【作者】 康静；

【导师】 成蓓；

【作者基本信息】 华中科技大学 ， 老年医学， 2010， 博士

【摘要】 背景内脂素(visfatin)是由内脏脂肪细胞高表达的一种脂肪细胞因子,与先前在淋巴细胞中发现的一种称为前B细胞克隆增强因子(PBEF)的蛋白结构一致,具有广泛的生物学效应。visfatin主要来源于脂肪组织中的巨噬细胞,同时在骨髓基质细胞、活化淋巴细胞、肝脏、脾脏、子宫、胸腺、胰腺、肌肉组织以及胎膜中均有表达。近年来的许多研究发现,visfatin与缺氧、易损斑块破裂、内皮功能紊乱、血管增生、炎症和糖脂代谢等密切相关,与动脉粥样硬化的发生发展过程密切相关。然而不同动脉粥样硬化性相关疾病患者血浆visfatin水平变化如何,本实验将做一探讨。目的通过检测动脉粥样硬化性相关疾病患者血浆visfatin水平,初步探讨血浆visfatin在动脉粥样硬化性相关疾病中的变化及其临床意义。方法将动脉粥样硬化性相关疾病(如冠心病、肥胖、代谢综合征、糖尿病、高血压等)患者和健康体检人群纳入研究对象,进行对照研究。通过酶联免疫吸附试验检测入选人群血浆visfatin水平,并同时抽血送检一般生化指标如空腹血糖(FPG)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C).低密度脂蛋白胆固醇(LDL-C)、C反应蛋白(CRP)、同型半胱氨酸(HCY)等,收集临床资料,分析一般临床特征,包括年龄、性别、身高、体重、BMI、血压等。结果①冠心病组(CAD组)血浆visfatin水平较对照组明显升高[(3.17±0.36)ng/ml] vs. [(0.64±0.18)ng/ml] (p<0.05); CAD组各亚组中,多支病变组血浆visfatin水平[(4.27±0.47)ng/ml]和双支病变组血浆visfatin水平[(3.16±0.39)ng/ml]明显高于单支病变组[(1.95±0.36)ng/ml] (p<0.05),多支病变组血浆visfatin水平较双支病变组升高[(4.27±0.47)ng/ml] vs. [(3.16±0.39)ng/ml] (p<0.05);CAD组血浆visfatin水平随颈动脉内膜中层厚度(IMT)的增加而升高(p<0.05)；CAD组冠状动脉病变支数越多,颈动脉粥样斑块发生率越高(p<0.05)。在CAD组,血浆visfatin水平与BMI、TG、LDL-C、CRP、HCY、IMT和冠状动脉病变支数呈明显正相关(P<0.05)。②单纯性肥胖组血浆visfatin水平[(2.03±0.29)ng/ml]和代谢综合征组(MS组)血浆visfatin水平[(4.47±0.37)ng/ml]明显高于对照组[(0.59±0.21)ng/ml] (p<0.05), MS组血浆visfatin水平高于单纯性肥胖组[(4.47±0.37)ng/ml] vs. [(2.03±0.29)ng/ml] (p<0.05);MS组血浆visfatin水平随MS组分数的增加而升高(P<0.05)。在MS组,血浆visfatin水平与BMI、腰围、TG、LDL-C、FPG和MS组分数呈明显正相关(p<0.05),与HDL-C呈负相关(p<0.05)。③糖耐量异常组(IGT组)血浆visfatin水平[(2.23±0.39)ng/ml]和糖尿病组(DM组)血浆visfatin水平[(4.17±0.38)ng/ml]明显高于对照组[(0.67±0.20)ng/ml](p<0.05),DM组血浆visfatin水平高于IGT组[(4.17±0.38)ng/ml]vs. [(2.23±0.39)ng/ml] (p<0.05)。在DM组,血浆visfatin水平与BMI、腰围、TG±FPG和HbA1C呈明显正相关(p<0.05)。④高血压组血浆visfatin水平[(1.17±0.36)ng/ml]和对照组[(0.97±0.48)ng/ml]比较,差异无显著性意义(p>0.05)。结论通过以上研究发现冠心病、肥胖、代谢综合征、糖耐量异常、糖尿病等动脉粥样硬化性相关疾病均可导致血浆visfatin水平升高,为进一步阐明visfatin在动脉粥样硬化发生发展过程中的作用奠定了一定的临床基础。visfatin的发现为研究动脉粥样硬化性相关疾病的发病机制增加了新的思路,为动脉粥样硬化性相关疾病的防治提供了一个新的靶点。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。泡沫细胞的形成是早期动脉粥样硬化的一个典型的病理特征,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与发展具有重要意义。巨噬细胞胆固醇代谢稳态的失衡贯穿于泡沫细胞形成的整个过程,参与巨噬细胞胆固醇代谢的两个关键酶,即酰基辅酶A：胆固醇酰基转移酶1 (Acyl-CoA:cholesterol acyltransferases, ACAT1)催化细胞内游离胆固醇形成胆固醇酯,ATP结合盒转运子A1 (ATP-binding cassette transporters Al, ABCA1)促进细胞内游离胆固醇的流出。若ACAT1表达上调,胞内胆固醇酯合成增加,ABCA1表达下调,胞内游离胆固醇流出减少,两者协同作用导致巨噬细胞胆固醇代谢稳态的失衡,从而促进泡沫细胞的形成。那么visfatin对参与胆固醇代谢的关键酶ACAT1和ABCA1的表达是否具有调控作用呢?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visfatin对酰基辅酶A：胆固醇酰基转移酶1(ACAT-1)和ATP结合盒转运子A1(ABCA1)表达的调控作用,明确visfatin致动脉粥样硬化的发病机制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,巨噬细胞在氧化低密度脂蛋白(ox-LDL)作用下进一步转变为泡沫细胞。实验分为两部分：①不同浓度visfatin干预组(4组)：即泡沫细胞对照组(ox-LDL)、不同浓度visfatin干预组(10-5 mol/L、10-6 mol/L、10-7mol/L)。巨噬细胞分别加入上述不同浓度的visfatin,预孵2h后再加入ox-LDL 100mg/L,作用24h。采用油红O染色法观察泡沫细胞内脂滴形成情况,RT-PCR法检测ACAT1 mRNA、ABCA1 mRNA水平,Western-blot法检测ACAT1蛋白、ABCA1蛋白表达。②不同时间visfatin干预组(4组)：即泡沫细胞对照组(ox-LDL).不同时间干预组(12h、24h、48h)。巨噬细胞加入visfatin 10-5 mol/L,预孵2h后再加入ox-LDL 100mg/L,分别作用12h、24h、48h。采用油红O染色法观察泡沫细胞内脂滴形成情况,RT-PCR法检测ACAT1 mRNA、ABCA1 mRNA水平,Western-blot法检测ACAT1蛋白、ABCA1蛋白表达。结果①与泡沫细胞对照组相比,visfatin干预组油红O染色显示脂滴形成增加,ACAT1 mRNA水平及蛋白表达显著升高(p<0.05), ABCA1 mRNA水平及蛋白表达显著降低(p<0.05)；②不同浓度visfatin干预组之间比较,随着visfatin浓度越高,ACAT1 mRNA水平及蛋白表达越高(p<0.05), ABCA1 mRNA水平及蛋白表达越低(p<0.05)；提示visfatin上调ACAT1 mRNA及蛋白表达,下调ABCA1 mRNA及蛋白表达,且上述效应呈浓度依赖性；③不同时间visfatin干预组之间比较,随着visfatin作用时间越长,ACAT1 mRNA水平及蛋白表达越高(p<0.05), ABCA1 mRNA水平及蛋白表达降低(24h组vs.12h组,p<0.05),但在48h组ABCA1 mRNA水平及蛋白表达略高于24h组。提示visfatin上调ACAT1表达呈时间依赖性,下调ABCA1表达不完全呈时间依赖性。结论visfatin可明显增加THP-1源性泡沫细胞内脂滴的形成,增加THP-1源性泡沫细胞形成过程中ACAT1 mRNA水平和蛋白表达,减少ABCA1 mRNA水平和蛋白表达。提示visfatin可在转录及翻译水平通过上调ACAT1表达,增加胞内胆固醇合成,同时下调ABCA1表达,抑制胞内游离胆固醇流出,两者协同作用促进泡沫细胞形成。背景过氧化物酶体增生物激活受体y (peroxisome proliferator activated-receptor gamma, PPARy)属于转录因子核受体超家族的一员,其被证实能在巨噬细胞核内表达,并能通过调控脂蛋白酶和CD36等基因表达而调节脂质稳态。多项对特异性敲除巨噬细胞中PPARy小鼠的研究发现,动脉壁的脂质稳态严重受损,并促进动脉粥样硬化的发展,证实PPARy在抗动脉粥样硬化中发挥着重要作用[3,4]。同时PPARy信号通路参与调控巨噬细胞内胆固醇代谢,通过对其下游靶基因,包括ACAT1和ABCA1的调控,维持胆固醇代谢的稳态。我们前期研究证实visfatin能上调ACAT1的表达,下调ABCA1的表达,导致巨噬细胞源性泡沫细胞的形成,那么PPARy信号通路是否参与了visfatin对ACAT1和ABCA1表达的调控作用呢?因此,在本实验中我们将观察PPARy信号通路在visfatin诱导泡沫细胞形成中的作用。目的探讨PPARy信号通路在visfatin诱导泡沫细胞形成中的作用。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,巨噬细胞在氧化低密度脂蛋白(ox-LDL)作用下进一步转变为泡沫细胞。实验分为两部分：①不同浓度visfatin干预组(4组)：即泡沫细胞对照组(ox-LDL)、不同浓度visfatin干预组(10-5 mol/L、10-6 mol/L、10-7mol/L)。巨噬细胞分别加入上述不同浓度的visfatin,预孵2h后再加入ox-LDL 100mg/L,作用24h。采用油红O染色法观察泡沫细胞内脂滴形成情况,RT-PCR法检测PPARy mRNA水平,Western-blot法检测PPARγ蛋白表达。②不同时间visfatin干预组(4组)：即泡沫细胞对照组(ox-LDL)、不同时间visfatin干预组(12h、24h、48h)。巨噬细胞加入visfatin 10-5 mol/L,预孵2h后再加入ox-LDL 100mg/L,分别作用12h、24h、48h。采用油红O染色法观察细胞内脂形成情况,RT-PCR法检测PPARy mRNA水平,Western-blot法检测PPARy蛋白表达。结果①与泡沫细胞对照组比较,visfatin干预组油红O染色显示脂滴形成增加,PPARy mRNA水平和蛋白表达明显降低(p<0.05)；②不同浓度visfatin干预组之间比较,随着visfatin浓度越高,PPARy mRNA及蛋白水平越低(p<0.05),提示visfatin下调PPARy mRNA及蛋白表达,且该效应呈浓度依赖性；③不同时间visfatin干预组之间比较,随着visfatin作用时间延长,24h组PPARγmRNA水平和蛋白表达较12h组降低(p<0.05),但在48h组PPARγmRNA水平和蛋白表达高于12h和24h组,提示visfatin下调PPARγ表达不完全呈时间依赖性。结论visfatin可明显增加THP-1源性泡沫细胞内脂滴的形成,降低THP-1源性泡沫细胞形成过程中PPARγmRNA水平和蛋白表达。提示visfatin可在转录及翻译水平下调PPARγ表达,表明PPARγ信号通路参与了visfatin诱导泡沫细胞的形成。更多还原

【Abstract】 Background Visfatin is a newly found novel adipokine having high expression in adipocyte. Its structure is the same as the previously identified human pre-B-cell colony enhancing factor (PBEF)in the Lymphoid System. Macrophages of adipose tissue is principle source. It is widely distributed in bone marrow stromal cells(BMSC), activated lymphocyte, liver, spleen, pancreas, uterus,thymus gland,muscle tissues,caul and so on. Vsfatin is related to oxygend eficit, vulnerable plaque rupture, endothelial disfunction, angiogenesis, inflammation and the metabolism of glucose and lipid. However, the exactly serum level of visfatin in different patients with atherosclerosis-related diseases and their correlation have not been clarified.Objective Investigating the serum level of visfatin in different patients and observing the relationship between the variation of visfatin and atherosclerosis-related diseases. Methods A comparative study was performed in patients with atherosclerosis-related diseases,such as coronary artery disease (CAD), carotid artery atherosclerosis, obesity, metabolism syndrome(MS), diabetes mellitus (DM) and Hypertension. Enzyme linked immunosorbent assay was used to detect plasma levels of visfatin, at the same time, generally selected biochemical parameters such as the plasma glucose(FPG), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), C-reactive protein(CRP) and homocysteine(HCY) were detected with submitted blood. Collecting clinical data, analyzing the general clinical feature such as age, sex, body mass index (BMI), blood pressure and so on.Results①The plasma levels of visfatin were significantly higher in the CAD group than in the controls [(3.17±0.36)ng/ml] vs. [(0.64±0.18)ng/ml] (p<0.05). In the CAD group,the higher extent of coronary artery lesions was,the higher the plasma visfatin (p<0.05); the greater the IMT was, the higher the plasma visfatin (p<0.05); the higher extent of coronary artery lesions was,the higher icedent of carotid atherosclerosis (p<0.05). In the CAD group, the plasma levels of visfatin were positively correlated with BMI, TG, LDL-C, CRP,HCY,IMT and the extent of coronary artery lesions (p<0.05).②The plasma levels of visfatin were significantly higher in the simple obesity group [(2.03±0.29)ng/ml] and MS group [(4.47±0.37)ng/ml] than in the controls [(0.59±0.21)ng/ml] (p<0.05). The plasma levels of visfatin were higher in the MS group than in the simple obesity group [(4.47±0.37)ng/ml] vs. [(2.03±0.29)ng/ml](p<0.05). In the MS group,the more ingredients of MS were,the higher the plasma visfatin (p<0.05), the plasma levels of visfatin were positively correlated with BMI, waistline, FPG, TG, LDL-C and the ingredients of MS(p<0.05), and negtively correlated with HDL-C(p<0.05).③The plasma levels of visfatin were significantly higher in the IGT group [(2.23±0.39)ng/ml] and the DM group [(4.17±0.38)ng/ml] than in the controls [(0.67±0.20)ng/ml] (p<0.05). The plasma levels of visfatin were higher in the DM group than in the IGT group[(4.17±0.38)ng/ml] vs. [(2.23±0.39)ng/ml](p<0.05). In the DM group, the plasma levels of visfatin were positively correlated with BMI, waistline, FPG, TG and HbA1C (p<0.05).④Comparison of Hypertension group and the controls, the plasma levels of visfatin were not significant changed [(1.17±0.36)ng/ml] vs. [(0.97±0.48)ng/ml] (p>0.05).Conclusions Serum level of visfatin in people with CAD,carotid artery atherosclerosis, obesity, MS and DM would lead to further increase. The data provide the natural phenomenon of visfatin, which may be the basis of further study about the function of visfatin. Visfatin may play an important role in the progress of atherosclerosis.The finding of visfatin will provide new idea in studying the atherosclerosis-related diseases, which will provide new target for the atherosclerosis-related diseases therapy.Background Acyl-CoA:cholesterol acyltransferases (ACAT1) could catalysis free cholesterol into cholesterol ester, and then form the foam cells. The foam cells formation would be promoted via up-regulating the expression of AC AT1. ATP-binding cassette transporter A1(ABCA1) could promote the efflux of free cholesterol from foam cells, and then inhibit the foam cells. The foam cells formation would be promoted via down-regulating the expression of ABCA1. Therefore, if visfatin has both functions about up-regulating ACAT1 and down-regulating ABCA1, the formation of foam cells would be promoted. The further mechanism of visfatin induce-atherosclerosis would be clarified. The data in this field has not been reported.Objective To investigate the effects of visfatin on the expression of ACAT1 and ABCA1 during the formation of 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-myristate 13-acetate (PMA). Macrophages were incubated with oxidized LDL (ox-LDL) to generate foam cells. The experiment divided into two parts:①The cells were divided into four groups:control group (ox-LDL), different concentration groups of visfatin (10-5 mol/L、10-6 mol/L、10-7 mol/L). Visfatin of different 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, the lipid droplet content were observed by Oil red staining method, the levels of ACAT1 mRNA and ABCA1 mRNA were detected by RT-PCR,the levels of ACAT1 protein and ABCA1 protein were detected by Western blotting.②The cells were divided into four groups:control group (ox-LDL), different time groups of visfatin (12h, 24h,48h). The concentration of visfatin(10-5 mol/L) were treated for 2 hours before ox-LDL(100mg/L) were added in. After being incubated for 12 hours,24 hours,48hours, the cells medium were changed, the lipid droplet content were observed by Oil red staining method, the ACAT1 mRNA and ABCA1 mRNA levels were detected by RT-PCR, the ACAT1 and ABCA1 protein levels were detected by Western blotting.Results①Comparison of control, in different concentration and time groups of visfatin, the lipid droplet contents were increased by Oil red staining method, the levels of ACAT1 mRNA and protein were increased (p<0.05), the levels of ABCA1 mRNA and protein were decreased (p<0.05).②Comparison of different concentration groups of visfatin, as concentration increasing, the levels of ACAT1 mRNA and protein were increased (p<0.05), the levels of ABCA1 mRNA and protein were decreased (p<0.05).These changes were in a dose-dependent manner.③Comparison of different time groups of visfatin, as time prolonging, the lipid droplet contents were changed by Oil red staining method, the levels of ACAT1 mRNA and protein were increased (p<0.05), these changes were in a time-dependent manner. The levels of ABCA1 mRNA and protein were decreased (24h group vs.12h group, p<0.05).Conclusion Visfatin could increase the formation of lipid droplet of foam cells derived from THP-1 macrophages. Visfatin might induced the formation of atherosclerosis via up-regulating the expression of ACAT-1 and down-regulating the expression of ABCA-1. Background Peroxisome proliferator-activated receptorγ(PPARγ) are key nuclear receptors that regulate macrophage cholesterol metabolism.Objective To investigate the roles of PPARγpathway during the formation of foam cells by visfatin induced.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-myristate 13-acetate (PMA). Macrophages were incubated with oxidized LDL (ox-LDL) to generate foam cells. The experiment divided into two parts:①The cells were divided into four groups:control group (ox-LDL), different concentration groups of visfatin (10-5mol/L、10-6 mol/L、10-7 mol/L). Visfatin of different 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, the lipid droplet content were observed by Oil red staining method, the levels of PPARγmRNA was detected by RT-PCR,the levels of PPARγprotein was detected by Western blotting.②The cells were divided into four groups:control group (ox-LDL), different time groups of visfatin (12h,24h,48h). The concentration of visfatin(10-5 mol/L) were treated for 2 hours before ox-LDL(100mg/L) were added in. After being incubated for 12 hours,24 hours,48hours, the cells medium were changed, the lipid droplet content were observed by Oil red staining method, the levels of PPARγmRNA was detected by RT-PCR,the levels of PPARγprotein was detected by Western blotting.Results①Comparison of control, in different concentration and time groups of visfatin, the lipid droplet contents were increased by Oil red staining method, the levels of PPARγmRNA and protein were decreased (p<0.05).②Comparison of different concentration groups of visfatin, as concentration increasing, the lipid droplet contents were increased by Oil red staining method, the levels of PPARγmRNA and protein were decreased (p<0.05).These changes were in a dose-dependent manner.③Comparison of different time groups of visfatin, as time prolonging, the levels of PPARγmRNA and protein were decreased (24h group vs.12h group, p<0.05).Conclusion Visfatin could increase the formation of lipid droplet of foam cells derived from THP-1 macrophages. Visfatin might induced the formation of atherosclerosis via PPARγpathway.更多还原