ApoA-Ⅰ介导内皮细胞1-磷酸鞘氨醇释放及其信号机制研究
发布时间：2018-12-13 19:03
【摘要】：背景与目的：1-磷酸鞘氨醇(sphingosine1-phosphate,S1P)是由鞘氨醇激酶1/2(sphingosine kinase1/2，SphK1/2)磷酸化鞘氨醇而合成。血管内皮细胞，，红细胞和血小板等是血液S1P的主要来源。脂蛋白是S1P在血浆中的主要结合载体，而绝大多数与高密度脂蛋白(high density lipoprotein, HDL)结合。HDL结合的S1P(HDL-S1P)也赋予HDL很多生物学功能，如抗血栓，保护内皮功能和抗动脉粥样硬化等作用。而S1P释放到细胞外通常受到细胞内或细胞外的刺激。载脂蛋白A-Ⅰ(apolipoproteinA-Ⅰ, apoA-Ⅰ)与ATP结合盒转运体A1(ATP-binding cassette A1, ABCA1)的结合是HDL生成的起始过程，也是胆固醇逆向转运的限速步骤。ApoA-Ⅰ也能结合清道夫受体B类Ⅰ型(scavenger receptor class B type Ⅰ，SR-BI)进行胆固醇和磷脂的双向转运。ApoA-Ⅰ除转运胆固醇外，还可转运其他磷脂成分。S1P即是一种信号鞘磷脂，赋予HDL诸多重要作用。作为HDL的主要载脂蛋白，apoA-Ⅰ是否能促进血管内皮细胞S1P释放，及其受体信号机制如何是本实验的研究目的。 方法：以apoA-Ⅰ不同时间和不同浓度处理培养人脐静脉内皮细胞(human umbilicalvein endothelial cells，HUVECs)。转染SphK，ABCA1和SR-B1siRNA48小时后，20μg/ml apoA-Ⅰ孵育5分钟。HUVECs分别预孵育N,N-Dimethylsphingosine(SphK1抑制剂)、U0126(ERK1/2抑制剂)、glybenclamide (ABCA1抑制剂)、BLT-1(SR-BI抑制剂)、AG490(JAK2抑制剂)、PP2(Src抑制剂)或PTX (G蛋白抑制剂)后，20μg/mlapoA-Ⅰ或1μM S1P孵育5分钟。HUVECs预孵育glybenclamide后，20μg/ml apoA-Ⅰ处理10分钟，5分钟时额外处理SEW2871(S1PR1激动剂)五分钟。高效液相色谱法(high performance liquid chromatography，HPLC)测定细胞内及培养基S1P浓度。免疫印迹方法(Western blot，WB)检测ERK1/2磷酸化水平。 结果：ApoA-Ⅰ促进内皮细胞S1P释放，在5分钟至10分钟之间达到峰值之后缓慢下降。apoA-Ⅰ能增加细胞内S1P水平，1至3分钟明显升高，而后下降。SphK1的抑制剂或siRNA均减少apoA-Ⅰ诱导的S1P释放。apoA-Ⅰ能激活内皮细胞ERK1/2磷酸化，且ERK1/2抑制剂减少apoA-Ⅰ诱导的S1P释放。ABCA1和SR-B-Ⅰ抑制剂或siRNA均能减少apoA-Ⅰ诱导的S1P释放和ERK1/2磷酸化水平。但是，JAK2或Src抑制并不能减少apoA-Ⅰ诱导的S1P释放和ERK1/2磷酸化水平。ABCA1，SR-B-Ⅰ抑制并不能减少S1P诱导的ERK1/2磷酸化，而S1PR1/3抑制剂能减少S1P诱导的ERK1/2磷酸化。在ABCA1被抑制后，S1PR1激动剂能增加细胞内S1P水平，但不能明显增加apoA-Ⅰ诱导的S1P释放。 结论: 1. ApoA-Ⅰ能促进内皮细胞S1P释放增加。 2. ApoA-Ⅰ通过内皮细胞释放的S1P，激活S1PR1/3-ERK1/2-SphK1途径间接增加细胞内S1P水平。 3. ApoA-Ⅰ介导的S1P释放依赖于ABCA1和SR-B-Ⅰ受体。 4.推测S1PR1/3-ERK1/2-SphK1信号通路为apoA-Ⅰ介导的S1P释放提供了正反馈途径。
[Abstract]:Background & AIM: 1-sphingosine phosphate (sphingosine1-phosphate,S1P) is synthesized from sphingosine kinase 1 / 2 (sphingosine kinase1/2,SphK1/2) phosphorylated sphingosine. Vascular endothelial cells, red blood cells and platelets are the main sources of blood S 1 P. Lipoprotein is the major binding carrier of S1P in plasma, and most of them bind to high density lipoprotein (high density lipoprotein, HDL). S1P (HDL-S1P), which binds to HDL, also endows HDL with many biological functions, such as antithrombotic. Protect endothelial function and anti-atherosclerosis and so on. The release of S 1 P out of cells is usually stimulated by intracellular or extracellular stimulation. The binding of apolipoproteinA- 鈪
本文编号：2377062
[Abstract]:Background & AIM: 1-sphingosine phosphate (sphingosine1-phosphate,S1P) is synthesized from sphingosine kinase 1 / 2 (sphingosine kinase1/2,SphK1/2) phosphorylated sphingosine. Vascular endothelial cells, red blood cells and platelets are the main sources of blood S 1 P. Lipoprotein is the major binding carrier of S1P in plasma, and most of them bind to high density lipoprotein (high density lipoprotein, HDL). S1P (HDL-S1P), which binds to HDL, also endows HDL with many biological functions, such as antithrombotic. Protect endothelial function and anti-atherosclerosis and so on. The release of S 1 P out of cells is usually stimulated by intracellular or extracellular stimulation. The binding of apolipoproteinA- 鈪
本文编号：2377062
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