木犀草素对微血管内皮细胞磷酸二酯酶4及黏附分子的作用研究

发布时间：2018-02-07 12:54

本文关键词： 磷酸二酯酶4 木犀草素黏附分子微血管内皮细胞大鼠　出处：《北京农学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的：中性粒细胞与内皮细胞的黏附是炎症反应的先决条件,也是影响炎症发生发展的关键因素。第二信使cAMP含量升高对内皮细胞黏附分子的过表达具有显著的抑制作用。由于对细胞内cAMP信号的决定性作用,磷酸二酯酶4(PDE4)被认为是新的炎症治疗靶点,其抑制剂被用于多种炎症性疾病的治疗。项目组前期研究表明木犀草素为高特异性的PDE4活性抑制剂,能抑制中性粒细胞与微血管内皮细胞的黏附,其机理与木犀草素抑制中性粒细胞PDE4活性进而影响其黏附分子的表达有关。本试验进一步研究木犀草素对微血管内皮细胞cAMP含量、PDE4及黏附分子表达的影响,深入阐明木犀草素的抗炎的有关分子机制。方法：1)取SD大鼠肺脏,以组织块细胞爬片法分离培养肺微血管内皮细胞。取第2代细胞采用CD31相关抗原免疫荧光染色等方法进行鉴定,用第3-5代肺微血管内皮细胞进行试验。2)以炎症因子fMLP激活肺微血管内皮细胞,以ELISA法检测cAMP水平,以HPLC法通过检测底物cAMP的含量计算PDE4活性,以实时荧光定量PCR法测定PDE4基因表达。3)采用流式细胞术检测肺微血管内皮细胞在炎症因子fMLP刺激前后黏附分子P-selectin、ICAM-1、VCAM-1表达的变化。结果：1)以组织块细胞爬片法培养得到的肺微血管内皮细胞,经CD31标记鉴定纯度可达95%以上,可用于进一步的试验。2)肺微血管内皮细胞cAMP-PDE、PDE4活性随反应液酶量的增加呈剂量依赖性升高。PDE4活性在肺微血管内皮细胞cAMP-PDE总活性中约占40%。3)进一步研究表明木犀草素对体外培养的肺微血管内皮细胞cAMP-PDE、PDE4活性呈剂量依赖性抑制作用,其抑制作用随木犀草素浓度升高而增强。木犀草素可抑制内皮细胞PDE4A、4B表达,促进4D表达。4)木犀草素对fMLP致肺微血管内皮细胞表面黏附分子VCAM-1升高具有显著的抑制作用；而对ICAM-1表达在低浓度有一定的抑制作用,而在高浓度则具有明显的促进作用；未检测到肺微血管内皮细胞P-selectin的表达。结论：木犀草素体外可抑制肺微血管内皮细胞PDE4活性,抑制内皮细胞PDE4A、4B基因表达,升高cAMP含量,抑制内皮细胞黏附分子VCAM-1表达,从而起到抗炎作用。
[Abstract]:Objective: adhesion of neutrophils to endothelial cells is a prerequisite for inflammatory response. The increase of cAMP content in the second messenger has a significant inhibitory effect on the overexpression of adhesion molecules in endothelial cells. Because of the decisive effect on the intracellular cAMP signal, it is also a key factor that affects the occurrence and development of inflammation. Phosphodiesterase 4 (PDE4) is considered to be a new target for inflammatory therapy and its inhibitors have been used in the treatment of various inflammatory diseases. Luteolin has been shown to be a highly specific inhibitor of PDE4 activity in previous studies in the project group. Inhibiting the adhesion of neutrophils to microvascular endothelial cells, The mechanism is related to luteolin inhibiting the PDE4 activity of neutrophils and affecting the expression of its adhesion molecules. The effect of luteolin on the content of cAMP and the expression of PDE4 and adhesion molecules in microvascular endothelial cells was further studied. The molecular mechanism of luteolin anti-inflammation was elucidated in depth. Methods the lungs of SD rats were taken from the lungs of SD rats. Lung microvascular endothelial cells were isolated and cultured by tissue mass cell climbing method. The second passage cells were identified by CD31 associated antigen immunofluorescence staining. The pulmonary microvascular endothelial cells were activated by inflammatory factor fMLP, the level of cAMP was measured by ELISA method, and the activity of PDE4 was calculated by HPLC method by detecting the content of cAMP. The expression of PDE4 gene in pulmonary microvascular endothelial cells was detected by flow cytometry before and after fMLP stimulation by real-time fluorescence quantitative PCR. The expression of adhesion molecule P-selectinine ICAM-1 / VCAM-1 was detected by flow cytometry. To the pulmonary microvascular endothelial cells, The purity can reach more than 95% by CD31 labeling. The activity of cAMP-PDEN-PDE4 in pulmonary microvascular endothelial cells increased in a dose-dependent manner with the increase of the amount of reactive enzyme. PDE4 activity accounted for about 40. 3% of the total activity of cAMP-PDE in pulmonary microvascular endothelial cells. The activity of cAMP-PDE4 in cultured pulmonary microvascular endothelial cells was inhibited in a dose-dependent manner. The inhibitory effect of luteolin was enhanced with the increase of luteolin concentration. Luteolin could inhibit the expression of PDE4An4B in endothelial cells and promote the expression of 4D.) luteolin could significantly inhibit the increase of VCAM-1 on the surface of pulmonary microvascular endothelial cells induced by fMLP. The expression of P-selectin in pulmonary microvascular endothelial cells was not detected. Conclusion: luteolin can inhibit the PDE4 activity of pulmonary microvascular endothelial cells in vitro. PDE4An4B gene expression was inhibited, cAMP content was increased, and VCAM-1 expression was inhibited in endothelial cells.
【学位授予单位】：北京农学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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