激动HepG2细胞β3-AR对泡沫细胞荷脂过程影响及PCI和CABG术后患者心血管危险因素达标现状的研究

发布时间：2018-06-14 23:13

本文选题：激动 + HepG2　；参考：《首都医科大学》2017年博士论文

【摘要】：背景动脉粥样硬化(atherosclerosis,AS)引起的心脑血管疾病是引起人类死亡的头号杀手。泡沫细胞的形成是AS早期的标志之一,也是AS发生、发展的关键步骤。泡沫细胞形成的主要原因是巨噬细胞内胆固醇流入和流出的失衡。因此,促进泡沫细胞内胆固醇流出能够抑制泡沫细胞的形成,延缓动脉粥样硬化病变的发生与发展。胆固醇逆转运(RCT)是指HDL将多余的胆固醇从周围组织(包括动脉粥样斑块)、细胞转运到肝脏进行机体的再循环或以胆酸的形式排出体外,这一过程包括组织细胞胆固醇的流出至细胞外受体(如HDL)、胆固醇的酯化以及清除。近来,如何促进巨噬细胞RCT成为抗动脉粥样硬化研究的热点。β3-AR最早被发现于1980年,主要在白色和棕色脂肪组织表达,近年研究发现其在心脏、脑、肺脏及肝脏等组织也有表达。本课题组近年的研究发现,激动Apo E-/-小鼠β3-AR能够改善血脂代谢紊乱,降低TC、VLDL/LDL-C和TG,升高HDL;促进胆固醇经胆汁及粪便排出;缩小胸主动脉动脉粥样硬化斑块面积,由此推断β3-AR可能通过促进胆固醇逆转运起到抗动脉粥样硬化作用。本研究使用β3-AR激动剂与拮抗剂刺激Hep G2细胞,观察其上清液对RAW264.7巨噬细胞源性泡沫细胞内脂质及胆固醇流出的影响,探索β3-AR对RCT的影响及其可能机制,为临床开发新型抗动脉硬化药物提供理论依据。目的通过激动或抑制Hep G2细胞的β3-AR,观察其分泌蛋白对巨噬细胞源性泡沫细胞荷脂过程的影响,探索β3-AR调节胆固醇逆转运(RCT)过程的可能机制。方法使用β3-AR激动剂(BRL37344)和β3-AR拮抗剂(SR52390A)分别刺激培养的Hep G2细胞,Elisa法检测上清液Apo A-I、Apo A-II、Apo B及β3-AR含量;实时定量PCR和蛋白印迹法分别检测细胞内Apo A-I、Apo A-II、Apo B、β3-AR、PPARγ及PPARα含量;以ac-LDL(50μg/m L)诱导巨噬细胞源性泡沫细胞形成,收集Hep G2各组细胞上清液,加入巨噬细胞源性泡沫细胞共同孵育,采用油红O染色进行细胞形态学观察,酶法测定细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)水平,3H标记的胆固醇测定胆固醇流出率,蛋白印迹法检测细胞中ABCA1和ABCG1的表达。结果1、对Hep G2细胞进行Elisa检测结果表明,与空白对照组相比,BRL37344组Apo A-I水平显著升高(P0.01),SR59230A组Apo A-I水平显著降低(P0.01);与空白对照组相比,BRL37344组Apo A-II含量显著降低(P0.01),SR59230A组Apo A-II水平无显著性差异(P0.05);Apo B水平在三组上清液中无显著性差异(P0.05)。2、RT-PCR结果表明,与空白对照组相比,BRL37344组Apo A-I m RNA水平显著升高(P0.01),SR59230A组Apo A-I m RNA水平显著降低(P0.01);与空白对照组相比,BRL37344组Apo A-II m RNA水平显著降低(P0.01),SR59230A组Apo A-II m RNA水平无显著性差异(P0.05);与空白对照组相比,BRL37344组β3-AR m RNA水平显著升高(P0.01),SR59230A组β3-AR m RNA水平显著降低(P0.01);Apo B水平在三组中无显著性差异(P0.05)。Apo A-I,Apo A-II和Apo B蛋白表达水平与m RNA表达情况相同。3、BRL37344刺激显著上调Hep G2细胞中PPARγ的表达(P0.01),但不增加Hep G2细胞中PPARα的表达(P0.05),SR59230A刺激使Hep G2细胞中PPARγ的表达水平显著下调(P0.01),对PPARα的表达无影响(P0.05)。BRL37344刺激引起的Apo AI蛋白表达上调效应可被PPARγ拮抗剂GW9662所阻断(P0.01)。4、酶法检测结果表明,与空白对照组相比,BRL37344组细胞内TC、CE水平显著降低(P均0.05),SR59230A组细胞内TC、CE水平显著升高(P均0.05);与空白对照组相比,BRL37344组细胞内FC水平显著升高(P0.01),SR59230A组细胞内FC水平无显著差异(P0.05)。5、与空白对照组相比,BRL37344刺激的Hep G2细胞上清液显著上调巨噬细胞内ABCA1蛋白的表达(P0.01);SR59230A组ABCA1蛋白的表达无显著性差异(P0.05);巨噬细胞内ABCG1蛋白表达在三组之间无显著性差异(P0.05)。结论1.激动β3-AR明显上调Hep G2细胞内Apo A-I蛋白水平表达;2.激动β3-AR显著上调Hep G2细胞内PPARγ蛋白的表达;3.激动β3-AR对Hep G2细胞内Apo A-I蛋白表达的上调可能依赖PPARγ途径;4.β3-AR激动后的Hep G2细胞上清液能够促进巨噬细胞源性泡沫细胞内胆固醇流出。5.β3-AR激动后的Hep G2细胞上清液可能通过ABCA1途径促进泡沫细胞内胆固醇流出。背景二级预防药物的使用能够为血运重建患者带来临床获益,比较经皮冠状动脉介入和冠状动脉搭桥患者术后二级预防药物的使用、血脂、血压及血糖达标率及终点事件发生率情况的研究鲜见报道。目的通过对PCI和CABG术后患者二级预防药物使用、血脂、血压及血糖达标率及终点事件发生率的分析比较,探讨PCI和CABG术后患者心血管相关危险因素控制情况是否存在差异及对预后的影响。方法回顾性分析2014年1月1日至2014年12月31日在我院行PCI和CABG患者共14230例,从临床电子病历数据库中提取资料,排除基线资料、用药情况缺失患者7707例,最终有6523例(PCI=4728,CABG=1795)患者纳入统计学分析。结果1.在未进行匹配的患者中,与CABG组相比,PCI组LDL-C1.8 mmol/L,LDL-C2.07 mmol/L及BP140/90mm Hg达标率更高(P均0.01),FBG和Hb A1C达标率在两组之间无显著性差异(P0.05)。倾向性评分匹配患者中LDL-C1.8 mmol/L、LDL-C2.07 mmol/L、BP140/90mm Hg、FBG及Hb A1C达标率与未匹配患者结果一致。2.在未匹配的PCI患者中,与年龄≥60岁的患者相比,年龄60岁的患者BP140/90mm Hg达标率显著升高(P0.01),但LDL-C2.07mmol/L达标率显著降低(P0.01);在未匹配CABG患者中,与年龄≥60岁的患者相比,年龄60岁的患者FBG7 mmol/L,Hb A1c7%及BP140/90mm Hg达标率显著升高(P均0.01)(P0.05)。3.在未匹配的PCI患者中,与女性患者相比,男性患者LDL-C1.8 mmol/L,FBG7 mmol/L及Hb A1c7%达标率显著升高(P均0.01);在未匹配的CABG患者中,女性和男性性LDL-C1.8 mmol/L,LDL-C2.07 mmol/L,BP140/90mm Hg,FBG7 mmol/L及Hb A1c7%达标率均无显著性差异(P均0.05)。4.在倾向性评分匹配后患者中,与CABG组相比,PCI组患者出院时使用他汀、ACEI/ARB、β受体阻滞剂的比例更高(P均0.01),合用依折麦布、贝特类降脂药物的患者比例更高(P均0.01),两组患者中阿司匹林使用率无显著性差异(P0.05)。5.在未匹配患者,PCI组复合终点事件发生率显著高于CABG组(P0.01);在倾向性评分匹配患者,PCI和CABG患者复合终点事件发生率无显著性差异(P0.05)。多变量COX回归分析发现LDL-C1.8 mmol/L和HBA1C7%是影响PCI和CABG患者复合终点事件发生的独立预测因子,患者LDL-C1.8mmol/L和HBA1C7%达标与复合终点事件发生风险降低显著相关。结论1.在总体和倾向性评分匹配的患者,PCI和CABG患者的血脂、血压达标率均存在差异,两组患者血脂、血糖及血压达标率都不高;2.在PCI和CABG术后患者中,年龄60岁和年龄≥60岁患者血脂、血压及血糖达标率存在差异,女性和男性患者血脂、血糖达标率存在差异;3.LDL-C1.8 mmol/L和HBA1C7%达标与复合终点事件发生风险降低显著相关。
[Abstract]:The cardiovascular and cerebrovascular diseases caused by atherosclerosis (AS) are the leading killer of human death. The formation of foam cells is one of the early signs of AS, and is also a key step for the development of AS. The main reason for the formation of foam cells is the imbalance of cholesterol inflow and outflow in macrophages. Therefore, foamer promotes foam. Intracellular cholesterol efflux inhibits the formation of foam cells and delays the occurrence and development of atherosclerotic lesions. The reverse cholesterol transport (RCT) refers to the process that HDL transfers excess cholesterol from the surrounding tissue (including atherosclerotic plaques) to the liver for the recirculation of the body or in the form of cholic acid. The flow of cholesterol in tissue cells to extracellular receptors (such as HDL), esterification and clearance of cholesterol. Recently, how to promote macrophage RCT has become a hot spot in anti atherosclerotic research. Beta 3-AR was first found in 1980, mainly in white and brown adipose tissue, and in recent years the study found its tissues in the heart, brain, lungs and liver. In recent studies, we have found that the excited Apo E-/- mouse beta 3-AR can improve blood lipid metabolism disorder, reduce TC, VLDL/LDL-C and TG, increase HDL, promote the excretion of cholesterol through bile and feces, reduce the area of atherosclerotic plaques in the thoracic aorta, and deduce that the beta 3-AR may be transported to the anti artery by promoting the reversal of cholesterol. This study used beta 3-AR agonist and antagonist to stimulate Hep G2 cells to observe the effect of supernatant on lipid and cholesterol efflux in RAW264.7 macrophage derived foam cells, explore the effect of beta 3-AR on RCT and its possible mechanism, and provide a theoretical basis for the development of a new type of anti arteriosclerosis drugs. The effect of the secretory protein on the lipid process of macrophage derived foam cells was observed by moving or inhibiting the Hep G2 cells, and the possible mechanism of the regulation of the cholesterol reverse transport (RCT) by beta 3-AR was explored. Methods the Hep G2 cells were stimulated by beta 3-AR agonist (BRL37344) and beta 3-AR antagonist (SR52390A), and the Elisa method was used to detect the supernatant. A-I, Apo A-II, Apo B and beta 3-AR content. Real-time quantitative PCR and Western blotting were used to detect the intracellular Apo A-I, Apo A-II, Apo purposes, beta, gamma and alpha content, and to induce macrophage derived foam cells by 50 micron. The staining was observed by cell morphology, the levels of intracellular total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE), 3H labeled cholesterol to determine the cholesterol efflux rate, and the expression of ABCA1 and ABCG1 in the cells were detected by Western blot. Results 1, Elisa detection of Hep G2 cells showed that compared with the blank control group, BRL373 was compared with the control group, BRL373. The level of Apo A-I in the 44 groups was significantly higher (P0.01), and the level of Apo A-I in the group SR59230A decreased significantly (P0.01), and the Apo A-II content in the BRL37344 group was significantly lower than that in the blank control group (P0.01). Compared with the group BRL37344, the level of Apo A-I m RNA increased significantly (P0.01), and Apo A-I m RNA level in SR59230A group decreased significantly (P0.01), and there was no significant difference between the group and the blank control group. The level of beta 3-AR m RNA in group SR59230A decreased significantly (P0.01), and there was no significant difference in Apo B level in the three groups (P0.05).Apo A-I. 05) SR59230A stimulation reduced the expression level of PPAR gamma in Hep G2 cells significantly (P0.01). The expression of Apo AI protein induced by P0.05.BRL37344 stimulation could be blocked by PPAR gamma antagonist GW9662. In group SR59230A, the level of TC and CE increased significantly (P 0.05). Compared with the blank control group, the level of FC in the BRL37344 group increased significantly (P0.01), and there was no significant difference in the level of FC in the SR59230A group (P0.05).5. Compared with the blank control group, the cell supernatant significantly up-regulated the macrophage protein. There was no significant difference in the expression of ABCA1 protein in group SR59230A (P0.05), and there was no significant difference between the three groups of ABCG1 protein expression in the three groups (P0.05). Conclusion 1. excitated beta 3-AR obviously up-regulated the expression of Apo A-I protein in Hep G2 cells, and 2. excited beta 3-AR significantly up-regulated the expression of protein in the cells; 3. The up-regulated expression of Apo A-I protein in Hep G2 cells may depend on the PPAR gamma pathway, and the Hep G2 cell supernatant after 4. beta 3-AR can promote the cholesterol efflux.5. beta 3-AR excitated in the macrophage derived foam cells, and the Hep G2 cell supernatant may promote the flow of cholesterol in the foam cell by means of the pathway. Background level two preventive drugs The use of two levels of preventive drugs after percutaneous coronary intervention and coronary artery bypass surgery in patients with coronary artery bypass and coronary artery bypass surgery is rarely reported. The purpose of this study is to report on the rate of blood lipid, blood pressure, blood glucose standard and the incidence of endpoint events. The purpose of this study is to use the two level prophylaxis for patients after PCI and CABG. The analysis and comparison of the rate of blood pressure and blood glucose standard and the incidence of endpoint events were compared to investigate whether there was a difference in the control of cardiovascular risk factors in patients with PCI and CABG and the impact on the prognosis. Methods a retrospective analysis of 14230 cases of PCI and CABG patients in our hospital from January 1, 2014 to December 31, 2014, from the clinical electronic medical record database, was reviewed. The data were extracted, the baseline data were excluded, 7707 cases of drug use were missing, and 6523 patients (PCI=4728, CABG=1795) were finally included in the statistical analysis. Results 1. of the patients who were not matched, the PCI group LDL-C1.8 mmol/L, LDL-C2.07 mmol/L and BP140/ 90mm Hg were higher (P both 0.01), and two groups were in the two groups. There was no significant difference (P0.05). The rate of LDL-C1.8 mmol/L, LDL-C2.07 mmol/L, BP140/90mm Hg, FBG and Hb A1C in the tendency score matched patients were consistent with the unmatched patients in the unmatched PCI patients. Compared with those aged 60 years old, the rate of 60 years old was significantly higher. The rate of mol/L standard was significantly lower (P0.01); in patients with unmatched CABG, the rate of FBG7 mmol/L, Hb A1c7% and BP140/90mm Hg (P 0.01) was significantly higher than those aged 60 years old (P0.05) (P0.05) (P0.05). Significantly increased (P 0.01); in unmatched CABG patients, there was no significant difference between female and male sex LDL-C1.8 mmol/L, LDL-C2.07 mmol/L, BP140/90mm Hg, FBG7 mmol/L and Hb A1c7% (all 0.05). The proportion of the patients was higher (P 0.01). The proportion of the combination of ezebeb and beet lipid lowering drugs was higher (P 0.01). There was no significant difference in the use rate of aspirin in the two groups (P0.05).5. in the unmatched patients, the incidence of compound end events in the PCI group was significantly higher than that in the CABG group (P0.01); the compound end point of the patients with PCI and CABG in the tendency score matched patients, and the patients with PCI and CABG. There was no significant difference in the incidence of events (P0.05). Multivariate COX regression analysis found that LDL-C1.8 mmol/L and HBA1C7% were independent predictors of complex terminal events in PCI and CABG patients. Patients' LDL-C1.8mmol/L and HBA1C7% standards were significantly correlated with the risk reduction of complex endpoint events. Conclusion 1. matched the overall and tendency score. Patients, PCI and CABG patients with blood lipids, blood pressure standard rates are different, two groups of blood lipids, blood sugar and blood pressure standard rate is not high; 2. in PCI and CABG patients, age 60 years old and age 60 years old patients blood lipid, blood pressure and blood glucose standard rate difference, female and male patients blood lipid, blood sugar standard rate difference exists, 3.LDL-C1.8 mmol/L And HBA1C7% compliance is significantly related to the risk reduction of composite endpoint events.
【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R543.5

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