sevoflurane pretreatment could siginificantly inhibit phosphorylation and degradation of IκBαand nucleus-bound translocation of p65 subunit compared with 10ng/ml TNF-a group (P<0.05). (3) sevoflurane exposure evoked an increased NO content in vascular endothelial cells and cell medium, followed by phosphorylation of eNOS in HUVECs (P<0.05). Compared with sevoflurane pretreatment group, L-NAME decreased p-eNOS/eNOS and NO content both in cells and medium, L-NAME aslo increased phosphorylation and degradation of IκBα, subsequent nucleus-bound translocation of NF-αB p65, mRNA and protein expression of ICAM-1 and VCAM-1 in HUVECs and the adhesion ratio of neutrophils to HUVECs (P＜0.05).Conclusion (1) Sevoflurane pretreatment inhibited the expression of ICAM-1, VCAM-1 induced by TNF-a and neutrophils adhesion to HUVECs. (2) Sevoflurane pretreatment down-regulated the activation of NF-κB signal pathway induced by TNF-a, which may play a major role in anti-inflammatory effects of sevoflurane pretreatment on vascular endothelial cells in vitro. (3) The anti-inflammatory effects of sevoflurane pretreatment could be mediated by eNOS phosphorylation and subsequent NO release, which inhibited NF-κB signal pathway activation and inflammatory response on TNF-a-induced vascular endothelial cell.更多还原