Leukotriene B4 stimulates polymorphonuclear leukocyte adhesion to cultured vascular endothelial cells.

Adhesion of polymorphonuclear leukocytes (PMN) to the endothelial lining of blood vessels is an essential component of the inflammatory response. We have examined the effects of various lipoxygenase metabolites of arachidonic acid on PMN adhesion to cultured vascular endothelial cells, using a quantitative monolayer adhesion assay. Our results indicated that leukotriene B4 (LTB4) could effectively stimulate PMN adhesion to endothelial cell surfaces, in contrast to the sulfidopeptide leukotrienes C4, D4, and E4, and the monohydroxyacid lipoxygenase products of leukocytes and platelets, 5S-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid and 12S-hydroxy-5,8-cis,10-trans,14-cis-eicosatetraenoic acid, respectively. LTB4-stimulation of PMN-endothelial adhesion did not appear to be dependent upon the generation of cyclooxygenase metabolites, nor was it inhibited by exogenous prostacyclin. Enhanced PMN adhesion was observed with endothelial cells that were cultured from different types of large vessels (arteries and veins) in several species. These findings suggest an important pathophysiologic role for LTB4 in regulating leukocyte-vessel wall interactions.     

The effect of cytomegalovirus infection on the adherence of polymorphonuclear leucocytes to endothelial cells

Adherence of polymorphonuclear leucocytes (PMN) to the endothelial lining of blood vessels is an essential component of the inflammatory response. In this study the effect of cytomegalovirus (CMV) infection on the adherence of PMNs has been examined using an in vitro model system. Human umbilical venous endothelial cells (HUVEC) were grown on fibronectin-coated plastics. CMV infection of HUVEC resulted in the appearance of viral antigens in a small percentage of the cells. At 24 h post-infection when no virus-induced cytopathic effect could be observed in the cell monolayers, the adherence of PMNs was significantly increased. The virus-induced adherence effect was cell bound and could not be induced by soluble components in the medium of the virus-infected cells. The augmentation of the PMN adherence to CMV-infected endothelium was sensitive to tunicamycin suggesting that the virus infection induces the expression of glycoproteins on the HUVEC membranes which are responsible for the PMN adherence. Thus CMV infection of the endothelium results in an increased adherence of PMNs. In the in vivo situation systemic viral infection can potentially lead to infection of blood vessel endothelium and thus can induce a damage of endothelium. This phenomenon could play a role in the atherogenesis process.     

丹参对兔急性胰腺炎早期中性粒细胞与内皮细胞黏附抑制作用的实验研究

目的：探讨中性粒细胞(PMN)与内皮细胞(EC)黏附在急性胰腺炎(AP)早期发病中的机制及丹参对AP的治疗作用。方法：采用兔AP模型，24只日本大耳兔随机分为生理盐水组和丹参治疗组。分别于术前、术后即刻及2、4、8、12、16和24h取血分离获得PMN，与体外培养的兔血管EC作用，测定PMN—EC黏附率及PMN表面黏附分子CD11a／CD18和CD11b／CDl8值。结果：术后即刻两组黏附分子CD11a／CD18、CD11b／CD18及PMN—EC黏附率均升高，生理盐水组术后4h达到高峰；丹参治疗组术后2、4、8、12、16和24h的CD11a／CD18、CD11b／CD18显著低于生理盐水组。结论：丹参能降低黏附分子CD11a／CD18、CD11b／CD18表达，抑制PMN—EC黏附，改善微循环及减轻PMN—EC黏附所致的组织损伤，有助于AP的早期治疗。     

Platelet adherence to cardiac and noncardiac endothelial cells in culture: lack of a prostacyclin effect

Cardiac tissues show a propensity to develop nonbacterial thrombotic endocarditis, a meshwork of platelets and fibrin. This lesion may cause a predisposition to subsequent colonization by circulating microorganisms, leading to infective endocarditis. We measured platelet adherence in vitro to cultured endothelial cells derived from the porcine aortic valve and ascending aorta. We found that valvular endothelial cells showed a twofold to threefold higher adherence than ascending aortic endothelial cells of chromium 51-labeled platelets in the presence of proteolytically active thrombin. This finding did not correlate with endothelial prostacyclin release: cardiac valve endothelial cells released more prostacyclin than did, ascending aortic cells, exogenous prostacyclin had no effect on thrombin-stimulated adherence, and aspirin inhibition of endothelial prostacyclin synthesis showed no effect on platelet adherence. Fixation of platelets abolished thrombin-stimulated adherence; fixation of endothelial cells had minimal effect. We suggest that these differences may contribute to the propensity of the cardiac valve to develop nonbacterial thrombotic endocarditis.     

Effects of erythromycin, josamycin and spiramycin on rat polymorphonuclear leukocyte chemotaxis

The effects of three macrolide antibiotics were studied on rat polymorphonuclear leukocyte chemotaxis. Rats were given 25 mg/kg twice a day of either erythromycin, josamycin or spiramycin by gastric intubation for 5 days. In all cases, chemotaxis was found to be impaired by 10-20% only. As macrolides are known to reach high intracellular concentrations within polymorphonuclear leukocytes, our results suggest that these antibiotics are unlikely to exert a deleterious influence on the chemotactic response of treated patients.     

Hydrogel surfaces to promote attachment and spreading of endothelial progenitor cells

Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin‐based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA‐based hydrogels provides an attractive surface for EPCs promoting spreading and the formation of an endothelial monolayer on the hydrogel surface. To increase EPC adhesion and spreading, we covalently immobilized CD34 antibody (Ab) on HA–heparin hydrogels, using standard EDC/NHS amine‐coupling strategies. We found that EPC adhesion and spreading on CD34 Ab‐immobilized HA–heparin hydrogels was significantly higher than their non‐modified analogues. Once adhered, EPCs spread and formed an endothelial layer on both non‐modified and CD34 Ab‐modified HA–heparin hydrogels after 3 days of culture. We did not observe significant adhesion and spreading when heparin was not included in the control hydrogels. In addition to EPCs, we also used human umbilical cord vein endothelial cells (HUVECs), which adhered and spread on HA–heparin hydrogels. Macrophages exhibited significantly less adhesion compared to EPCs on the same hydrogels. This composite material could possibly be used to develop surface coatings for artificial cardiovascular implants, due to its specificity for EPC and endothelial cells on an otherwise non‐thrombogenic surface. Copyright © 2012 John Wiley & Sons, Ltd.     

己酮可可碱对内毒素诱导大鼠心肌细胞细胞间黏附分子-1表达的影响及其机制

目的观察己酮可可碱（PTX）对内毒素（LPS）诱导大鼠心肌细胞间黏附分子-1（ICAM-1）表达的影响，探讨PTX对心肌的保护作用及其机制。方法将培养的心肌细胞分为3组：空白对照组、LPS损伤组和药物保护组。空白对照组心肌细胞不予任何处理。LPS组心肌细胞中一部分用100μg／L LPS刺激2、4、6和8h，另一部分用10、50和100μg／L LPS刺激6h．采用酶联免疫吸附法（ELISA）检测心肌细胞ICAM-1的表达。免疫细胞化学法检测核转录因子-kB（NF-kB）p65的表达，蛋白质免疫印迹法（Western blot）检测NF-kB抑制蛋白-α（IkB—α）表达；并观察LPS处理前加入50、100、200mg／LPTX对心肌细胞ICAM-1表达的影响。结果心肌细胞ICAM-1表达与LPS刺激呈时相-剂量依赖方式；LPS刺激可迅速活化NF-kBp65，于1h时达高峰；IkB-α表达先降低后升高；加入PTX后大鼠心肌细胞ICAM-1、NF-kBp65表达下降，IkB-α升高。结论PTX可通过抑制ICAM-1上游NF-kB途径对LPS造成的心肌损伤起保护作用。     

黄芪多糖对人心脏微血管内皮细胞增殖及再灌注后内皮细胞与中性粒细胞黏附的影响

目的:观察黄芪多糖对人心脏微血管内皮细胞的增殖效应及其对再灌注损伤人心脏微血管内皮细胞与人外周血中性粒细胞黏附作用的影响。方法:实验于2006-07/12在北京中医药大学东直门医院中医内科重点实验室完成。①实验材料:原代人心脏微血管内皮细胞(ScienCell公司);黄芪多糖(惠州市东方植物保健科技有限公司)。②实验过程及评估:实验使用体外培养的人心脏微血管内皮细胞第4,5代细胞,采用倒置相差显微镜下观察细胞形态和生长情况;以四甲基偶氮唑盐法检测经不同浓度黄芪多糖处理24h,以及经黄芪多糖100,50,25mg/L分别处理4,6,8h后的增殖变化;以体外缺糖缺氧2h模拟体内缺血,复糖复氧6h模拟再灌注复制人心脏微血管内皮细胞再灌注损伤模型;虎红法测定经缺氧再复氧处理后的人心脏微血管内皮细胞与外周血中性粒细胞的黏附;免疫细胞化学法和图象定量分析系统观察细胞间黏附分子1的蛋白表达变化。结果:①人心脏微血管内皮细胞形态和生长情况:人心脏微血管内皮细胞复苏48～72h后细胞呈铺路石样生长,在15个细胞倍增周期内,其形态、生物、生理学特性稳定。②不同浓度黄芪多糖对人心脏微血管内皮细胞增殖的影响:黄芪多糖浓度≥5g/L时,对细胞生长具有抑制作用(P<0.05或0.01);浓度为2.5g/L时,对细胞的增殖无影响;浓度在1.25g/L～18.78mg/L时,可明显促进细胞的增殖(P<0.05或0.01)。③黄芪多糖处理不同时间对人心脏微血管内皮细胞增殖的影响:经黄芪多糖100,50,25mg/L3个剂量分别处理4,6,8h后,其增殖与正常对照组比无明显变化。④黄芪多糖对人心脏微血管内皮细胞与中性粒细胞间黏附作用及人心脏微血管内皮细胞细胞间黏附分子1蛋白表达的影响:黄芪多糖50mg/L可显著抑制再灌注损伤人心脏微血管内皮细胞与外周血中性粒细胞的黏附(P<0.01);100mg/L可明显降低人心脏微血管内皮细胞的细胞间黏附分子1蛋白表达(P<0.05)。结论:①黄芪多糖在一定浓度范围内可促进人心脏微血管内皮细胞的增殖,但需较长的作用时间才能发挥促增殖效应。②黄芪多糖对缺血再灌注损伤人心脏微血管内皮细胞具有保护作用,其作用机制可能与促进再灌注损伤人心脏微血管内皮细胞增殖修复,降低人心脏微血管内皮细胞表面细胞间黏附分子1蛋白表达,进而抑制与外周血中性粒细胞的黏附有关。     

红细胞保存时间对受者中性粒细胞炎症释放水平的影响

目的 探讨研究红细胞随保存时间的延长对受者中性粒细胞炎症释放水平的影响.方法 分别将红细胞在保存的第1、14、21天（D1、D14、D21）分离上清,-20℃留存备用,然后将上述冻存的上清与健康受者中性粒细胞体外培养12 h后,再加入细菌脂多糖后培养12、24 h收集上清-20℃留存待检,最后采用酶联免疫分析法（ELISA）方法检测其白介素-6（IL-6）水平.结果 在红细胞在保存的第1、14、21天（D1、D14、D21）收集的未加中性粒细胞的红细胞上清,检测发现：无IL-6表达,而在细菌脂多糖的诱导下,IL-6浓度水平随时间变化逐渐增加,差异有统计学意义（P〈0.05）.观察组D1、D14和D21 IL-6水平分别高于对照组相应时间点的,差异有统计学意义（P〈0.05）.结论 在细菌脂多糖的刺激下,输注保存时间长的红细胞增强了中性粒细胞的炎症反应,保存时间一定程度上可预测大量输血后引发的相关免疫反应.     

骨髓间质干细胞与心肌细胞移植

对冠心病心力衰竭患者而言心肌细胞移植、血管生成和基因治疗是提高心功能新的治疗措施。骨髓间质干细胞(mesenchymalstemcells,MSCs)是多能干细胞,易于培养,增殖扩增能力强,易于基因转染,具有多向分化能力,在体内、外诱导能分化为心肌细胞,将其作为细胞移植的种子细胞应用于心肌组织工程已成为研究热点。MSCs心肌细胞移植的动物实验研究与临床研究已有长足的进展。应用MSCs移植治疗心肌梗死被证明是有效和安全的,是提高心功能新的治疗措施。对于MSCs的表面特征标志、分化机制及提高心脏功能的机制、移植细胞的长期存活、转归及远期疗效等尚需进一步的研究。     

Beneficial effect of methylprednisolone on cardiac myocytes in a rat model of severe brain injury

Cardiac injury, occurred after traumatic brain injury (TBI), has been recognized for more than a century. Bcl-2 is a key regulatory component of the mitochondrial cell death pathway, and its overexpression is cytoprotective in many cell types. The therapeutic agents, which induce the expression of bcl-2 protein, might provide a new therapy to prevent cardiac myocyte damage following TBI. In this study, we investigated whether methylprednisolone sodium succinate (MPSS) influences the expression of bcl-2 in the heart. Wistar-Albino female rats underwent TBI (300 g/cm) generated by the weight-drop method, and were left untreated (n = 6) or treated with either MPSS (30 mg/kg) (n = 6) or vehicle (albumin solution) (n = 6). The heart was isolated from each animal with TBI. For comparison, the hearts were isolated from sham-operated (n = 6) and control rats (n = 6). The relative expression of bcl-2 mRNA in the heart was quantitated by real-time polymerase chain reaction. We also assessed lipid peroxidation in the heart tissue by determining the concentration of thiobarbituric acid-reactive substances (TBARs) as an indicator of tissue damage. The bcl-2 expression level was significantly higher in the hearts of MPSS-treated rats compared to that of other TBI groups (p < 0.0001). Moreover, TBI increased the lipid peroxidation in the heart, which was significantly reduced by the treatment with MPSS (p < 0.0001). These findings provide evidence for the efficacy of MPSS in protection of cardiac myocytes to achieve optimal heart donation after TBI in heart transplantation.     

Effects and mechanism of pentoxifylline on the expression of intercellular adhesion molecule-1 in the rat cardiac myocytes after lipopolysaccharide challenge]

OBJECTIVE: To investigate the effects of pentoxifylline (PTX) on the expression of intercellular adhesion molecule-1 (ICAM-1) of rat cardiac myocytes after lipopolysaccharide (LPS) challenge in vitro, and to evaluate the protective effect of PTX on cardiac myocytes and its mechanism. METHODS: Cultured cardiac myocytes were randomly divided into three groups: control group, LPS group and PTX protection group. No drug was given in the control group. In LPS group, a part of cardiac myocytes were stimulated with 100 microg/L of LPS for 2, 4, 6, 8 hours, respectively, and the others were stimulated with 10, 50, 100 microg/L LPS, respectively, for 6 hours. Expression of ICAM-1, nuclear factor-KappaB (NF-KappaB) p65 and NF-KappaB inhibitor-alpha (IKappaB-alpha) in cardiac myocytes was respectively measured with enzyme-linked immunoadsorbent assay (ELISA), immunohistochemistry, and Western blot. The expression of ICAM-1 in cardiac myocyte was also determined after the addition of 50, 100, 200 mg/L of PTX before LPS challenge. RESULTS: The intensity of expression of ICAM-1 was highly dependent on the stimulation of LPS in dose depending and time-depending manner. The stimulation quickly resulted in activation of NF-KappaB p65 to translocate into nuclei. This process peaked at 1 hours and then declined. The expression of IKappaB-alpha was depressed at first, then elevated. With the addition of PTX, the expression of ICAM-1 and NF-KappaB p65 was depressed, and the expression of IKappaB-alpha was elevated. CONCLUSION: PTX may have a protective effect on the cardiac myocytes against LPS injury through inhibiting the pathway of NF-KappaB, which regulates the production of ICAM-1.     

Hyperglycemia and lipopolysaccharide decrease depression effect of interleukin 8 production by hypothermia: an experimental study with endothelial cells

Objective This study assessed whether hyperglycemia and lipopolysaccharide (LPS) decrease the depression effect of interleukin (IL) 8 production by hypothermia in endothelial cells. Design and setting Laboratory study in a university laboratory. Subjects Human umbilical vein endothelial cells (HUVECs). Interventions HUVECs were cultivated in various concentrations of glucose (5.5 or 16.5 mM = 100 or 300 mg/dl) with or without LPS stimulation for 5, 12, or 24 h at either 30° or 37 °C. Results After culturing, IL-8 mRNA expressions and IL-8 levels were measured. At 37 °C, hyperglycemia significantly increased basal IL-8 mRNA at 12 h and basal IL-8 at 24 h. At 37 °C hyperglycemia significantly increased LPS-stimulated IL-8 mRNA at 12 h and LPS-stimulated IL-8 at 12 and 24 h. At 30 °C basal IL-8mRNA, basal IL-8, and LPS-stimulated IL-8 were significantly decreased by hypothermia, but these hypothermic effects were not observed in LPS-stimulated IL-8 mRNA. Furthermore even at 30 °C hyperglycemia significantly increased LPS-stimulated IL-8 mRNA at all time points and LPS stimulated IL-8 at 24 h. Conclusions Hypothermia (30 °C) decreases the production of IL-8 in HUVECs but does not decrease the expression of IL-8 mRNA. When hypothermia is followed by hyperglycemia and LPS stimulation, such a combination may expose the patients to a high risk of secondary tissue damage during therapeutic hypothermia.     

蜂胶乔松素对脂多糖诱导人脐静脉内皮细胞的保护作用

目的:观察蜂胶乔松素对脂多糖诱导人脐静脉内皮细胞的影响,探讨其对人脐静脉内皮细胞可能的保护作用。方法:实验于2006-03/10在泰山医学院生命科学研究所(省重点实验室)完成。①实验材料:取出生1h内新生儿脐带,患者知情同意。②实验分组及方法:培养人脐静脉内皮细胞,建立脂多糖损伤模型(以10mg/L的脂多糖培养液培养细胞12h),实验分为空白对照组(加等量D-Hank’s液)、脂多糖组(10mg/L)、乔松素组(加10mg/L脂多糖预孵育12h后,按50,100,200mg/L分别加入乔松素),各组设8个复孔,共同孵育24h。③实验评估:光镜下观察细胞形态,MTT法观察乔松素对人脐静脉内皮细胞活性的影响,ELISA方法检测培养上清中血管假血友病因子的含量,TUNEL检测细胞凋亡率。结果:①细胞形态:空白对照组细胞紧密贴壁,呈铺路石状生长。脂多糖组可见多数细胞呈圆形;乔松素组见上述细胞较脂多糖组明显减少。②乔松素对人脐静脉内皮细胞活性、凋亡及血管假血友病因子含量的影响:与对照组比较,脂多糖组能明显诱导人脐静脉内皮细胞的凋亡(P<0.01),不同浓度乔松素组可改善内皮细胞形态,组织活性明显升高(P<0.05),同时抑制内皮细胞血管假血友病因子的释放(P<0.05),使脂多糖诱导的人脐静脉内皮细胞凋亡细胞数明显减少(P<0.05)。结论:乔松素能增强人脐静脉内皮细胞活性,抑制脂多糖诱导人脐静脉内皮细胞的凋亡,从而发挥可能的内皮细胞保护功能。     

体外诱导大鼠骨髓间充质干细胞向心肌细胞定向分化的研究

Objective To investigate the role of cardiac myocytes in the differentiation of bone marrow mesenchymal stem cells (MSCs) to cardiac myocytes and the biological properties in the course. Methods The bone marrow of the extremities of the rats was flushed, and bone marrow MSCs were obtained by method of density gradient centrifugation. They were cultured. The second passage of cultured MSCs were labelled with bromodeoxyuridine (BrdU). The cardiac myocytes were obtained from the apex of rat heart with trypsin digestion method, and they were cocultured with labeled MSCs. The developmental changes of bone marrow MSCs were observed under light microscope with immunohistochemical staining for BrdU and a-sarcomeric actin on the 3rd day, anti electron microscopic examination on the 5th day. Results MSCs proliferated fast in primary culture and subculture, positive rate was (90. 34± 2. 31)%, and there was statistical difference when it compared with control group [(4.07±1.35)%, P<0. 01]. The morphology of MSCs changed significantly after coculture. There were new cells nuclei of which were positively stained for BrdU and its cytoplasm positive for actin. Under transmissive electron microscope, sarcomeres like structure and abnormal Z line were observed in cytoplasm. Conclusion Cardiac myocytes can effectively induce bone marrow MSCs to differentiate into cardiac myocytes by coculturing.     

体外诱导大鼠骨髓间充质干细胞向心肌细胞定向分化的研究

目的 探讨心肌细胞对骨髓间充质干细胞(MSCs)向心肌细胞定向诱导分化的作用及其生物学特性.方法 取大鼠四肢骨髓,用细胞分离液通过密度梯度离心分离MSCs,并将MSCs进行原代培养和传代培养,将传2代MSCs用5-溴脱氧尿核苷(BrdU)进行标记.采用胰蛋白酶消化法取乳鼠心尖部心肌细胞进行培养,并与标记的MSCs混合培养,光镜下观察其动态变化,并于第3日行免疫组化染色,第5日行电镜观察.结果 原代和传代的MSCs增殖迅速,其阳性率达(90.34±2.31)%,与平行对照组[(4.07±1.35)%]比较差异有统计学意义(P<0.01).与心肌细胞混合后,光镜下观察MSCs形态出现明显的变化,第3日BrdU标记阳性的MSCs胞质中肌动蛋白(actin)表达阳性.电镜下观察原始细胞内出现了不规则的肌小节样结构.结论 心肌细胞与MSCs的接触可有效诱导MSCs向心肌细胞定向分化.     

体外诱导大鼠骨髓间充质干细胞向心肌细胞定向分化的研究

Objective To investigate the role of cardiac myocytes in the differentiation of bone marrow mesenchymal stem cells (MSCs) to cardiac myocytes and the biological properties in the course. Methods The bone marrow of the extremities of the rats was flushed, and bone marrow MSCs were obtained by method of density gradient centrifugation. They were cultured. The second passage of cultured MSCs were labelled with bromodeoxyuridine (BrdU). The cardiac myocytes were obtained from the apex of rat heart with trypsin digestion method, and they were cocultured with labeled MSCs. The developmental changes of bone marrow MSCs were observed under light microscope with immunohistochemical staining for BrdU and a-sarcomeric actin on the 3rd day, anti electron microscopic examination on the 5th day. Results MSCs proliferated fast in primary culture and subculture, positive rate was (90. 34± 2. 31)%, and there was statistical difference when it compared with control group [(4.07±1.35)%, P<0. 01]. The morphology of MSCs changed significantly after coculture. There were new cells nuclei of which were positively stained for BrdU and its cytoplasm positive for actin. Under transmissive electron microscope, sarcomeres like structure and abnormal Z line were observed in cytoplasm. Conclusion Cardiac myocytes can effectively induce bone marrow MSCs to differentiate into cardiac myocytes by coculturing.     

体外诱导大鼠骨髓间充质干细胞向心肌细胞定向分化的研究

Objective To investigate the role of cardiac myocytes in the differentiation of bone marrow mesenchymal stem cells (MSCs) to cardiac myocytes and the biological properties in the course. Methods The bone marrow of the extremities of the rats was flushed, and bone marrow MSCs were obtained by method of density gradient centrifugation. They were cultured. The second passage of cultured MSCs were labelled with bromodeoxyuridine (BrdU). The cardiac myocytes were obtained from the apex of rat heart with trypsin digestion method, and they were cocultured with labeled MSCs. The developmental changes of bone marrow MSCs were observed under light microscope with immunohistochemical staining for BrdU and a-sarcomeric actin on the 3rd day, anti electron microscopic examination on the 5th day. Results MSCs proliferated fast in primary culture and subculture, positive rate was (90. 34± 2. 31)%, and there was statistical difference when it compared with control group [(4.07±1.35)%, P<0. 01]. The morphology of MSCs changed significantly after coculture. There were new cells nuclei of which were positively stained for BrdU and its cytoplasm positive for actin. Under transmissive electron microscope, sarcomeres like structure and abnormal Z line were observed in cytoplasm. Conclusion Cardiac myocytes can effectively induce bone marrow MSCs to differentiate into cardiac myocytes by coculturing.