【作者】 王健；

【导师】 李春阳； 曾晓雄；

【作者基本信息】 南京农业大学 ， 发酵工程， 2014， 硕士

【摘要】 研究背景心血管疾病是危害人类健康的头号杀手,而80-90%心血管疾病患者都伴随着高血压。“阻止高血压饮食计划(DASH)"中重要的一条就是增加水果蔬菜的摄入量。细胞因子肿瘤坏死因子-α(TNF-α)作为重要的炎症因子,可以诱导内皮细胞表达相应蛋白,能显著增强血管内皮细胞单核细胞趋化蛋白-1(MCP-1)、细胞间粘附分子-1(ICAM-1)和血管细胞间粘附分子-1(VCAM-1)蛋白和mRNA的表达,促进炎症发生及发展进程。蓝莓作为功能性食品,其果实中花青素的含量很高,具有抗炎症、增强心脏功能、改善血管循环、防止脑神经衰老、明目及抗癌等独特功效。研究发现蓝莓花青素中锦葵色素的含量很高,因此本文以氯化锦葵色素、氯化锦葵色素-3-葡萄糖苷及氯化锦葵色素-3-半乳糖苷为研究对象,研究它们对TNF-α诱导的体外人脐静脉内皮细胞炎症的保护作用及糖苷协同作用,并从分子基础上研究其作用机制,为心血管疾病的研究提供一定的理论基础。研究目的：1.体外分离人脐静脉内皮细胞(HUVEC),采用TNF-α干预细胞,建立内皮细胞功能损伤所致的炎症模型,观察不同浓度氯化锦葵色素、氯化锦葵色素-3-葡萄糖苷及氯化锦葵色素-3-半乳糖苷对内皮细胞的保护作用。2.采用ELISA、Western Blot及RT-PCR方法检测不同锦葵色素对细胞中MCP-1、CAM-1及VCAM-1的表达情况,探讨它们保护HUVEC的分子机制。3.观察细胞中及细胞核中IκBα蛋白表达情况及NF-κB核转录情况,从分子基础上解释不同氯化锦葵色素对HUVEC炎症的保护作用及糖苷协同作用。研究方法：1.采用酶解法体外分离新鲜的婴儿脐带,得到人脐静脉内皮细胞,用M199完全培养基进行培养、传代及冻存；取3-6代人脐静脉肉皮细胞进行试验。2.采用不同浓度的肿瘤坏死因子TNF-α诱导HUVEC 4h及采用最佳浓度的TNF-α刺激细胞不同时间,取其细胞,分别采用ELISA检测细胞上清中MCP-1、 ICAM-1及VCAM-1蛋白表达水平；采用Western Blot检测细胞中ICAM-1及VCAM-1蛋白表达水平,根据分析结果确定TNF-α诱导HUVEC最佳剂量及时效。3.采用不同浓度的氯化锦葵色素、氯化锦葵色素-3-葡萄糖苷及氯化锦葵色素-3-半乳糖及选择最佳浓度的糖苷预处理细胞18h,加入10μg/L TNF-α刺激6 h,分别采用MTT法检测细胞活力；采用ELISA检测细胞上清中MCP-1及VCAM-1蛋白表达水平；采用Western Blot检测细胞中ICAM-1及VCAM-1蛋白表达水平,采用荧光定量RT-PCR检测细胞MCP-1及VCAM-1 mRNA的表达水平。4.采用最佳浓度的锦葵色素糖苷作用细胞,采用Western Blot检测细胞中IκBα蛋白表达变化,采用核转录-转运试剂盒检测细胞质中NF-κB核转录情况,从分子基础上研究抗炎症作用的机制。研究结果：1.TNF-α诱导HUVEC表达MCP-1α ICAM-1及VCAM-1蛋白正常细胞少量表达MCP-1αICAM-1及VCAM-1蛋白,在受到炎症因子TNF-α刺激时,各蛋白的表达量上升。不同浓度TNF-α刺激HUVEC得到细胞上清及细胞,通过ELISA及Western Blot检测MCP-1、ICAM-1及VCAM-1蛋白呈浓度依赖性变化,低浓度时蛋白表达量不显著,随着浓度增加,蛋白表达量增加,具有显著性,尤其在浓度为10斗g／L时,相比对照组MCP-1、ICAM-1及VCAM-1蛋白表达具有显著性意义(P<0.01)。采用最佳浓度10μg/L TNF-α刺激细胞不同时间,表达量呈时间依赖性变化；短刺激时间对细胞中MCP-1、ICAM-1及VCAM-1蛋白表达量影响较小,随着刺激时间的增加,表达量亦增长,尤其在刺激时间6h,细胞上清中可溶性MCP-1、 ICAM-1及VCAM-1蛋白表达量达到峰值,分别是对照组的3.762±0.041、8.436±0.367、8.554±0.069倍,具有显著性差异(P<0.01)。细胞中ICAM-1/β-Actin及VCAM-1/β-Actin相对表达分别为对照组的2.268±0.012(P<0.001)、1.616±0.022(P<0.01)倍；在刺激时间为6h时,两者蛋白表达量达到峰值。2.氯化锦葵色素抑制TTNF-α诱导的HUVEC由MCP-1、ICAM-1及VCAM-1蛋白的表达MTT法检测细胞活力显示：当1、10、50、100 μmol/L Mv-Cl、Mv-3-glc-Cl及Mv-3-gal-Cl相比空白组的抑制率为92.6%、87.6%、70.1%、64.8%,92.6%、87.6%、70.1%、64.8%及86.3%、85.1%、70.3%、61.5%,与TNF-α共同作用时,,相比TNF-α刺激组具有显著性意义(P<0.01),说明Mv-Cl、Mv-3-glc-Cl及Mv-3-gal-Cl不同程度的对内皮细胞具有一定的保护作用,由于浓度的增加,对细胞活力具有一定的影响。采用ELISA、Western Blot及RT-RCR检测细胞中MCP-1、ICAM-1及VCAM-1蛋白及mRNA表达情况,正常情况下,正常细胞少量表达MCP-1、ICAM-1及VCAM-1蛋白及mRNA,当细胞受到炎症因子TNF-α刺激时,三种因子的表达量上升,当不同浓度Mv-Cl(1、10、50、100μmol/L)作用细胞时,蛋白及mRNA的表达量相比TNF-α刺激组具有显著下降,尤其是浓度为50、100 μmol/L时,三种因子的抑制率达到90%以上,接近于正常组,说明随着浓度的增加,Mv-Cl对TNF-α诱导的内皮细胞炎症具有保护作用.3.氯化锦葵色素-3-O-葡萄糖苷及半乳糖苷抑制TNF-α诱导的HUVEC中MCP-1、 ICAM-1及VCAM-1蛋白的表达通过ELISA、Western Blot及RT-PCR方法检测细胞中MCP-1、ICAM-1及VCAM-1蛋白及mRNA变化,得出：不同浓度的Mv-3-glc-Cl、Mv-3-gal-Cl对TNF-α引起的炎症反应呈浓度依赖式保护作用。对于细胞中MCP-1及VCAM-1蛋白或mRNA, Mv-3-glc-Cl比Mv-3-gal-Cl抑制效果更明显,但在低浓度刺激时,对于细胞中ICAM-1, Mv-3-gal-Cl比Mv-3-glc-Cl作用效果明显,但随着浓度增加,葡萄糖苷式作用比半乳糖苷式发挥更强的保护作用。4.糖苷氯化锦葵色素协同抑制TNF-α诱导的HUVEC中MCP-1、ICAM-1及VCAM-1蛋白的表达及分子机理研究通过ELISA法及Werstern Blot及RT-PCR检测发现预处理的Mv-3-glc-Cl、 Mv-3-gal-Cl和(Mv-3-glc-Cl+Mv-3-gal-Cl)对MCP-1、ICAM-1和VCAM-1蛋白及mRNA表达相比单独刺激TNF-α组具有明显的抑制作用,并且具有协同作用。通过Western Blot检测细胞质中IκBα蛋白得出,正常细胞受到TNF-α介导时,IκBα蛋白含量降低,验证了IκBα被降解,,从而表达MCP-1, ICAM-1和VCAM-1;而当孵育不同浓度氯化锦葵色素时,IKBα相对降解量相比TNF-α组减少。此外,通过免疫荧光检测NF-κBp65核转运情况也得到相应结果,正常细胞NF-κB捕捉的荧光强度很弱。单独TNF-α组NF-κB捕捉的荧光强度最强,随着Mv-3-glc-Cl、 Mv-3-gal-Cl和(Mv-3-glc-Cl+Mv-3-gal-Cl)的加入,NF-κB荧光强度相对于对照组渐渐变弱,证实了氯化锦葵色素能阻止NF-κB的激活,起到抑制炎症因子的作用,从而起到抗炎症的作用。更多还原

【Abstract】 Cardiovascular diseases are the leading cause of death and disability worldwide, and 80-90% cardiovascular patients had hypertension. One of important plans on "Dietary Approaches to Stop Hypertension (DASH)" is to increase fruit and vegetable intake. Tumor necrosis factor-alpha(TNF-a), a prototypic proinflammatory cytokine commonly found in atherosclerotic lesions, can have direct effects on vascular endothelial cells to induce protein and mRNA of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1), etc., which are important in the onset and progress of inflammation. Blueberry could enhance heart function, improve blood circulation, relaxation, promote blood vessels, and prevent hypertension. Blueberry anthocyanins are the most effective antioxidants found in nature. Malvidin (Mv) is one of the most wide-spread anthocyanidins. Therefore, we guessed that antioxidant pigment Malvidin maybe have potential inflammation inhibitory activity. The objective of the study was to study the anti-inflammatory properties of Malvidin chloride (Mv-Cl), Malvidin-3-glucoside chloride (Mv-3-glc-Cl), Malvidin-3-galactoside chloride (Mv-3-gal-Cl) on TNF-a-induced inflammation in human vascular umbilical endothelial cells (HUVECs). In addition, we investigated the synergistic anti-inflammatory effect of Mv-3-glc-Cl and Mv-3-gal-Cl in human vascular umbilical endothelial cells.This provides a theoretical basis for the proinflammatory cytokines induced endothelial dysfunction and inflammation to result cardiovascular disease.Objective:1. The HUVECs are cultured in vitro. Expose endothelial cells to TNF-a to establish cell model of inflammation. To investigate Mv-Cl, Mv-3-glc-Cl, and Mv-3-gal-Cl respectively whether they can protect the injury of endothelial cells.2. To detect the MCP-1, ICAM-1 and VCAM-1 protein and mRNA content by ELISA, western Blot and PT-PCR. To explore the molecular mechanisms of their anti-inflammatory effect to endothelial cells.To detect the expression of IκBα and NF-κB nuclear transcription situation, and primarily analyze the protective mechanism of different Malvidin towards the TNF-a induced inflammatory response in HUVECs.Methods:1. The human umbilical vein endothelial cells were isolated by enzymatic digestionfrom the fresh baby umbilical cord. The endothelial cells were cultured with M199 complete medium, passaged and frozen. The second to sixth passage cells were used for experiments.2. The HUVECs were treated with different concentrations of tumor necrosis factor (TNF-a) for 4 h, and treated different time with optimal concentrations of TNF-a. MCP-1, ICAM-1 and VCAM-1 protein levels in cell supernatants were measured by ELISA. The ICAM-1 and VCAM-1 protein levels in cells were tested by Western Blot. According to the results of the analysis, the optimal agent efficiency and treatment time of TNF-a to induce inflammmatary response in HUVECs were obtained.-.3. The HUVECs were pretreated with different concentrations of Mv-Cl, Mv-3-glc-Cl and Mv-3-gal-Cl for 18 h, respectively, followed by TNF-α (10 μg/L) stimulation for 6 h. DMSO was used as control. Cell viability was measured by MTT assay. MCP-1, ICAM-1 and VCAM-1 protein levels in cell supernatants were measured by ELISA. The cell ICAM-1 and VCAM-1 protein levels were tested by Western Blot. The changes on mRNA expression level of MCP-1 and ICAM-1 in cells were detected by RT-PCR.4. The protein of κBα was assessed by Western-blot. The activity of NF-κB was evaluated by immunofluorescence. We analyzed the molecular basis of the anti-inflammatory effect mechanism.Results:1. The effect of TNF-a on the expression of MCP-1, ICAM-1 and VCAM-1protein in cell surface and cellsIn unstimulated cells, the surface expression of MCP-1, ICAM-1 and VCAM-1 protein was very low. The preoteins in cell supernatant and cells obtained were assessed by ELISA and Western Blot, respectively. Incubation with different concentrations of TNF-a, their expression increased in a dose-dependent manner. The protein expression change was not significant at low concentrations. The protein expression increased significantly as the concentration increases. Especially the protein expression level of MCP-1, ICAM-1, and VCAM-1 got the highest values when HUVECs were induced by 10μg/L of TNF-a (P< 0.01).2. The HUVECs were stimulated with 10 μg/L TNF-a for different time. We found that the expression of MCP-1, ICAM-1, and VCAM-1 proteins was less affected with short time. ELIS A results on the cell-free endothelial supernatants showed that MCP-1, ICAM-1, and VCAM-1 proteins released into medium all significantly (3.762 ± 0.041, 8.436 ± 0.367,8.554 ± 0.069 fold, respectively) increased after TNF-a stimulated 6 h (P< 0.01). The ICAM-1/β-Actin and VCAM-1/β-Actin relative expression to the control group were 2.268 ± 0.012 (P< 0.001),1.616 ± 0.022 (P< 0.01) folds in cells at 6 h. Thus, the protein expression level of MCP-1, ICAM-1 and VCAM-1 got the highest values when HUVECs were stimulated 6 h by 10 μg/L TNF-a. Effect of Mv-Cl on TNF-a-induced MCP-1, ICAM-1, and VCAM-1 protein expression in HUVECsMTT tells that Mv-Cl, Mv-3-glc-Cl and Mv-3-gal-Cl of 1,10,50,100 μmol/L could inhibit 92.6%,87.6%,70.1%,64.8%; 92.6%,87.6%,70.1%,64.8%and 86.3%, 85.1%,70.3%,61.5% cells compared with unstimulated cells(P< 0.01). It indicated that Mv-Cl has a protective effect to TNF-a-induced cells.The changes on protein and mRNA expression level of MCP-1, ICAM-1, and VCAM-1 in cells were detected by ELIS A, Western Blot and RT-PCR. In unstimulated cells, the protein and mRNA expression of MCP-1, ICAM-1, and VCAM-1 protein were very low. Incubation with TNF-a, their expression increased. Pretreatment with 1,10,50,100 μmol/L Mv-Cl, TNF-a-induced MCP-1, ICAM-1, and VCAM-1 expression in the cells were inhibited. Even,50 and 100 μmol/L of Mv-Cl could inhibit more than 90% increased three proteins, whose level was simial to the control, which indicated that high concentration of Mv-Cl might protect the inflammotary cells.3. Effect of Mv-3-glc-Cl and Mv-3-gal-Cl on TNF-a-induced MCP-1, ICAM-1, and VCAM-1 expression in HUVECsThe changes on protein and mRNA expression level of MCP-1, ICAM-1, and VCAM-1 in cells were detected by ELISA, Western Blot, and RT-PCR. Mv-3-glc-Cl and Mv-3-gal-Cl had different inhibitory effects on the protein and mRNA levels of endothelial MCP-1, ICAM-1, and VCAM-1 in a concentration-dependent manner. For most condition, Mv-3-glc-Cl showed stronger effect than Mv-3-gal-Cl, except that low concentration of Mv-3-glc-Cl (1 and 10 μmol/L) had no significant inhibition on TNF-a-induced ICAM-1 expression. Mv-3-glc-Cl had stronger effect than Mv-3-gal-Cl at high concentration.4. Synergistic effect of Mv-3-glc-Cl and Mv-3-gal-Cl and molecular mechanism of their anti-inflammationThe changes on protein and mRNA expression level of MCP-1, ICAM-1, and VCAM-1 in cells were detected by ELISA, Western Blot, and RT-PCR. The results showed that the synergistic effect existed between Mv-3-glc-Cl and Mv-3-gal-Cl. They could inhibited TNF-a-induced increases of MCP-1, ICAM-1, and VCAM-1 production both in the protein and mRNA levels. TNF-a induced IκB degradation, and subsequently increased the expression of MCP-1, ICAM-1, and VCAM-1. The inhibitory effect of Mv-3-glc-Cl, Mv-3-gal-Cl and (Mv-3-glc-Cl+Mv-3-gal-Cl) (10 μmol/1) on TNF-a-induced expression of IκB degradation was decreased. The activation of NF-κB requires the translocation of the p65 subunit of NF-κB from the cytoplasm to the nucleus. Immunocytochemistry was performed by using NFκB and fluorescein isothiocyanate (FITC)-conjugated antibody. In un-stimulated cells, the levels of p65 in the nucleus were very low. Upon simulation by TNF-a, the levels of p65 in the nucleus were increased. On the other hand, pretreated with Mv-3-glc-Cl, Mv-3-gal-Cl, and their mixture, the fluorescence intensity levels of p65 were decreased in the nucleus. The high concentration of Mv-3-glc-Cl, Mv-3-gal-Cl, and their mixture might completely inhibit the nuclear translocation of p65, with the p65 protein level in the cell nucleus similar to the control, which suggested the anti-inflammation mechanism of Malvidin by the NF-κB pathway. These results indicated the potential role of Mv in preventing chronic inflammation in many diseases.更多还原