本文關(guān)鍵詞： 磷酸二酯酶4 木犀草素 黏附分子 微血管內(nèi)皮細(xì)胞 大鼠　出處：《北京農(nóng)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：中性粒細(xì)胞與內(nèi)皮細(xì)胞的黏附是炎癥反應(yīng)的先決條件,也是影響炎癥發(fā)生發(fā)展的關(guān)鍵因素。第二信使cAMP含量升高對內(nèi)皮細(xì)胞黏附分子的過表達(dá)具有顯著的抑制作用。由于對細(xì)胞內(nèi)cAMP信號的決定性作用,磷酸二酯酶4(PDE4)被認(rèn)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(xì)胞與微血管內(nèi)皮細(xì)胞的黏附,其機理與木犀草素抑制中性粒細(xì)胞PDE4活性進(jìn)而影響其黏附分子的表達(dá)有關(guān)。本試驗進(jìn)一步研究木犀草素對微血管內(nèi)皮細(xì)胞cAMP含量、PDE4及黏附分子表達(dá)的影響,深入闡明木犀草素的抗炎的有關(guān)分子機制。方法：1)取SD大鼠肺臟,以組織塊細(xì)胞爬片法分離培養(yǎng)肺微血管內(nèi)皮細(xì)胞。取第2代細(xì)胞采用CD31相關(guān)抗原免疫熒光染色等方法進(jìn)行鑒定,用第3-5代肺微血管內(nèi)皮細(xì)胞進(jìn)行試驗。2)以炎癥因子fMLP激活肺微血管內(nèi)皮細(xì)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(dá)。3)采用流式細(xì)胞術(shù)檢測肺微血管內(nèi)皮細(xì)胞在炎癥因子fMLP刺激前后黏附分子P-selectin、ICAM-1、VCAM-1表達(dá)的變化。結(jié)果：1)以組織塊細(xì)胞爬片法培養(yǎng)得到的肺微血管內(nèi)皮細(xì)胞,經(jīng)CD31標(biāo)記鑒定純度可達(dá)95%以上,可用于進(jìn)一步的試驗。2)肺微血管內(nèi)皮細(xì)胞cAMP-PDE、PDE4活性隨反應(yīng)液酶量的增加呈劑量依賴性升高。PDE4活性在肺微血管內(nèi)皮細(xì)胞cAMP-PDE總活性中約占40%。3)進(jìn)一步研究表明木犀草素對體外培養(yǎng)的肺微血管內(nèi)皮細(xì)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(nèi)皮細(xì)胞PDE4A、4B表達(dá),促進(jìn)4D表達(dá)。4)木犀草素對fMLP致肺微血管內(nèi)皮細(xì)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(dá)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(jìn)作用；未檢測到肺微血管內(nèi)皮細(xì)胞P-selectin的表達(dá)。結(jié)論：木犀草素體外可抑制肺微血管內(nèi)皮細(xì)胞PDE4活性,抑制內(nèi)皮細(xì)胞PDE4A、4B基因表達(dá),升高cAMP含量,抑制內(nèi)皮細(xì)胞黏附分子VCAM-1表達(dá),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(xué)位授予單位】：北京農(nóng)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳武,姜代勛,楊柳,胡燕,張春霞,于同泉,路蘋;豬嗜中性粒細(xì)胞cAMP磷酸二酯酶的提取與活性檢測[J];北京農(nóng)學(xué)院學(xué)報;2003年04期

2 李培鋒;李成應(yīng);希日莫;關(guān)紅;李利平;;秦公散對LPS活化的內(nèi)皮細(xì)胞表達(dá)黏附分子和分泌一氧化氮的影響[J];動物醫(yī)學(xué)進(jìn)展;2010年11期

3 胥楠,陳曉理;炎癥過程中內(nèi)皮細(xì)胞的功能變化及其后果[J];國外醫(yī)學(xué)(外科學(xué)分冊);2005年01期

4 閻高峰;李學(xué)剛;袁呂江;;木犀草素生物活性研究[J];糧食與油脂;2006年03期

5 楊穎;宋曙輝;徐桂花;;黃酮類化合物木犀草素研究進(jìn)展[J];糧食與油脂;2009年09期

6 戴麗;端莉梅;姚志清;;木犀草素對脂多糖誘導(dǎo)下小鼠急性肺損傷的保護(hù)作用及其機制研究[J];河北醫(yī)藥;2015年03期

7 何秀娟;呂U，

本文編號：1494425