替罗非班通过上调miR-22保护ox-LDL诱导的脐静脉内皮细胞损伤

目的探讨替罗非班对氧化型低密度脂蛋白(ox-LDL)诱导的脐静脉内皮细胞(EA.hy926)损伤的影响和可能机制。方法采用低、中、高剂量的替罗非班作用于ox-LDL诱导的EA.hy926细胞,采用细胞计数法(CCK-8)、流式细胞术分别检测细胞活力和凋亡。实时荧光定量PCR(qRT-PCR)检测miR-22表达水平。酶联免疫吸附法(ELISA)检测细胞培养上清液中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)水平。转染miR-22模拟物上调EA.hy926细胞中miR-22表达水平,采用上述方法检测上调miR-22对ox-LDL诱导的EA.hy926细胞损伤的影响。结果替罗非班作用于ox-LDL诱导的EA.hy926细胞后,细胞存活率显著升高,凋亡率显著降低,细胞培养上清液中TNF-α和IL-6含量显著降低,miR-22表达显著升高(P0.05)。上调miR-22表达后,ox-LDL诱导的EA.hy926细胞培养上清液中TNF-α和IL-6含量显著降低,细胞存活率显著升高,凋亡率显著降低(P0.05)。结论替罗非班能够减轻ox-LDL诱导的脐静脉内皮细胞凋亡和炎症反应,其机制可能与上调miR-22表达有关。     

系统性血管炎抗内皮细胞抗体与抗中性粒细胞胞质抗体的相关性初探

目的以来源于大小血管内皮细胞的两种永生细胞株为底物,检测系统性血管炎血清中抗内皮细胞抗体(AECA),分析其与抗中性粒细胞胞质抗体(ANCA)的相关性。方法细胞酶联免疫吸附试验(Cyto-ELISA)法检测血清中AECA;间接免疫荧光法(IIF)及抗抗体结合内皮细胞表面的蛋白酶3(PR3)、抗MPO-ELISA检测血清中ANCA;IIF及抗PR3、抗MPO-ELISA检测细胞株中PR3及MPO;反转录—聚合酶链反应(RT-PCR)法检测细胞株中PR3及MPOmRNA。结果AECAEA(EA.hy926为底物所测AECA)阳性率33.6%(41/122),AECAHMEC(HMEC-1为底物所测AECA)37.7%(46/122),ANCA35.3%(43/122),AECAEA或AECAHMEC与ANCA串联诊断系统性血管炎敏感性分别为59.8%(73/122)或60.7%(74/122)。AECAEA与ANCA,AECAHMEC与ANCA分别行配对字2检验,差异均无显著性(P>0.05),符合率仅分别为49.2%及51.6%。EA.hy926和HMEC-1中蛋白水平及mRNA水平均无PR3和MPO的表达。结论EA.hy926与HMEC-1中无PR3、MPO蛋白水平的表达,ANCA与AECA可能是两种相互独立的抗体,串联检测可提高诊断敏感性。     

重组人内皮细胞抗原成分α烯醇化酶的表达及其在弥漫性结缔组织疾病中的意义

目的 对蛋白质组学分离和鉴定的抗内皮细胞抗体(AECA)相关抗原-α烯醇化酶进行基因重组和表达,榆测其特异性抗体在血管炎和其他自身免疫性疾病患者血清中的阳性率.方法 聚合酶链反应(PCR)扩增人enol基冈CDS片段并与表达载体重组,在大肠杆菌中表达和纯化重组人α烯醇化酶.用质谱鉴定后的重组蛋白作为抗原,用点印迹法和酶联免疫吸附试验(ELISA)法分别检测健康人和不同结缔组织病患者血清中抗人α烯醇化酶抗体水平.结果 克隆了人enol基因cDNA片段.将重组的人α烯醇化酶在大肠杆菌中进行原核表达和纯化.用重组蛋白作为抗原,点印迹法检测健康对照和不同结缔组织病患者血清中抗人α烯醇化酶抗体,证实:①系统性血管炎(SV)、系统性红斑狼疮(SLE)、干燥综合征(SS)和类风湿关节炎(RA)患者血清中抗α烯醇化酶抗体的阳性率分别为76.7%、78.3%、63.6%和78.9%,均明显高于多发性肌炎/皮肌炎(PM/DM)组和健康对照组(P<0.01).②系统性血管炎中自塞病(BD)、多发性大动脉炎(TA)、韦格纳肉芽肿病(WG)、显微镜下多动脉炎(MPA)和Churg-Strauss综合征(CSS)患者血清中抗α烯醇化酶抗体阳性率各为74.0%、81.5%、62.5%、92.3%和80.0%.③SV和SLE患者血清中抗α烯醇化酶抗体的阳性率与SS和RA患者血清中该抗体的阳性率差异无统计学意义(P>0.05).④不同患者血清中抗α烯醇化酶抗体强阳性者所占比例分别为:SV 51.7%,SLE 33.3%,SS42.9%,RA 20.0%.RA组与SV组差异有统计学意义(P<0.05).结论 SV和SLE、SS、RA患者血清中均存在不同水平的抗α烯醇化酶抗体,α烯醇化酶是AECA识别的自身抗体之一.     

阿托伐他汀钙对EA.hy926细胞全基因表达谱的影响及生物信息学分析

目的探讨阿托伐他汀钙对体外培养人脐静脉内皮细胞EA.hy926细胞基因表达谱的影响及作用的分子机制。方法取EA.hy926细胞分实验组和对照组,实验组采用10μmol/L阿托伐他汀钙体外干预人脐静脉内皮细胞EA.hy926 24h,用Affymetrix HG-U133plus 2.0全基因组表达芯片检测阿托伐他汀钙对EA.hy926细胞基因表达谱的影响。并运用基因富集分析(GSEA)软件、DAVID基因功能聚类分析软件及Cmap数据库对芯片数据进行分析,对相关靶基因进行实时定量PCR验证。结果与对照组比较,实验组基因芯片分析筛选出差异表达倍数>2倍的基因649个,其中上调基因295个,下调基因354个。经DAVID及GSEA基因富集分析显示,阿托伐他汀钙广泛下调了细胞周期调控相关基因,上调了Kruppel样转录因子等血管保护基因,Cmap数据库分析显示,阿托伐他汀钙与组蛋白去乙酰化酶抑制剂及白藜芦醇呈正相关的联强度高。结论阿托伐他汀钙从多角度发挥抗动脉粥样硬化作用,作用机制可能与组蛋白去乙酰化酶抑制剂及白藜芦醇相似。     

淫羊藿苷对S-亚硝基化谷胱甘肽诱导损伤的内皮细胞的保护作用

目的探讨淫羊藿苷对S-亚硝基化谷胱甘肽诱导损伤的内皮细胞的保护作用。方法内皮细胞(EA.hy926)预先给予不同浓度ICA(高浓度:10μmol/L,中浓度:1μmol/L,低浓度:0.1μmol/L),之后给予S-亚硝基化谷胱甘肽(1mmol/L)损伤,建立模型。采用:噻唑兰染色法检测细胞存活率,通过对乳酸脱氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶、活性氧、细胞色素C、Caspase-3等相关指标的测定进行分析。结果淫羊藿苷可明显提高内皮细胞内超氧化物歧化酶以及谷胱甘肽过氧化物酶的活性,抑制活性氧的产生,降低细胞色素C的释放以及Caspase-3的活性,从而减少细胞的凋亡,差异有统计学意义(P<0.01),对S-亚硝基化谷胱甘肽诱导损伤的内皮细胞起到保护作用。结论淫羊藿苷对S-亚硝基化谷胱甘肽诱导损伤的内皮细胞具有保护作用,其机制可能与淫羊藿苷可提高细胞内超氧化物歧化酶以及谷胱甘肽过氧化物酶的活性,抑制活性氧的产生和减少细胞色素C的释放有关。     

p53在神经酰胺诱导内皮细胞凋亡中的作用

目的：探讨p53在外源性神经酰胺（C2-ceramide）诱导人脐静脉内皮细胞凋亡中的作用。方法：体外培养脐静脉内皮细胞,用不同浓度的C2-ceramide进行处理,Tunel检测细胞凋亡；逆转录-聚合酶链反应（RT-PCR）和Western blot检测p53的表达；并用p53的抑制剂PFT-α（pifithrin alpha,PFT-α）预处理细胞,再加入C2-ceramide与对照组比较观察细胞凋亡指数。结果：C2-ceramide呈剂量依赖性诱导内皮细胞凋亡；p53的表达随着C2-ceramide浓度的增加呈现降低的趋势；PFT-α不能抑制C2-ceramide诱导的凋亡。结论：神经酰胺能诱导内皮细胞的凋亡,可能不依赖于p53途径。     

棕榈酸对EA.hy926细胞炎症反应相关基因表达的影响及机制

目的观察棕榈酸(PA)对内皮细胞株EA.hy926细胞炎症反应的影响,检测相关基因表达的变化并探讨其可能的信号途径。方法体外培养内皮细胞株EA.hy926,分为白蛋白对照组和PA处理组。改良MTT法检测PA孵育对EA.hy926细胞活性的影响,实时荧光定量PCR法检测白细胞介素1(IL-1)、白细胞介素6(IL-6)、白细胞介素8(IL-8)、肿瘤坏死因子α(TNF-α)和人单核细胞趋化蛋白1(MCP-1)mRNA水平,ELISA法检测IL-6、IL-8和TNF-α浓度,Western blot检测磷酸化核因子κB(NF-κB)p65、NF-κB p65、核因子κB抑制蛋白(IκBα)水平。结果与白蛋白对照组相比,20、50、100μmol/L PA处理组的IL-1、IL-6、IL-8 mRNA水平显著升高(P0.05),50、100μmol/L PA处理组的TNF-α、MCP-1 mRNA水平显著升高(P0.05),20、50、100μmol/L PA处理组细胞培养上清液中IL-6和IL-8的浓度显著升高(P0.05),IκB蛋白水平显著降低(P0.05),NF-κB p65磷酸化水平显著升高(P0.05)。结论 PA能够导致EA.hy926细胞炎症反应,其机制可能与激活NF-κB信号途径有关。     

α-硫辛酸对糖基化终产物诱导血管内皮细胞凋亡的影响

目的观察α-硫辛酸对糖基化终产物诱导血管内皮细胞凋亡的影响。方法体外培养人脐静脉内皮细胞,以人血清清蛋白作为阴性对照,用含不同浓度梯度(100、200、400mg/L)糖基化终产物的培养液对内皮细胞体外培养60min及α-硫辛酸200μg/ml作用30min后再加入200mg/L糖基化终产物作用内皮细胞60min,采用ELISA法检测内皮细胞8-OHdG水平,瑞氏-吉姆萨染色及Hoechst33258染色观察内皮细胞形态学变化。结果糖基化终产物组内皮细胞8-OHdG水平较对照组明显增加(P0.05),糖基化终产物以剂量依赖方式诱导培养的内皮细胞凋亡,α-硫辛酸干预后内皮细胞8-OHdG水平显著降低,明显减轻内皮细胞凋亡的形态学改变。结论α-硫辛酸抑制糖基化终产物诱导内皮细胞凋亡,其作用机制可能是通过降低细胞氧化应激水平。     

苦荞麦总黄酮对软脂酸诱导的EA.hy926细胞Bcl-2表达的影响

目的研究苦荞麦总黄酮对软脂酸诱导的人脐静脉血管内皮细胞株(EA.hy926)Bcl-2表达的影响。方法苦荞麦总黄酮作用于高浓度软脂酸刺激下的EA.hy926细胞,RT-PCR技术检测Bcl-2 mRNA表达水平,免疫组织化学方法检测Bcl-2蛋白的表达情况。结果与对照组相比,模型组细胞Bcl-2 mRNA及蛋白表达显著降低(P<0.01);与模型组相比较,苦荞麦总黄酮组与二甲双胍组细胞Bcl-2 mRNA和蛋白表达水平明显升高(P<0.01);苦荞麦总黄酮组与二甲双胍组之间无显著差异(P>0.05)。结论苦荞麦总黄酮可以促进EA.hy926细胞Bcl-2基因及蛋白的表达发挥抑制凋亡的作用。     

活化血小板释放产物对内皮细胞的损伤及丹酚酸B的保护效应

目的探讨活化血小板释放产物(PLTaRP)对内皮细胞的损伤作用及丹酚酸B的保护效应。方法分离健康人外周血血小板,以二磷酸腺苷激活后提取PLTaRP,并与EA.hy926人脐静脉内皮细胞株共培养,分为对照组、模型组、阳性对照组、低剂量组、中剂量组、高剂量组,检测各组细胞活力及乳酸脱氢酶(LDH)释放水平。DAPI染色观察细胞核形态,比较各组细胞凋亡率。结果与对照组比较,模型组12 h内LDH含量持续升高,并呈时间依赖趋势(P<0.01),模型组、阳性对照组、低剂量组和中剂量组细胞存活率明显减低(P<0.05,P<0.01),高剂量组细胞存活率差异无统计学意义(P>0.05)。与模型组比较,低、中、高剂量组LDH含量明显降低(P<0.01)。结论 PLTaRP可刺激EA.hy926细胞株LDH释放增加,并呈时间依赖趋势,刺激12 h可使细胞活力明显下降。丹酚酸B可减少PLTaRP造成的LDH释放。高剂量丹酚酸B可明显抑制细胞凋亡。     

Induction of endothelial cell apoptosis by heat-shock protein 60-reactive antibodies from anti-endothelial cell autoantibody-positive systemic lupus erythematosus patients

OBJECTIVE: To determine whether anti-endothelial cell autoantibodies (AECAs) from systemic lupus erythematosus (SLE) patients with the antiphospholipid syndrome are involved in the initial endothelial cell (EC) membrane perturbation effect that is postulated to provide a target for antiphospholipid antibody (aPL) binding and, hence, to trigger the thrombotic cascade. To identify the AECA antigenic target on ECs and to determine the mechanism whereby the EC membrane is disrupted. METHODS: AECAs from SLE patients were assayed for binding to ECs by flow cytometry. Positive AECAs were assayed by immunoblotting, and a consensus antigen was identified by mass spectrometry. This candidate antigen was tested in recombinant form for AECA recognition. AECAs were affinity-purified on this antigen and incubated with ECs to determine their physiologic effects. Anti-Hsp60 antibody titers were determined by enzyme-linked immunosorbent assay. The relationship of anti-Hsp60 status and lupus anticoagulant (LAC) status to thrombotic manifestations between disease onset and the last followup visit were analyzed. RESULTS: Most of the SLE sera (73%) possessed IgG that bound to the surface of ECs. These positive IgG shared reactivity against a 60-kd EC surface polypeptide that was identified as human Hsp60. The presence of Hsp60 at the EC surface was established using anti-Hsp60 antibodies from commercial sources or affinity-purified from SLE sera that bound ECs. Incubation of ECs with these anti-Hsp60 antibodies induced apoptosis in a time- and dose-dependent manner, as determined by Hoechst 33342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Anti-Hsp60 antibodies were not restricted to SLE patients, but were found in patients with other autoimmune diseases. However, anti-Hsp60 antibodies were significantly associated with an increased frequency of thrombosis when present in combination with LAC in the SLE patients. CONCLUSION: The presence of Hsp60 at the surface of ECs serves as a target for the anti-Hsp60 antibodies in SLE sera. These anti-Hsp60 antibodies bind to ECs and induce apoptosis, particularly phosphatidylserine exposure, thus providing a target for the binding of aPL and inducing the subsequent thrombotic cascade.     

Anti-endothelial cell antibodies from lupus patients bind to apoptotic endothelial cells promoting macrophage phagocytosis but do not induce apoptosis

OBJECTIVE: Anti-endothelial cell antibodies (AECA) have been reported to induce apoptosis. We investigated the induction of apoptosis by these autoantibodies and their involvement in the removal of apoptotic cells. METHODS: AECA isolated from patients with active systemic lupus erythematosus (SLE) were incubated with human umbilical vein endothelial cells (HUVECs). AECA-positive sera were identified using a cell-based ELISA. Apoptosis was measured by morphology and phosphatidylserine externalization using flow cytometry with fluorescein isothiocyanate (FITC)-conjugated annexin V. Flow cytometry was used to investigate AECA binding to apoptotic cells using FITC-conjugated anti-human immunoglobulin G (IgG). Apoptotic endothelial cells were stained with a red dye (PKH26) and co-cultured with macrophages, and phagocytosis was visualized under phase contrast microscopy. RESULTS: AECA from patients with SLE did not induce apoptosis compared with normal IgG (nIgG) at any time point, as assessed by morphology (at 24 h, P = 0.167) or phosphatidylserine externalization (at 24 h, P = 0.098). However, there was increased binding of AECA to apoptotic endothelial cells (48.8 +/- 11.9 compared with 25.8 +/- 6.7% AECA binding to freshly isolated cells, P< 0.001). These opsonized endothelial cells showed greater phagocytosis by macrophages (mean phagocytic index 24.9 +/- 4.5%) when cells opsonized with nIgG were compared with AECA (34.8 +/- 3.4% n = 5, P = 0.01). CONCLUSION: In conclusion, AECA bind to apoptotic endothelial cells but do not induce endothelial cell apoptosis. Macrophage phagocytosis is increased by opsonization of apoptotic endothelial cells by AECA, a proinflammatory mechanism of cell removal.     

Induction of endothelial cell apoptosis by heat‐shock protein 60–reactive antibodies from anti–endothelial cell autoantibody–positive systemic lupus erythematosus patients

Objective

To determine whether anti–endothelial cell autoantibodies (AECAs) from systemic lupus erythematosus (SLE) patients with the antiphospholipid syndrome are involved in the initial endothelial cell (EC) membrane perturbation effect that is postulated to provide a target for antiphospholipid antibody (aPL) binding and, hence, to trigger the thrombotic cascade. To identify the AECA antigenic target on ECs and to determine the mechanism whereby the EC membrane is disrupted.

Methods

AECAs from SLE patients were assayed for binding to ECs by flow cytometry. Positive AECAs were assayed by immunoblotting, and a consensus antigen was identified by mass spectrometry. This candidate antigen was tested in recombinant form for AECA recognition. AECAs were affinity‐purified on this antigen and incubated with ECs to determine their physiologic effects. Anti‐Hsp60 antibody titers were determined by enzyme‐linked immunosorbent assay. The relationship of anti‐Hsp60 status and lupus anticoagulant (LAC) status to thrombotic manifestations between disease onset and the last followup visit were analyzed.

Results

Most of the SLE sera (73%) possessed IgG that bound to the surface of ECs. These positive IgG shared reactivity against a 60‐kd EC surface polypeptide that was identified as human Hsp60. The presence of Hsp60 at the EC surface was established using anti‐Hsp60 antibodies from commercial sources or affinity‐purified from SLE sera that bound ECs. Incubation of ECs with these anti‐Hsp60 antibodies induced apoptosis in a time‐ and dose‐dependent manner, as determined by Hoechst 33342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Anti‐Hsp60 antibodies were not restricted to SLE patients, but were found in patients with other autoimmune diseases. However, anti‐Hsp60 antibodies were significantly associated with an increased frequency of thrombosis when present in combination with LAC in the SLE patients.

Conclusion

The presence of Hsp60 at the surface of ECs serves as a target for the anti‐Hsp60 antibodies in SLE sera. These anti‐Hsp60 antibodies bind to ECs and induce apoptosis, particularly phosphatidylserine exposure, thus providing a target for the binding of aPL and inducing the subsequent thrombotic cascade.

     

转染Zbtb7a基因对人胃癌SGC7901细胞增殖、凋亡的影响及其机制

目的 观察转染Zbtb7a基因对人胃癌细胞系SGC7901增殖及凋亡的影响,并探讨其机制.方法 将pcDNA3.1-Zbtb7a和pSilencer 3.1-H1-mk用Lipofectamine2000转染SGC7901细胞,采用RT-PCR和Western Blot法检测MK mRNA及蛋白表达,CCK-8试剂盒和平板克隆法检测细胞增殖,流式细胞仪Annexin V-PI染色检测细胞凋亡.结果 转染Zbtb7a后,SGC7901细胞中MK表达水平明显升高,细胞增殖能力明显增强（P＜0.05）,并且0.5ng/mL TRAIL诱导的细胞凋亡受到抑制（P＜0.05）.Zbtb7a高表达后干扰MK的表达,细胞增殖及克隆能力则明显降低（P＜0.05）,TRAIL诱导的细胞凋亡数也明显增加（P＜0.05）.结论 Zbtb7a可以通过上调MK的表达,促进SGC-7901细胞增殖以及抑制TRAI诱导的细胞凋亡.     

Thrombogenic role of cells undergoing apoptosis

Apoptosis is involved in many biological processes, especially during chemotherapy in cancer patients. Chemotherapy is also associated with an increased risk of thrombosis. The relationship between thrombogenicity and apoptosis was studied in various human tumour cell lines and non-tumour cell lines. Apoptosis was induced by the chemotherapeutic agent camptothecin and by Fas ligand, then quantified by staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. A significant correlation between thrombin generation and degree of apoptosis was observed (P < 0.0005). Addition of anti-tissue factor antibody in excess or of tissue factor pathway inhibitor partially inhibited thrombin generation, suggesting that tissue factor activation was responsible for this process. A statistical correlation between tissue factor activity and degree of apoptosis was also found (P < 0.005). Both thrombin generation and tissue factor activity were blocked by the addition of annexin V, which binds and inhibits phosphatidylserine. This indicates that the exteriorization and exposure of phosphatidylserine on the cell surface membrane during apoptosis were essential for both thrombin generation and tissue factor activation.     

Enhancement of basophil apoptosis by olopatadine and theophylline.

Regulation of basophil survival is an important aspect in the pathogenesis of allergic inflammation associated with local accumulation of basophils. However, pharmacologic modulation of basophil survival is largely unknown except for the apoptosis-enhancing effect of glucocorticoids. We tested the effects of two anti-allergic and anti-asthmatic drugs, olopatadine and theophylline, on basophil survival. Basophils were highly purified from normal human peripheral blood. Apoptosis was analyzed by flow cytometry using annexin V staining or another staining method that detected alterations in the mitochondrial transmembrane potential. In addition to the conventional method using annexin V, basophil apoptosis was successfully established by analysis of the mitochondrial transmembrane potential. Olopatadine decreased the number of live basophils, and they induced apoptosis of basophils during culture. The decline in live basophils was induced by olopatadine even when low doses of IL-3 were included in the culture medium. Theophylline also affected basophil apoptosis and induced a decrease in the number of live basophils. Basophil apoptosis was enhanced by both olopatadine and theophylline. This effect may partly explain the pharmacologic basis of why these drugs are effective on allergic diseases.     

TL1A在高糖诱导的人脐静脉内皮细胞凋亡中的作用及机制探讨

目的 研究肿瘤坏死因子配体相关分子1A（TL1A）在高糖诱导的人脐静脉内皮细胞凋亡中的作用,并探讨机制.方法 采用RT-PCR、Western blot法检测不同浓度（5.6、11.2、22.4、33.6 mmol/L）葡萄糖与人脐静脉内皮细胞共同孵育48 h以及22.4 mmol/L葡萄糖作用于内皮细胞12、24、48、96 h后,TL1A mRNA及蛋白质在人脐静脉内皮细胞中的表达情况.在22.4 mmol/L葡萄糖培养的人脐静脉内皮细胞中加入不同浓度的TL1A单克隆抗体（1、3、9 μg/mL）,48 h后进行Annexin V/PI双染色流式细胞凋亡计数,以纯化重组人TL1A作为内源性TL1A的对照.结果 在人脐静脉内皮细胞中,随着葡萄糖浓度升高及作用时间延长,TL1A mRNA和蛋白水增高（P＜0.05）.流式细胞计数显示,高糖培养人脐静脉内皮细胞经TL1A单克降抗体处理后凋亡明显减少（P＜0.05）.结论 高浓度葡萄糖诱导体外培养的人脐静脉内皮细胞TL1A的表达呈浓度、时间依赖性增高;TL1A表达增多可能是高糖诱导人脐静脉内皮细胞凋亡的重要因素.