烧伤大鼠早期切痂对内皮细胞间粘附分子-1及E-选择素 mRNA表达影响

目的　研究烧伤血清对体外培养的内皮细胞的细胞间粘附分子１（ＩＣＡＭ１）及Ｅ选择素（Ｅｓｅｌｅｃｔｉｎ）ｍＲＮＡ表达的影响,探讨实施休克期切痂上述指标的变化。　方法　Ｗｉｓｔａｒ 大鼠１７６ 只,３０％ Ⅲ度烫伤,收集不同时间点血清,采用逆转录ＰＣＲ 方法分别观察伤后不同时相点血清刺激内皮细胞ＩＣＡＭ１、Ｅｓｅｌｅｃｔｉｎ ｍＲＮＡ表达的规律。　结果　烫伤后血清可刺激内皮细胞ＩＣＡＭ１ 、ＥｓｅｌｅｃｔｉｎｍＲＮＡ表达上调,而且表达量与伤后时间呈正相关。伤后８ 及２４ 小时切痂动物血清不引起内皮细胞的上述变化,９６ 小时切痂（１－６０±０－１７）、（１－２０ ±０－０９） 效果不及休克期切痂组（０－９３ ±０－１４） 、（０－９３ ±０－２２）。　结论　及早切痂去除了坏死组织,避免了内皮细胞大量合成ＩＣＡＭ１、Ｅｓｅｌｅｃｔｉｎ 等粘附分子,减轻了由粘附分子介导的中性粒细胞对内皮细胞的进一步损害。     

急性胆管炎时肝组织ICAM-1和E-selectin mRNA的表达变化

目的　观察胆道感染大鼠肝组织细胞粘附分子１（ＩＣＡＭ１） 和Ｅｓｅｌｅｃｔｉｎ 的表达变化,探讨这些粘附分子在胆道感染肝脏损害病生机理中的作用。方法　应用原位分子杂交和斑点杂交技术观察大鼠胆道感染时ＩＣＡＭ１ 和Ｅｓｅｌｅｃｔｉｎ ｍＲＮＡ在肝组织的定位及转录表达强度。结果　急性胆管炎３ 小时后ＩＣＡＭ１ 和Ｅｓｅｌｅｃｔｉｎ ｍＲＮＡ表达开始增强,ＩＣＡＭ１ １２ 小时表达最强,Ｅｓｅｌｅｃｔｉｎ ６ 小时表达最强。ＩＣＡＭ１ 的阳性反应细胞主要为肝窦内皮细胞、枯否细胞和肝小叶中央静脉,Ｅｓｅｌｅｃｔｉｎ 的阳性反应细胞主要为肝窦内皮细胞和肝小叶中央静脉。胆道感染后３ 小时肝脏ＩＣＡＭ１ 和Ｅｓｅｌｅｃｔｉｎ ｍＲＮＡ含量开始进行性增高,ＩＣＡＭ１ １２ 小时达高峰,Ｅｓｅｌｅｃｔｉｎ ６ 小时达高峰。结论　胆道感染初期ＩＣＡＭ１和Ｅｓｅｌｅｃｔｉｎ 表达均明显增强,为ＰＭＮ 与肝窦内皮细胞粘附和迁移提供了分子基础,可能在胆道感染肝脏损害的病生机理中具有重要意义。     

脓毒症小鼠肝损伤时的粘附分子基因表达

目的 探讨脓毒症小鼠肝损伤时肝窦内皮细胞粘附分子基因表达变化。方法 用盲肠结扎穿孔（ＰＬＰ）法制造小鼠脓毒症模型,假手术组（ｓｈａｍ）接受同样的手术操作但不行ＣＬＰ。分别在致伤后３、１２ｈ分离肝窦内皮细胞,用ＲＴ－ＰＣＲ的方法定量检测Ｅ－ｓｅｌｅｃｔｉｎ、ＩＣＡＭ－１的基因表达,同时还测定了肝组织中ＭＰＯ活性及微血管通透性的改变。结果 ＣＬＰ后３ｈ肝窦内皮细胞Ｅ－ｓｅｌｅｃｔｉｎ基因的表达明显增高     

血小板活化因子及其受体对颈髓损伤后血脊髓屏障损害的分子机制研究

目的：探讨血小板活化因子（ＰＡＦ）及其受体对颈髓损伤后血脊髓屏障损害的分子机制。方法：采用蛛网膜下腔注射ＰＡＦ及静脉注射ＰＡＦ受体拮抗剂ＢＮ５２０２１，应用地高辛标记ｃＤＮＡ探针原位杂交技术检测颈髓损伤后颈髓血管内皮细胞细胞间粘附分子－１ｍＲＮＡ（ＩＣＡＭ－１ｍＲＮＡ）和内皮细胞白细胞粘附分子－１ｍＲＮＡ（ＥＬＡＭ－１ｍＲＮＡ）表达。观察ＰＡＦ及其受体拮抗剂对颈髓损伤后血脊髓屏障、ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达的影响。结果：伤后颈髓血管内皮细胞ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达、伊文思蓝含量、水含量呈不同程度的增加；ＰＡＦ可使伤后血管内皮细胞ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达、伊文思蓝含量、水含量增加更为显著，ＰＡＦ受体拮抗剂可抑制ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达，降低颈髓组织伊文思蓝含量及水含量。结论：ＰＡＦ通过增加伤后颈髓血管内皮细胞粘附分子的表达是导致血脊髓屏障损害的重要分子基础。ＰＡＦ受体对伤后血管内皮细胞细胞粘附分子的表达具有调控作用。     

肝细胞肝癌细胞间粘附分子-1 mRNA表达的研究

寻找甲胎蛋白（ＡＦＰ）以外更新、更敏感的血清标记物是肝癌研究中的重要课题。近年有关细胞间粘附分子－１（ＩＣＡＭ－１）和肝细胞肝癌（ＨＣＣ）的研究提示,ＩＣＡＭ－１在ＨＣＣ病人组织及血清中（ｃＩＣＡＭ－１）呈高水平表达＾〔１,３〕。本研究通过检测ＨＣＣ组织中ＩＣＡＭ－１信使核糖核酸（ＩＣＡＭ－１ｍＲＮＡ）的表达,探讨血清中高水平ＩＣＡＭ－１与ＨＣＣ组织中ＩＣＡＭ－１ｍＲＮＡ的相互关系,为ｃＩＣＡＭ－测定在ＨＣＣ早期诊断及术后复发监测中的作用寻找理论依据。     

肠缺血—再灌流大鼠小肠血管内皮细胞ICAM—1表达的变化及？…

目的 探讨肠缺血－再灌流过程中肠局部血管内皮细胞的细胞间粘附分子－１（ｉｎｔｅｒｃｅｌｕｌａｒ ａｄｈｅｓｉｏ ｍｏｌｅｃｕｌｅ－１，ＩＣＡＭ－１）ｍＲＮＡ和蛋白表达的变化与中性粒细胞聚集的关系及其在肠损伤中的作用。方法 采用肠系膜上动脉夹闭的方法制作大鼠肠缺血－再灌流模型，应用逆转录－聚合酶链反应及免疫组织化学的方法观察小肠ＩＣＡＭ－１表达的变化，同时检测组织髓过氧化物酶活性及反映肠功能的指标Ｄ     

大鼠肾脏缺血及保存再灌注后E-selectin的表达情况

目的了解大鼠肾脏缺血及保存再灌注后Ｅｓｅｌｅｃｔｉｎ的表达情况。方法以ＥｓｅｌｅｃｔｉｎｃＤＮＡ为探针,应用Ｎｏｒｔｈｅｒｎｂｌｏｔ法分别测定缺血１５、３０分钟及保存２４、４８小时以及缺血、保存再灌注后８、１６小时大鼠肾脏组织ＥｓｅｌｅｃｔｉｎｍＲＮＡ的表达情况。结果实验显示缺血及保存的肾脏未见ＥｓｅｌｅｃｔｉｎｍＲＮＡ表达,而缺血及保存再灌注后肾脏均见ＥｓｅｌｅｃｔｉｎｍＲＮＡ表达,且保存肾脏在再灌注后早期ＥｓｅｌｅｃｔｉｎｍＲＮＡ表达受到暂时性抑制。结论缺血及保存后再灌注是肾脏组织ＥｓｅｌｅｃｔｉｎｍＲＮＡ表达的原因,保存再灌注后表达的暂时性抑制可能与低温有关。     

NF—kB及其能脏疾病关系研究进展

ＮＦ－ｋＢ是参与基因转录的蛋白质分子，广泛存在于机体各种组织细胞中。当细胞受到外界面素刺激时，胞质内ＮＦ－ｋＢ与抑制蛋白（ＩｋＰｓ）分离并活化，进入细胞核内与ＤＮＡ结合启动基因转录，调节包括细胞因子和炎症介质（如ＩＬ－２、ＩＬ－２ａ、ＩＬ－６、ＩＬ－８，ＶＣＡＭ、ＩＣＡＭ、Ｅ－ｓｅ－ｋｅｃｔｉｎ、ＩＦＮ－β、ＭＣＰ－１、ＲＡＮＴＥＳ等）在内的众多蛋白质表达，从而参与调节组织细胞的生理、病理反应。在     

CsA,MPA和RPM对CD28通路共刺激后淋巴细胞IL—4mRNA表达？…

目的 观察环孢素Ａ（ＣｓＡ）霉酚酸（ＭＰＡ）和雷帕霉素（ＲＰＭ）对ＣＤ２８通路共刺激后淋巴细胞ＩＬ－４ｍＲＮＡ表达的影响。方法 以ＰＣＲ扩增方法获得了ＩＬ－４特异的ｃＤＮＡ片段，并将其克隆至ＰＧＥＭ－３Ｚ载体中，以此作为探针对ＲＴ－ＰＣＲ产物进行杂交定量，比较三种免疫抑制剂对ＣＤ２８通路共刺激后淋巴细胞ＩＬ－４ｍＲＮＡ表达的影响。结果 以抗ＣＤ３ｍＡｂ＋抗ＣＤ２８ｍＡｂ共刺激后，发现淋巴细胞稳定地     

粘附分子表达在脓毒症小鼠肺损伤中的作用

目的 探讨肺微血管内皮细胞粘附分子表达在脓毒症小鼠肺损伤中的作用。方法 盲肠结扎穿孔（ＣＬＰ）制造小鼠脓毒症模型,分别在致伤后３、１２ｈ分离肺微血管内皮细胞,用逆转录－聚合酶链（ＲＴ－ＰＣＲ）方法定量检测Ｅ－选择素、细胞间粘附分子（ＩＣＡＭ）－１和血管细胞粘附分子（ＶＣＡＭ）－１的基因表达,同时测定肺组织中髓过氧化物酶（ＭＰＯ）活性的改变。结果 ＣＬＰ后３ｈ肺微血管内皮细胞Ｅ－选择素、ＩＣＡＭ－１     

不同时期切痂对烫伤大鼠骨骼肌和脂肪组织葡萄糖转运蛋白-4 mRNA表达水平的影响

目的 -探讨大面积烫伤后实施休克期切痂对大鼠骨骼肌、脂肪组织葡萄糖转运蛋白4(GLUT4)mRNA表达水平的影响。方法采用30％Ⅲ度TBSA烫伤大鼠模型,分别于伤后8、24、96h切痂,测定血清胰岛素、胰高血糖素、皮质醇及血糖水平,采用RT-PCR法检测烫伤和切痂后大鼠骨骼肌、脂肪组织GLUT4 mRNA表达水平。结果休克期切痂使血糖、胰高血糖素、皮质醇水平下降;伤后骨骼肌、脂肪组织GLuT4 mRNA表达水平下降,休克期切痂可促使其回升。结论大面积烫伤后大鼠骨骼肌、脂肪组织GIMT4 mRNA表达减少,休克期切痂可促使其恢复,有利于糖代谢的改善,减轻高代谢状态。     

大鼠烫伤后不同时间切痂骨骼肌解偶联蛋白2、3 mRNA表达水平的改变

目的观察烫伤大鼠伤后不同时间切痂其骨骼肌解偶联蛋白(UCP)2、UCP3 mRNA表达水平的异同. 方法选用120只雄性Wistar大鼠,其中8只作为正常对照组;余下112只造成30%TBSAⅢ度烫伤后分成4组A组不切痂,分别于伤后8、24、96、120、168 h处死;B组伤后8 h切痂,于伤后24、96、120、168 h处死;C组伤后24 h切痂,伤后96、120、168 h处死;D组伤后96 h切痂,伤后120、168 h处死.测定各组大鼠各时相点的血清瘦素、肿瘤坏死因子(TNF)α含量以及腓肠肌UCP2、UCP3 mRNA表达水平. 结果 (1)血清瘦素水平A组大鼠伤后24～168 h均低于正常对照组(P<0.01),B、C、D组伤后120 h和(或)168 h均高于A组(P<0.01).(2)血清TNF-α水平A组伤后各时相点均高于正常对照组(P<0.01),B组伤后各时相点均低于A组(P<0.05或0.01).C组伤后168 h低于A组(P<0.05).(3)腓肠肌UCP2 mRNA的表达量A组大鼠在烫伤后8 h即已明显升高(P<0.01),24 h到达高峰,以后逐渐下降.B、C组伤后168 h时分别为0.32±0.20、0.35±0.15,明显低于同时相点A组 0.71±0.12(P<0.05).各组腓肠肌UCP3 mRNA表达的变化趋势与UCP2类似. 结论大鼠严重烫伤后UCP2、UCP3 mRNA表达上调可能是代谢率升高的重要因素之一,休克期切痂可降低这一表达,降低代谢率.     

休克期切痂对烫伤大鼠γ干扰素、白细胞介素4 mRNA表达的影响

目的探讨烫伤大鼠伤后不同时间切痂对Th1细胞因子γ干扰素(IFN-γ)和Th2细胞因子白细胞介素4(IL-4)循环水平的变化及其mRNA在脾脏T淋巴细胞表达的影响。方法160只健康雄性Wistar大鼠背部30％Ⅲ度烫伤后,随机分为单纯烫伤对照组和伤后8h、24h、96h切痂组,分别在伤后4、12、24、48、96、120、168h活杀动物,采集血液和脾脏标本。ELISA法检测血浆内IFN-γ、IL-4浓度;Ficoll分离液法、贴壁法和尼龙毛柱吸附法提取纯化脾脏T淋巴细胞,一步法提取细胞总RNA、RT-PCR法扩增目的基因、琼脂糖凝胶电泳分析IFN-γ和IL-4 mRNA的表达。结果烫伤4h大鼠循环内上述细胞因子的浓度大幅升高,同时其mRNA表达迅速上调。Th1型细胞因子IFN-γ及其mRNA表达在烫伤后24h达峰值,其后逐渐下降;Th2型细胞因子IL4的循环浓度及mRNA表达进行性增加;伤后7d出现明显偏向Th2型反应的现象。切痂组两个细胞因子浓度增加及其mRNA表达上调的变化幅度小于单纯烫伤对照组,其中8h切痂组的变化最小,24h和96h切痂组次之。结论休克期切痂有利于抑制严重烧伤后Th2型细胞因子的过度表达。     

E-selectin early overexpression induced by allogeneic activation in isolated mouse lung

BACKGROUND: The interaction between host lymphocytes and graft endothelial cells plays an important role in graft rejection. METHODS: Using our model of isolated ventilated lung from female mouse perfused with fresh blood from either isogeneic or allogeneic male mouse for 3 hours without noticeable ischemia, we have investigated the kinetics of the early events after endothelial cell triggering by E-selectin engagement. RESULTS: Isogeneic perfusion induced nonspecific endothelial cell activation, which was characterized by up-regulation of E-selectin, intercellular adhesion molecule (ICAM)-1, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, and lymphotoxin-alpha (mRNAs by real-time polymerase chain reaction). Allogeneic perfusion was characterized after 3 hours by an additional loose adhesion of lymphocytes mediated by the E-selectin and related to the allogeneic activation of endothelial cells. These in turn expressed the I-A molecule (immunostaining). ICAM-1 and lymphocyte function-associated antigen (LFA)-3 mRNA levels were significantly increased in lung extracts after 2 hours, then vascular cell adhesion molecule (VCAM)-1 and TNF-alpha mRNAs after 3 hours without evidence of TNF-alpha production (enzyme-linked immunoadsorbent assay). The major participation of the E-selectin in early allogeneic activation by way of the protein kinase (PK)C pathway was confirmed by using a neutralizing anti-CD62E monoclonal antibody or the inhibitory PKC 19-31 fragment. CONCLUSIONS: Altogether, our results demonstrate that E-selectin expression (1) is not a consequence of TNF-alpha triggering, (2) up-regulates its own expression and expression of I-A, VCAM-1, TNF-alpha, and lymphotoxin-alpha mRNAs, and (3) down-regulates expression of LFA-3 and ICAM-1 mRNAs. In conclusion, in our physiologic model, the E-selectin highly participates in the loose adhesion of allogeneic lymphocytes and in the early activation of endothelial cell and therefore in structural and functional lung alterations.     

P-cresol,a uremic toxin,decreases endothelial cell response to inflammatory cytokines

BACKGROUND: Infectious diseases are among the most morbid events in uremia. The uremic toxin p-cresol may play a role in the immunodeficiency of uremia by depressing phagocyte functional capacity. Leukocyte adhesion to endothelium, a key event in the immune response, is mediated by endothelial adhesion molecules. These include intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, which are induced by various inflammatory cytokines. We asked whether p-cresol alters endothelial adhesion molecule expression and modifies endothelial/leukocyte adhesion. METHODS: Human umbilical vein endothelial cells (HUVEC) were incubated with p-cresol in the presence or absence of tumor necrosis factor (TNF) or interleukin-1beta (IL-1beta). Thereafter, the endothelial molecules ICAM-1, VCAM-1, and E-selectin were quantitated and the monocyte (THP-1) adhesion to HUVEC measured. RESULTS: P-cresol decreased cytokine-induced protein and mRNA expression of ICAM-1 and VCAM-1. In addition, p-cresol significantly decreased the adhesion of THP-1 to cytokine-stimulated HUVEC. CONCLUSIONS: P-cresol may play a role in the immune defect of uremic patients by inhibiting cytokine-induced endothelial adhesion molecule expression and endothelium/monocyte adhesion.     

VCAM-1, ICAM-1, and E-selectin in IgA nephropathy and Schönlein-Henoch syndrome: differences between tissue expression and serum concentration

The tissue expressions of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and endothelial leukocyte adhesion molecule 1 (E-selectin-1) were investigated in biopsy specimens from 28 patients with different stages of IgA nephropathy (IgAN) and 20 patients with acute renal failure (ARF) or chronic renal diseases (amyloidosis, Alport's glomerulopathy) by immunohistochemistry. The results were compared with the serum levels of the three adhesion molecules. VCAM-1 expression was significantly increased on parietal/tubular epithelial cells in IgAN and ARF. Significantly elevated circulating VCAM-1 levels were measured in IgAN and amyloidosis, but did not correlate with renal function (creatinine clearance). Significantly increased glomerular endothelial/epithelial ICAM-1 expression was found in IgAN and ARF. Intense mesangial ICAM-1 expression was found in mild stages of IgAN and in Sch?nlein-Henoch syndrome. Circulating ICAM-1 was not significantly elevated in IgAN and different renal diseases. VCAM-1 and ICAM-1 expressions of interstitial infiltrating cells were significantly higher in severe than in mild IgAN and associated with an increased infiltration of inflammatory leukocytes. Patients with IgAN and different renal diseases had decreased mesangial and almost absent interstitial E-selectin expression as compared with controls. The circulating E-selectin levels were significantly elevated in ARF. In conclusion, the tissue expression of adhesion molecules in IgAN reflects a continuous inflammatory renal activity. However, only increased circulating VCAM-1 serum levels correlated significantly with the histological state of renal inflammation and could be used as a disease marker.     

Tissue-specific induction of E-selectin in glomeruli is augmented following diabetes mellitus

We recently demonstrated that induction of adhesion molecules is tissue, cell type, and blood vessel size specific. We examined here whether the glomeruli, a peculiar vascular system, express adhesion molecules in a specific manner in the murine kidney. In addition, since serum levels of soluble adhesion molecules have been reported to be elevated in diabetic patients, we examined the influence of diabetes mellitus on the induction of adhesion molecules in the kidney. Analysis of E-selectin mRNA expression by in situ hybridization indicated that it was selectively induced in glomeruli by intravenous administration of interleukin-1beta, while ICAM-1 mRNA expression was seen diffusely in endothelium lining the small arteries and capillaries or in glomeruli, and VCAM-1 mRNA expression was most prominent in endothelial cells of larger blood vessels. Induction of E-selectin mRNA expression in glomeruli by proinflammatory stimuli was augmented in streptozotocin-induced diabetic mice as compared with control mice, while ICAM-1 or VCAM-1 mRNA induction was only slightly influenced. Furthermore, immunohistochemical analysis showed that selective expression of E-selectin in glomeruli was augmented predominantly in epithelial cells, depending on the duration of diabetes mellitus, in KK-Ay mice. These findings suggest that glomerulus-specific expression of E-selectin is related to the development of diabetic nephropathy.     

罗格列酮对大鼠急性胰腺炎肺损伤黏附分子表达的作用

目的 探讨过氧化物酶体增殖物激活受体-γ(PPAR-γ)激动剂罗格列酮(ROSI)对大鼠急性胰腺炎肺损伤(APALI)黏附分子的作用及其机制.方法 雄性Wistar大鼠54只,随机分为假手术组(SO组)、急性胰腺炎组(SAP组)和罗格列酮预处理组(ROSI组).胆胰管逆行注射5%牛磺胆酸钠制备急性胰腺炎模型.ROSI组造模前30 min经股静脉注射10%二甲基亚砜(DMSO)溶解的罗格列酮(6 mg/kg);SO组、SAP组则注射等量10%DMSO.术后3 h、6 h、12 h分批剖杀大鼠,每个时间点6只.检测血清淀粉酶(AMY)、肺组织髓过氧化物酶(MPO)、肺湿干比(W/D),取肺组织行病理学检查;逆转录聚合酶链反应(RT-PCR)检测肺组织细胞间黏附分子-1(ICAM-1)、P-选择素和E_选择素mRNA表达水平.结果 SAP组各时间点AMY、MPO、W/D和肺组织病理评分均较S0组升高(P<0.05);ROSI组上述指标较SAP组下降,AMY、MPO、W/D和病理评分在6 h、12 h差异有统计学意义(P<0.05).SAP组ICAM-1、P-选择素和E-选择素mRNA表达在12 h达高峰,均较SO组12 h升高(P<0.05),ROSI组上述指标mRNA表达在12 h点均较SAP组下降(P<0.05).结论 罗格列酮通过抑制肺组织IcAM-1、P-选择素和E-选择素的表达,减轻急性胰腺炎肺损伤程度.