apoai介导内皮细胞1磷酸鞘氨醇释放及其信号机制分析 word格式.docxVIP

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-BI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-BI 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-BI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。关键词：高密度脂蛋白1-磷酸鞘氨醇鞘氨醇激酶1 载脂蛋白A-I ATP结合盒转运体A1 B类I型清道夫受体3ApoA-IinducesS1Preleasefromendothelialcellsandsignalingmechanism.ABSTRACTObjective:Sphingosine-1-phosphate(S1P)isabioactivelysophospholipidgeneratedby thesphingosinekinase1/2(SphK1/2)-catalysedphosphorylationofsphingosine.Red bloodcells,plateletandvascularendothelialcellsmaybethemajorcellularsourcesof S1Pinplasma.Morethan65%oftheS1Pinbloodisassociatedwiththelipoproteins LDL,VLDL,andHDL,withthemajorityoflipoprotein-associatedS1P(54%)boundto HDL.FindingsfromagrowingnumberofstudiesindicatethatS1Pisamediatorfor manycardiovasculareffectsofHDL,includingantithrombotic,protectionofendothelial functionandinhibit/reverseatherosclerosis.Akeyenzymesphingosinekinase(SphK) canphosphorylateitssubstratesphingosinetogenerateS1P,thenS1Pisreleasedfrom cellsthroughdifferentcarriers.ThenascentHDLcreatedbyATP-bindingcassetteA1 (ABCA1)-mediatedeffluxofcellularphospholipid(PL)andfree(unesterified) cholesterol(FC)toapolipoproteinA-I(apoA-I)isarate-limitingstepof cholesterol-efflux.ApoA-IwasalsoallowedtobindontoscavengerreceptorclassB memberI(SR-BI),whichcanpromotebidirectionalunesterifiedlipidsmovement.We willdiscussanapoA-IinduceS1PreleasefromendothelialcellsandwhetherABCA1 and SR-BIortheir cellarsignal participated in this process in this experiment.Methods:HUVECsweretreatedwith20μg/mlapoA-Ifordifferenttime(5,10,20,40,80 minutes),orincubatedwithdifferent