【作者】 万晶晶；

【导师】 成蓓；

【作者基本信息】 华中科技大学 ， 老年医学， 2009， 博士

【摘要】 第一部分Ghrelin对巨噬细胞源性泡沫细胞ATP结合盒转运子A1和G1表达的调控作用背景：ATP结合盒转运子A1和G1（ATP-binding cassette transporter A1／G1，ABCA1／ABCG1）是两种胞膜蛋白。在动脉壁，ABCA1是单核巨噬细胞内胆固醇流出的重要途径，其基因突变可引起胞内胆固醇流出障碍；ABCG1也是单核巨噬细胞内胆固醇流出的途径之一，有助于清除脂质，防止泡沫细胞形成，从而抑制动脉粥样硬化（atherosclerosis，AS）的形成。Ghrelin是一种促生长激素释放肽，与糖脂代谢及相关疾病有密切联系，诸多研究提示Ghrelin在AS的发展中扮演着有益的角色，但Ghrelin对巨噬细胞源性泡沫细胞形成的调控作用及机制尚未见报道。目的：本研究探讨了Ghrelin对单核／巨噬细胞泡沫化过程中胞内脂滴含量的影响，同时研究单核／巨噬细胞分化成为泡沫细胞过程中Ghrelin对ABCA1和ABCG1表达的调控作用，以揭示其抗AS的效应及其机制。方法：体外培养人源单核细胞系THP-1，由佛波酯（phorbol myristate acetate，PMA）作用使其分化为巨噬细胞，后者可在在氧化低密度脂蛋白（ox-LDL）存在条件下进一步转变为泡沫细胞，油红O染色法鉴定泡沫细胞形成。巨噬细胞加入不同浓度的Ghrelin(10^-7mol／L，10^-6 mol／L，10^-5mol／L)预孵2小时后再加入ox-LDL共同孵育不同时间（12h，24h，48h），随后各组均加入10mg/L的apoA-I，CO₂培养箱中孵育12小时。油红O染色法观察细胞内脂滴含量，运用PT-PCR法检测ABCA1和ABCG1mRNA水平，Western-blot法检测ABCA1和ABCG1蛋白表达，采用酶法，通过荧光分光光度计检测细胞内外胆固醇含量并计算胆固醇流出率。结果：Ghrelin可明显减少细胞内脂滴的形成，显著增加胞内游离胆固醇流出率，随着Ghrelin浓度增高，其抑制胞内胆固醇酯含量和增加胆固醇流出率的作用增强，10^-7mol／L，10^-6mol／L和10^-5mol／L Ghrelin干预组胆固醇流出率分别为（29．3±1．1）％，（36．0±1．4）％和（41．6±2．1）％，差异具有统计学意义（P＜0．05）；胆固醇酯含量分别为34．5±3．0、27．8±2．3、24．3±2．3，与泡沫细胞组相比有显著差异（P＜0．05）。但不同作用时间组无明显差别。Ghrelin能显著增加单核／巨噬细胞泡沫化过程中ABCA1和ABCG1 mRNA水平和蛋白表达，不同浓度干预组ABCA1／G1表达有显著差异（P＜0．05），不同时间组ABCA1／G1表达无统计学差异（P＞0．05）。结论：Ghrelin能上调ABCA1和ABCG1 mRNA与蛋白的表达，从而促进胞内胆固醇流出，抑制巨噬细胞源性泡沫细胞的形成，并可能因此而延缓动脉粥样硬化的发生；Ghrelin的上述作用呈浓度依赖性而不呈时间依赖性。第二部分Ghrelin通过生长激素促分泌素受体抑制巨噬细胞源性泡沫细胞形成背景：Ghrelin是生长激素促分泌素受体（growth hormone secretagogue receptor，GHS-R）的配体，能通过与该受体结合发挥促生长激素分泌的效应。巨噬细胞膜上也同样表达GHS-R，目前研究表明在动脉粥样硬化斑块处Ghrelin受体的密度明显上调。我们前面的研究提示Ghrelin能通过下调酰基辅酶A：胆固醇酰基转移酶1（acyl-CoA：cholesterol acyltransferases，ACAT1）的表达、上调ABCA1和ABCG1的表达而促进胆固醇外流，从而抑制胆固醇酯聚集于巨噬细胞使之泡沫化。据此，我们推测上述表象是Ghrelin通过与GHS-R发生受体-配体相互作用而发挥效应。目的：以人单核细胞系THP-1源性泡沫细胞为研究对象，探讨不同程度拮抗GHS-R受体后，Ghrelin对单核巨噬细胞源性泡沫细胞形成过程中人ACAT-1和ABCA1／G1基因和蛋白表达的调控作用。检测不同程度拮抗GHS-R受体后，Ghrelin对单核巨噬细胞源性泡沫细胞形成的影响，其胞内胆固醇酯含量及胆固醇流出率变化。方法：体外培养THP-1，由佛波酯（PMA）诱导分化为巨噬细胞，继而在氧化低密度脂蛋白（ox-LDL）存在条件下进一步转变为泡沫细胞，油红O染色法鉴定泡沫细胞形成。单核细胞分化为巨噬细胞后，与不同浓度(10^-5mol／L，5×10^-5mol／L，10^-4mol／L)GHS-R特异性拮抗剂[D-Lys3]-GHRP-6预孵2h后，再与Ghrelin(浓度为10^-5mol／L)共育，2h后加入ox-LDL作用24h。随后各组均加入10mg／L的apoA-I，CO₂培养箱中孵育12小时。运用PT-PCR法和Western-blot法分别检测Ghrelin干预后及拮抗生长激素促分泌素受体后ACAT-1、ABCA1和ABCG1 mRNA水平与ACAT-1蛋白表达，采用酶法，通过荧光分光光度计检测细胞内外胆固醇含量和游离胆固醇含量，并计算胆固醇酯含量和胆固醇流出率。结果：油红O染色结果显示GHS-R拮抗剂[D-Lys3]-GHRP-6阻断Ghrelin对巨噬细胞泡沫化的抑制作用。Ghrelin能显著降低单核／巨噬细胞泡沫化过程中ACAT-1mRNA水平和蛋白表达（P＜0．01），不同浓度(10^-5mol／L，5×10^-5mol／L和10^-4mol／L)[D-Lys3]-GHRP-6干预后，ACAT-1 mRNA水平逐渐增高，分别为1．14±0．04，1．58±0．03和2．4±0．16，ACAT-1蛋白表达也逐渐增高，分别为1．25±0．09，1．77±0．11和2．3±0．09，与Ghrelin组（mRNA：0．89±0．05，蛋白：0．86±0．08）相比，差异具有统计学意义（P＜0．05）。同时，Ghrelin能显著增加单核／巨噬细胞泡沫化过程中ABCA1／G1 mRNA水平和蛋白表达（P＜0．01）。拮抗GHS-R后，Ghrelin抑制泡沫细胞形成的效应被阻断，且GHS-R特异性拮抗剂浓度越高，该阻断作用越明显（P＜0．05）。胆固醇含量检测结果表明，不同浓度[D-Lys3]-GHRP-6干预组较Ghrelin组胞内胆固醇酯含量分别升高了（16．7±1．2）％，（54．5±4．5）％和（73．4±7．9）％，阻断了Ghrelin抑制胆固醇酯形成的作用作用（P＜0．05）。胆固醇流出率结果则表明，[D-Lys3]-GHRP-6也阻断了Ghrelin对胆固醇流出的促进作用（P＜0．05）。结论：Ghrelin可能通过GHS-R途径下调ACAT-1转录翻译水平，及上调ABCA1／G1转录翻译水平，从而抑制胆固醇聚集于巨噬细胞，并同时促进胞内胆固醇的流出，避免游离胆固醇滞留于细胞。若拮抗Ghrelin的受体GHS-R，则Ghrelin的这种有益的效应被阻断，而不能发挥抑制巨噬细胞泡沫化的作用。第三部分过氧化物酶体增生物激活受体γ信号通路参与Ghrelin抑制巨噬细胞泡沫化过程背景：过氧化物酶体增生物激活受体γ（peroxisome proliferator-activated receptorγ，PPARγ）是PPAR超家族的亚型之一，属于核受体转录蛋白。PPARγ表达于巨噬细胞核内，并被证实能调节细胞和动脉壁脂质稳态，提示其在抗动脉粥样硬化中发挥着重要作用。我们早期研究显示PPARγ的活化能使ACAT1的表达受抑制。而我们前面的研究证实Ghrelin能下调ACAT1表达，因此，我们推测PPARγ信号通路参与了Ghrelin对ACAT1的调控过程。我们进一步推测PPARγ也同时参与了Ghrelin对ABCA1和ABCG1的调控作用。目的：以人单核／巨噬细胞源性泡沫细胞为研究对象，探讨Ghrelin对人单核细胞系（THP-1）源性泡沫细胞PPARγ表达的调控作用；探讨不同程度地抑制PPARγ后，胞内胆固醇酯含量及胆固醇流出率的变化；探讨不同程度抑制PPARγ对Ghrelin调控ACAT-1、ABCA1和ABCG1 mRNA与蛋白表达的影响，从而确定PPARγ信号通路是否为Ghrelin抑制巨噬细胞泡沫化的机制之一。方法：体外培养THP-1，在佛波酯（phorbol myristate acetate，PMA）的诱导下分化为巨噬细胞，与不同浓度(10^-7mol／L，10^-6mol／L，10^-5mol／L)ghrelin预孵后加入氧化低密度脂蛋白（ox-LDL）共育24h。运用PT-PCR法和Western-blot法分别检测不同浓度Ghrelin(10^-7mol／L，10^-6mol／L，10^-5mol／L)作用后PPARγmRNA与蛋白的表达。巨噬细胞与不同剂量（10μmol／L，20μmol／L，50μmol／L）PPARγ特异性抑制剂GW9662预孵2h后，加入Ghrelin(10^-5mol／L)作用2h，再与终浓度为100mg／Lox-LDL共育24h，随后各组均加入10mg／L的apoA-I，CO₂培养箱中孵育12小时。油红O染色法观察细胞内脂滴含量；PT-PCR法和Western-blot法分别检测Ghrelin干预后PPARγmRNA与蛋白的表达，以及不同程度抑制PPARγ后ACAT-1、ABCA1和ABCG1mRNA水平与ACAT-1蛋白表达，采用酶-荧光分光光度计法检测细胞内外胆固醇含量和游离胆固醇含量，并计算胆固醇流出率。结果：与泡沫细胞组相比，不同浓度组(10^-7mol／L，10^-6mol／L，10^-5mol／L)Ghrelin能显著增加单核／巨噬细胞泡沫化过程中PPARγmRNA与蛋白表达，且干预的Ghrelin浓度越高，PPARγ表达越高，其mRNA表达分别为0．87±0．06，1．44±0．15，1．57±0．11，其蛋白表达分别为0．82±0．06，1．46±0．11，1．73±0．14，与泡沫细胞组相比，差异均具有统计学意义（P＜0．05）。抑制PPARγ后，Ghrelin抑制泡沫细胞形成的效应被阻断，且PPARγ抑制剂浓度越高，该阻断作用越明显。PPARγ的抑制剂GW9662还拮抗了Ghrelin对巨噬细胞泡沫化过程中ACAT-1和ABCA1／G1 mRNA水平和蛋白表达的调控作用，随着PPARγ抑制剂剂量的增高，其阻断Ghrelin下调ACAT-1和上调ABCA1／G1的效应越显著（P＜0．05）。胆固醇流出率结果显示，GW9662对照组和泡沫细胞组胞内胆固醇酯含量及胆固醇流出率无显著差别；不同剂量GW9662干预后，胞内胆固醇含量较Ghrelin组升高了（18．3±4．3）％，（30．6±11．4）％，（52．6±8．4）％；而胆固醇流出率则分别下降了（14．3±3．2）％，（27．3±4．1）％，（47．7±4．6）％，差异均有统计学意义。（P＜0．05）。结论：Ghrelin能浓度依赖性上调单核／巨噬细胞泡沫化过程中PPARγmRNA与蛋白表达，并进而调控PPARγ下游途径ACAT-1、ABCA1和ABCG1的转录和翻译水平，从而抑制巨噬细胞源性泡沫细胞的形成，并可能通过该信号转导通路发挥抗动脉粥样硬化的作用。更多还原

【Abstract】 PartⅠRegulation of Ghrelin on the Expression of ATP-bindingCassette Transporters A1/G1 in Macrophage-Derived Foam CellsBackground:ATP-binding cassette transporter A1（ABCA1）and ATP-bindingcassette transporter G1（ABCG1）are involved in cholesterol removal from the macrophagefoam cells,and form the critical part of a process termed as reverse cholesterol transport,and therefore play critical roles in foam cell formation.Ghrelin,an endocrine peptide newlyidentified mainly in stomach epithelium,stimulates food intake in humans.Recently,manyresults imply a beneficial role of this hormone in the development of atherosclerosis.Theaim of this study is to investigate the regulation of ghrelin on the expression ofATP-binding Cassette transporters A1 and G1（ABCA1/ABCG1）during foam cellsformation.Methods:The human monocytic leukemia cell line（THP-1）was chosen in our study.The differentiation of THP-1 cells into macrophages was induced by using phorbolmyristate acetate（PMA）.Macrophages were then incubated with oxidized LDL（ox-LDL）to generate foam cells.Ghrelin of different concentrations were treated at different timepoints during foam cells formation.The ABCA1/ABCG1 protein and mRNA expressionwere detected by Western blotting and RT-PCR.The effect of variance of cholesterolcontent was measured by zymochemistry via-fiuorospectrophotometer. Results:Ghrelin reduced the content of lipid droplet in foam cells,and increased theefflux of intracellular cholesterol significantly.Ghrelin increased ABCA1 protein mass andmRNA level in a dose-dependent manner.Compared with untreated cells,treatment ofTHP-1 derived foam cells with 10^-7mol/L ghrelin resulted in a significant 1.6-fold increasein ABCA1 protein expression.And 2- and 2.8-fold increase at 10^-6and 10^-5mol/L ghrelin.The changements of ABCA1 mRNA level were the same as ABCA1.Ghrelin also increasedABCG1 protein mass and mRNA level in a dose-dependent manner.Conclusion:Ghrelin might inhibit foam cells formation by up-regulating theexpression of ABCA1 and ABCG1.PartⅡGhrelin inhibit foam cell formation via a GrowthHormone Secretagogue Receptor-Dependent PathwayBackground:Ghrelin,a endogenous ligand of the growth hormone secretagoguereceptor（GHS-R）,revealed cardioprotective effects in both experimental models andhuman.There is far less imformation information on mechanisms that produceantiatherogenic effects.Acyl-coenzyme A:cholesterol acyltransferase 1（ACAT-1）andABCA1 have been implicated in regulating cellular cholesterol homeostasis and thereforeplay critical roles in foam cell formation.We demonstrated that ghrelin coulddown-regulated the expression of ACAT-1 and up-regulated ABCA1/G1 simultaneously,and hypothesize that ghrelin can inhibit foam cell formation by regulating the expression ofACAT-1 and ABCA1 via the signaling pathway of GHS-R.The aim of this part of study isto investigate the expression of ACAT-1 and ABCA1/G1 with ghrelin and variousconcentration of GHS-R antagonist in macrophage derived foam cells.Methods:The human monocytic leukemia cell line（THP-1）was chosen in our study.The differentiation of THP-1 cells into macrophages was induced by phorbol 12-myristate13-acetate（PMA）.Macrophages were then incubated with oxidized LDL（ox-LDL）togenerate foam cells.Ghrelin and [D-Lys3]-GHRP-6,the special antagonist of growth hormone secretagogue receptor（GHS-R）,were treated during foam cells formation.TheACAT-1 and ABCA1/G1 protein and mRNA levels were detected by Western blotting andRT-PCR.The effect of variance of cholesterol content was measured by zymochemistryvia-fluorospectrophotometer.Results:Ghrelin reduced the content of cholesteryl ester in foam cells obviously.ACAT-1 protein and mRNA levels were also decreased.The antagonist of GHS-R inhibitedthe effects of ghrelin on ACAT-1 expression in a dose-dependent manner.Ghrelin increasedthe cholesterol efflux obviously.ABCA1/G1 protein and mRNA levels were also increased.The antagonist of GHS-R inhibited the effects of ghrelin on ABCA1 and ABCG1expression in a dose-dependent manner.Conclusions:Ghrelin might interfere foam cells formation by simultaneouslydown-regulating the expression of ACAT-1 and up-regulating the expression of ABCA1/G1via a GHS-R pathway.PartⅢStudy of the Peroxisome Proliferator-activated Receptor ysignaling pathway in the Inhibition of Foam Cell Formation by GhrelinBackground:To investigate the effects of ghrelin on the expression of peroxisomeproliferator-activated receptorγin foam cells formation.And to investigate the effects ofghrelin on the expression of ACAT-1 and ABCA1/G1 in THP-1 derived foam cells.Methods:The human monocytic leukemia cell line（THP-1）was chosen in our study.The differentiation of THP-1 cells into macrophages was induced by using PMA.Macrophages were then incubated with oxidized LDL（ox-LDL）to generate foam cells.The PPARγmRNA level and protein expression were detected by RT-PCR and Westernblotting.Macrophage were incubated with ghrelin and antagonist of PPARγ,and then wereincubated with oxidized LDL（ox-LDL）to generate foam cells.The ACAT-1 and ABCA1/G1 mRNA level and protein expression were detected by RT-PCR and Westernblotting.The effect of variance of cholesterol content was measured by zymochemistry viafluorospectrophotometer.Results:Ghrelin increased PPARγprotein expression in a dose-dependent manner.The changements of PPARγmRNA level were the same as protein expression.Ghrelinreduced cholesteryl ester obviously.ACAT-1 protein and mRNA levels were also decreased.The antagonist of PPARγinhibited the effects of ghrelin on ACAT-1 expression in adose-dependent manner.Ghrelin increased the cholesterol efflux obviously.ABCA1/G1protein and mRNA levels were also increased.The antagonist of PPARγinhibited theeffects of ghrelin on ABCA1/G1 expression in a dose-dependent manner.Conclusion:Ghrelin could increase the expression of PPARγin a dose-dependentmanner during foam cell formation.The PPARγinhibitor could interfere the expression ofACAT-1 and ABCA1/G1 regulated by ghrelin.The PPARγsignaling pathway mightparticipate in the inhibition of foam cells formation by ghrelin.更多还原