【作者】 王彦富；

【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】 第一部分不同动脉粥样硬化相关人群外周血ghrelin水平变化及意义背景Ghrelin与其受体结合后具有广泛生物学效应。已知ghrelin受体mRNA在心血管组织分布广泛，已有的研究显示ghrelin可通过抑制炎症、抗凋亡、调节脂质代谢等不同途径对心血管起到保护作用，然而其不同动脉粥样硬化相关人群外周血ghrelin水平变化如何，其变化是否与疾病相关等方面的研究尚未见报道。目的通过检测不同人群外周血ghrelin水平，初步探讨ghrelin水平变化情况及其临床意义。方法及结果将不同年龄、冠心病、肥胖、糖尿病患者纳入研究对象，进行对照研究。通过酶联免疫吸附试验检测入选者外周血ghrelin水平，并同时抽血送检入选者一般生化指标如TC、TG、HDLc等，搜集临床资料，分析一般临床特征包括：年龄、性别、吸烟、BMI、高血压等。检测发现，与青年组ghrelin水平（2．51±0．77）ng／ml相比，老年组ghrelin水平（1．43±0．64）ng／ml显著降低，p=0．009；老年组与中年组ghrelin水平（2．24±0．32）ng／ml相比，ghrelin降低程度亦呈现显著统计学差异，p=0．036，随着年龄增长，外周血ghrelin水平显著降低，两者之间显著相关，r_p=-0．564，p=0．015；经校正后，冠心病组外周血ghrelin水平为（1．82±0．34）ng／ml，与对照组（3．00±0．29）ng／ml相比，p=0．025，具有显著统计学差异；肥胖组（BMI≥28Kg／m²）外周血ghrelin浓度（1．87±0．89）ng／ml显著低于对照组（2．77±1．49）ng／ml，p=0．047；糖尿病患者外周血ghrelin水平（2．41±0．74）ng／ml显著低于对照组（3．35±13．02）mg／ml，p=0．013。结论由上可知，随着年龄增加，外周血ghrelin水平呈现降低趋势；冠心病、肥胖、糖尿病均可导致外周血ghrelin水平进一步降低。为今后进一步阐明ghrelin的功能奠定了一定基础。第二部分Ghrelin可通过抑制Th17细胞促炎作用延缓动脉粥样硬化进展背景Th17细胞是近年来发现的一种CD4⁺效应T细胞，可特异性产生IL-17，在炎症性疾病中起着重要调节作用。已知，IL-17可促进单核巨噬细胞分泌炎症因子TNF-α，IL-6并与其发挥协同作用，加强炎症反应。由于AS是一种慢性炎症性疾病，且与其密切相关的IL-17、IL-6及TNFα在厌食-恶液质综合症发病中起着重要作用，ghrelin作为重要的食欲调节激素与上述炎症因子的关系如何及其对AS的影响是否与上述炎症因子有关尚未见报道。目的通过检测颈动脉粥样硬化患者外周血ghrelin及Th17细胞含量的变化及观察应用ghrelin干预患者PBMCs后上述炎症因子的表达变化，初步探讨ghrelin延缓AS的病理机制。方法经颈动脉超声检查将入选对象分为两组，即AS组37例和对照组31例（以颈动脉内-中膜厚度-IMT≥1．2mm为标准分组）。采用流式细胞分析法，检测各组患者外周血Th17细胞占CD4⁺T细胞百分比；应用酶联免疫吸附法（ELISA）检测患者外周血Ghrelin含量，同时检测患者外周血浆Th17相关因子：白介素（IL）-17、IL-6、肿瘤坏死因子（TNF）-α水平；体外培养AS组患者外周血单个核细胞，经Ghrelin干预后观察上述炎症因子水平变化。结果AS组患者外周血Th17／CD4⁺T细胞百分比为（1．92±0．52）％，明显高于对照组（0．59±0．32）％，P＜0．05；外周血Ghrelin浓度分别为（2．19±0．84）ng／ml、（3．54±1．04）ng／ml，差异具有显著性，P＜0．05；Th17相关炎症细胞因子（IL-17、IL-6、TNF-α）水平在AS组分别为（101．29±25．00）pg／ml、（7．67±3．58）pg／ml和（17．98±3．16）pg／ml，显著高于对照组（76．97±27．64）pg／ml、（3．89±3．50）pg／ml、（6．38±1．27）pg／ml，具有显著统计学意义，P＜0．05；应用Ghrelin干预患者PBMCs后发现，上述炎症因子分泌明显受抑，且呈浓度依赖性，组间相比差异具有显著性，P＜0．05；患者血浆Ghrelin浓度与颈动脉内膜厚度呈显著负相关（r=-0．498，P＜0．05），与Th17相关炎症因子（IL-17、IL-6、TNF-α）水平呈负相关（相关系数分别为r=-0．544、-0．363、-0．565，P＜0．05）。结论Ghrelin通过抑制Th17相关炎症因子表达延缓动脉粥样硬化进展。第三部分Ghrelin对THP-1源性巨噬细胞泡沫化过程中酰基辅酶A：胆固醇酰基转移酶1表达的影响背景目前关于动脉粥样硬化的发病机制存在着诸多学说，如损伤-反应学说、炎症学说等。然而，我们注意到，所有学说最终都归结为泡沫细胞形成，因此，研究泡沫细胞形成机制，对于有效预防动脉粥样硬化的发生、发展具有重要意义。人酰基辅酶A：胆固醇酰基转移酶1（Acyl-CoA：cholesterol acyltransferases，ACAT-1）催化细胞内游离胆固醇形成胆固醇酯，进而形成泡沫细胞，在泡沫细胞形成过程中起着重要作用，抑制巨噬细胞内ACAT1功能将有助于减少泡沫细胞形成，起到抗AS作用。我们在前期实验中发现ghrelin本身可通过上调膜转运蛋白ATP结合盒转运子A1促进泡沫细胞内游离胆固醇流出，抑制泡沫细胞形成。因此，如果ghrelin同时具有下调ACAT1的功能，抑制游离胆固醇的酯化过程，将有助于我们进一步明确ghrelin延缓AS进展的机制。此方面的研究尚未见报道。目的研究单核／巨噬细胞分化成为泡沫细胞过程中Ghrelin对人酰基辅酶A：胆固醇酰基转移酶1（ACAT-1）表达以及细胞内胆固醇酯的影响。方法体外培养人源单核细胞系（THP-1），由佛波酯（PMA）作用将其诱导分化为巨噬细胞，后者可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。实验分为对照组（Ox-LDL）、不同浓度ghrelin干预组(10^-5、10^-6、10^-7 mol／L)以及不同时间干预组（Ghrelin 10-5 mol／L+Ox-LDL100mg／L）。巨噬细胞加入不同浓度的Ghrelin预孵两小时后再加入Ox-LDL（100mg／L），作用24h；换液，各组均加入10mg／L的apoA-I，并在含有0．3％BSA的RPMI1640培养基、CO₂培养箱中孵育12h，采用酶法，通过荧光分光光度计检测细胞内胆固醇酯含量化，采用油红O染色法观察细胞内脂滴含量，运用RT-PCR法检测ACAT-1 mRNA水平，Western-blot法检测ACAT-1蛋白表达。结果Ghrelin可明显减少THP-1源性泡沫细胞内脂滴的形成；Ghrelin可显著降低细胞内胆固醇酯含量，随着Ghrelin浓度升高，胞内胆固醇酯含量分别降低了（14．6±0．5）％，（28．3±1．5）％和（45．4±1．0）％，组间相比差异显著，且胞内胆固醇含量与ACAT-1蛋白及mRNA表达明显相关，相关系数r分别为0．968、0．943；Ghrelin能显著降低单核／巨噬细胞泡沫化过程中ACAT-1 mRNA水平和蛋白表达，组间相比具有显著统计学差异，且此干预作用呈浓度依赖性；Ghrelin不同时间干预组之间油红O染色结果显示脂滴变化不明显，ACAT1表达无明显差异，p＞0．05。结论Ghrelin可在转录及翻译水平通过下调ACAT-1表达，减少胞内胆固醇蓄积，抑制泡沫细胞形成；Ghrelin对ACAT1的抑制效应呈现浓度依赖性，无时间依赖性。更多还原

【Abstract】 PartⅠSignificance of serum ghrelin level in differentpeople with atherosclerosisBackground Many different biological effects could be found following the binding ofghrelin with its receptor.The receptor mRNA of ghrelin is widely distributed incardiovascular tissue.From the foregone documents of ghrelin,we found that ghrelin act tosafeguard the function of cardiovascular tissue via inhibiting inflammation,anti-apoptotic,regulating lipid metabolism and so on.However,the exactly serum level of ghrelin indifferent people with atherosclerosis have not been clarified,and whether a correlation existbetween the different concentration of ghrelin and CAD or DM etc.is still unknown.Objective Investigating the serum level of ghrelin in different people and observing therelationship between the variation of ghrelin and diseases.Methods and results A comparative study was performed in patients with different age,obesity,coronary artery disease （CAD） and diabetes mellitus （DM）.Enzyme linkedimmunosorbent assay was used to detect plasma levels of ghrelin,at the same time,generally selected biochemical parameters such as total cholesterol （TC）,triglyceride （TG）,high density lipoprotein cholesterol （HDLc）,etc.were detected with submitted blood,collecting clinical data,analyzing the general clinical feature:age,sex,smoking,body massindex （BMI）,hypertension and so on.We found that,compared with young people,serumlevel of ghrelin in older age-group reduced from （2.51±0.77）ng/ml to （1.43±0.64）ng/ml, p=0.009.Compared with middle-aged people （2.24₀.32）ng/ml,p=0.036,showedstatistically significant differences.The serum level of ghrelin decreased significantly withage.Further analysis indicated the positive linear correlation between ghrelin level and age,rp=-0.564,p=0.015.Compared with control group,serum level of ghrelin in obesity people（BMI≧28Kg/m²） reduced from （2.77±1.49）ng/ml to （1.87±0.89）ng/ml,and there aresignificant difference,p=0.047.After correction of age,serum level of ghrelin in CADpeople reduced from （3.00±0.29）ng/ml to （1.82±0.34）ng/ml,p=0.025,the differencebetween them was significant in statistics.Compared with control group,serum level ofghrelin in DM people reduced from （3.35±13.02）ng/ml to （2.41±0.74）ng/ml,and thedifference is significant,p=0.013.Conclusions Peripheral blood serum level of ghrelin show a tendency of decline with age.Serum level of ghrelin in people with obesity,CAD and DM would lead to further decrease.The data provide the natural phenomenon of ghrelin,which may be the basis of furtherstudy about the function of ghrelin.PartⅡThe progression of atherosclerosis could be retarded byghrelin via inhibiting the proinflammatory function of Th17 cellsBackground Recently,Th17 cell has been described as one neotype CD4+ T cell,which could produce interleukin-17 （IL-17） especially and serve as the important regulatorsof inflammatory diseases.IL-17 could promote the secretion of TNF-α,IL-6 frommononuclear macrophages;and by combining these two inflammatory factors,IL-17 couldenhance the inflammatory reaction.It’s well known that atherosclerosis is a chronicinflammatory disease,IL-17,TNF-αand IL-6,which close related with atherosclerosis,play an important role in the onset of Anorexia-Cachexia Syndrome.However,what is therelationship between ghrelin,the important appetite regulation hormone,and theinflammatory factors described above? Whether the protective effect of ghrelin on the progress of atherosclerosis is concerned with these inflammatory factors? Few documentshave described it.Objective To investigating the serum level of ghrelin and Th17 cells content and toobserve the changing of the inflammatory factors described above in the PBMCs afterbeing interfered by ghrelin.Study the mechanism of Ghrelin on retarding the developmentof atherosclerosis.Methods The objectives were divided into two groups after detecting the intima-mediathickness （IMT）,i.e.atherosclerosis （AS） group,including 37 patients （IMT≧1.2mm）and control group,including 31 patients （IMT＜1.2mm）.Flow cytometry was used to detectthe percentage of Th17 in CD4+T cells;Plasma concentrations of Ghrelin and Th17-relatedcytokines （IL-17、IL-6、TNF-α） were measured by enzyme-linked immunosorbentassay（ELISA）.Peripheral blood mononuclear cells （PBMCs） of patients with AS werecultured in vitro,and the concentration of inflammation factors,above-mentioned,weredetected after being interfered by Ghrelin.Results The percentage of Th17/CD4⁺ T was found to be significantly higher in AS group[（1.92±0.52）%] than that in control group [（0.59±0.32）%]（P＜0.05）.The concentration ofGhrelin markedly decreased in AS group [（2.19±0.84）ng/ml] （P＜0.05）;and Th17-relatedcytokines （IL-17、IL-6、TNF-α） markedly increased in AS group [（101.29±25.00）pg/ml,（7.67±3.58）pg/ml,（17.98±3.16）pg/ml] compared with control group [（76.97±27.64）pg/ml,（3.89±3.50）pg/ml,（6.38±1.27）pg/ml] （P＜0.05）;Ghrelin could inhibit the expression of thecytokines,and this inhibiting effect was in a dose-dependent manner （P＜0.05）.Therelationship between Ghrelin and IMT,cytokines were negative.The correlation coefficientbetween Ghrelin and IMT,IL-17、IL-6 TNF-αwere r=-0.498,-0.544,-0.363 and -0.565,respectively （P＜0.05）.Conclusion Ghrelin retards the development of AS via inhibiting the expression ofTh17-related cytokines. PartⅢEffects of Ghrelin on the Expression ofAcyl Coenzyme A:Cholesterol Acyltransferases-1 during Foam Cells FormationBackground Acyl-CoA:cholesterol acyltransferases （ACAT-1） could catalysis freecholesterol into cholesterol ester,and then form the foam cells.The foam cells formationwould be inhibited via down-regulating the expression of ACAT1.As we all know,ghrelincould inhibit the formation of foam cells via promoting the efflux of free cholesterol fromfoam cells by up-regulating the expression of ATP-binding cassette transporter A1.Therefore,if ghrelin has both functions about up-regulating ABCA1 and down-regulatingACAT1,the formation of foam cells would be inhibited.The further mechanism of ghrelinanti-atherosclerosis would be clarified.The data in this field has not been reported.Objective To investigate the effects of ghrelin on the expression of ACAT-1 and thecholesterol content of foam cells during the formation of foam cells.Methods The human monocytic leukemia cell line （THP-1） was chosen in our study.Thedifferentiation of THP-1 cells into macrophages was induced by phorbol 12-myristate13-acetate （PMA）.Macrophages were incubated with oxidized LDL （Ox-LDL） to generatefoam cells.The cells were divided into four groups to carry out control study:control group（Ox-LDL）,different concentration group of ghrelin (10^-5、10^-6、10^-7 mol/L).Ghrelin ofdifferent concentrations were treated for 2 hours before Ox-LDL（100mg/L） were added in.After being incubated for 24 hours,the cells medium were changed,and then apoA-Ⅰ（10mg/L） were added in with 0.3%BSA.After 12 hours incubation,the cells were collectedfor detection.The effect of variance of cholesterol content was measured by zymochemistryvia-fluorospectrophotometer,the lipid droplet content were observed by Oil red stainingmethod,the ACAT-1 protein and mRNA levels were detected by Western blotting andRT-PCR.Results Ghrelin could reduce the formation of lipid droplet of foam cells derived fromTHP-1 macrophages;Ghrelin reduced the content of cholesterol ester in foam cellsobviously,the cholesterol ester content of foam cells decreased （14.6±0.5）%,（28.3±1.5）%and （45.4±1.0）%,separately with increasing concentration of ghrelin.Further analysisindicated the positive linear correlation between the protein and mRNA expression of ACAT1 and cholesterol content,r=0.942,0.935,separately.ACAT-1 protein and mRNAlevels were also decreased.Ghrelin could reduce ACAT-1 protein mass and mRNA level ina dose-dependent manner.Not change was observed about the amount of lipid dropletstained by Oil Red O as time prolonging.The changing about the expression of ACAT1 wasnot significant,p＞0.05.Conclusion Ghrelin might retard the formation of atherosclerosis via down-regulating theexpression of ACAT-1.The inhibiting effect of ghrelin on the expression of ACAT1 was ina dose-dependent,not a time-dependent manner.更多还原