本文關鍵詞： 磷酸二酯酶4 木犀草素 黏附分子 微血管內皮細胞 大鼠　出處：《北京農學院》2015年碩士論文　論文類型：學位論文

【摘要】：目的：中性粒細胞與內皮細胞的黏附是炎癥反應的先決條件,也是影響炎癥發(fā)生發(fā)展的關鍵因素。第二信使cAMP含量升高對內皮細胞黏附分子的過表達具有顯著的抑制作用。由于對細胞內cAMP信號的決定性作用,磷酸二酯酶4(PDE4)被認為是新的炎癥治療靶點,其抑制劑被用于多種炎癥性疾病的治療。項目組前期研究表明木犀草素為高特異性的PDE4活性抑制劑,能抑制中性粒細胞與微血管內皮細胞的黏附,其機理與木犀草素抑制中性粒細胞PDE4活性進而影響其黏附分子的表達有關。本試驗進一步研究木犀草素對微血管內皮細胞cAMP含量、PDE4及黏附分子表達的影響,深入闡明木犀草素的抗炎的有關分子機制。方法：1)取SD大鼠肺臟,以組織塊細胞爬片法分離培養(yǎng)肺微血管內皮細胞。取第2代細胞采用CD31相關抗原免疫熒光染色等方法進行鑒定,用第3-5代肺微血管內皮細胞進行試驗。2)以炎癥因子fMLP激活肺微血管內皮細胞,以ELISA法檢測cAMP水平,以HPLC法通過檢測底物cAMP的含量計算PDE4活性,以實時熒光定量PCR法測定PDE4基因表達。3)采用流式細胞術檢測肺微血管內皮細胞在炎癥因子fMLP刺激前后黏附分子P-selectin、ICAM-1、VCAM-1表達的變化。結果：1)以組織塊細胞爬片法培養(yǎng)得到的肺微血管內皮細胞,經(jīng)CD31標記鑒定純度可達95%以上,可用于進一步的試驗。2)肺微血管內皮細胞cAMP-PDE、PDE4活性隨反應液酶量的增加呈劑量依賴性升高。PDE4活性在肺微血管內皮細胞cAMP-PDE總活性中約占40%。3)進一步研究表明木犀草素對體外培養(yǎng)的肺微血管內皮細胞cAMP-PDE、PDE4活性呈劑量依賴性抑制作用,其抑制作用隨木犀草素濃度升高而增強。木犀草素可抑制內皮細胞PDE4A、4B表達,促進4D表達。4)木犀草素對fMLP致肺微血管內皮細胞表面黏附分子VCAM-1升高具有顯著的抑制作用；而對ICAM-1表達在低濃度有一定的抑制作用,而在高濃度則具有明顯的促進作用；未檢測到肺微血管內皮細胞P-selectin的表達。結論：木犀草素體外可抑制肺微血管內皮細胞PDE4活性,抑制內皮細胞PDE4A、4B基因表達,升高cAMP含量,抑制內皮細胞黏附分子VCAM-1表達,從而起到抗炎作用。
[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.
【學位授予單位】：北京農學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S853.7

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