本文選題：激動 + HepG2�。� 參考：《首都醫(yī)科大學》2017年博士論文

【摘要】：背景動脈粥樣硬化(atherosclerosis,AS)引起的心腦血管疾病是引起人類死亡的頭號殺手。泡沫細胞的形成是AS早期的標志之一,也是AS發(fā)生、發(fā)展的關鍵步驟。泡沫細胞形成的主要原因是巨噬細胞內膽固醇流入和流出的失衡。因此,促進泡沫細胞內膽固醇流出能夠抑制泡沫細胞的形成,延緩動脈粥樣硬化病變的發(fā)生與發(fā)展。膽固醇逆轉運(RCT)是指HDL將多余的膽固醇從周圍組織(包括動脈粥樣斑塊)、細胞轉運到肝臟進行機體的再循環(huán)或以膽酸的形式排出體外,這一過程包括組織細胞膽固醇的流出至細胞外受體(如HDL)、膽固醇的酯化以及清除。近來,如何促進巨噬細胞RCT成為抗動脈粥樣硬化研究的熱點。β3-AR最早被發(fā)現(xiàn)于1980年,主要在白色和棕色脂肪組織表達,近年研究發(fā)現(xiàn)其在心臟、腦、肺臟及肝臟等組織也有表達。本課題組近年的研究發(fā)現(xiàn),激動Apo E-/-小鼠β3-AR能夠改善血脂代謝紊亂,降低TC、VLDL/LDL-C和TG,升高HDL;促進膽固醇經膽汁及糞便排出;縮小胸主動脈動脈粥樣硬化斑塊面積,由此推斷β3-AR可能通過促進膽固醇逆轉運起到抗動脈粥樣硬化作用。本研究使用β3-AR激動劑與拮抗劑刺激Hep G2細胞,觀察其上清液對RAW264.7巨噬細胞源性泡沫細胞內脂質及膽固醇流出的影響,探索β3-AR對RCT的影響及其可能機制,為臨床開發(fā)新型抗動脈硬化藥物提供理論依據。目的通過激動或抑制Hep G2細胞的β3-AR,觀察其分泌蛋白對巨噬細胞源性泡沫細胞荷脂過程的影響,探索β3-AR調節(jié)膽固醇逆轉運(RCT)過程的可能機制。方法使用β3-AR激動劑(BRL37344)和β3-AR拮抗劑(SR52390A)分別刺激培養(yǎng)的Hep G2細胞,Elisa法檢測上清液Apo A-I、Apo A-II、Apo B及β3-AR含量;實時定量PCR和蛋白印跡法分別檢測細胞內Apo A-I、Apo A-II、Apo B、β3-AR、PPARγ及PPARα含量;以ac-LDL(50μg/m L)誘導巨噬細胞源性泡沫細胞形成,收集Hep G2各組細胞上清液,加入巨噬細胞源性泡沫細胞共同孵育,采用油紅O染色進行細胞形態(tài)學觀察,酶法測定細胞內總膽固醇(TC)、游離膽固醇(FC)和膽固醇酯(CE)水平,3H標記的膽固醇測定膽固醇流出率,蛋白印跡法檢測細胞中ABCA1和ABCG1的表達。結果1、對Hep G2細胞進行Elisa檢測結果表明,與空白對照組相比,BRL37344組Apo A-I水平顯著升高(P0.01),SR59230A組Apo A-I水平顯著降低(P0.01);與空白對照組相比,BRL37344組Apo A-II含量顯著降低(P0.01),SR59230A組Apo A-II水平無顯著性差異(P0.05);Apo B水平在三組上清液中無顯著性差異(P0.05)。2、RT-PCR結果表明,與空白對照組相比,BRL37344組Apo A-I m RNA水平顯著升高(P0.01),SR59230A組Apo A-I m RNA水平顯著降低(P0.01);與空白對照組相比,BRL37344組Apo A-II m RNA水平顯著降低(P0.01),SR59230A組Apo A-II m RNA水平無顯著性差異(P0.05);與空白對照組相比,BRL37344組β3-AR m RNA水平顯著升高(P0.01),SR59230A組β3-AR m RNA水平顯著降低(P0.01);Apo B水平在三組中無顯著性差異(P0.05)。Apo A-I,Apo A-II和Apo B蛋白表達水平與m RNA表達情況相同。3、BRL37344刺激顯著上調Hep G2細胞中PPARγ的表達(P0.01),但不增加Hep G2細胞中PPARα的表達(P0.05),SR59230A刺激使Hep G2細胞中PPARγ的表達水平顯著下調(P0.01),對PPARα的表達無影響(P0.05)。BRL37344刺激引起的Apo AI蛋白表達上調效應可被PPARγ拮抗劑GW9662所阻斷(P0.01)。4、酶法檢測結果表明,與空白對照組相比,BRL37344組細胞內TC、CE水平顯著降低(P均0.05),SR59230A組細胞內TC、CE水平顯著升高(P均0.05);與空白對照組相比,BRL37344組細胞內FC水平顯著升高(P0.01),SR59230A組細胞內FC水平無顯著差異(P0.05)。5、與空白對照組相比,BRL37344刺激的Hep G2細胞上清液顯著上調巨噬細胞內ABCA1蛋白的表達(P0.01);SR59230A組ABCA1蛋白的表達無顯著性差異(P0.05);巨噬細胞內ABCG1蛋白表達在三組之間無顯著性差異(P0.05)。結論1.激動β3-AR明顯上調Hep G2細胞內Apo A-I蛋白水平表達;2.激動β3-AR顯著上調Hep G2細胞內PPARγ蛋白的表達;3.激動β3-AR對Hep G2細胞內Apo A-I蛋白表達的上調可能依賴PPARγ途徑;4.β3-AR激動后的Hep G2細胞上清液能夠促進巨噬細胞源性泡沫細胞內膽固醇流出。5.β3-AR激動后的Hep G2細胞上清液可能通過ABCA1途徑促進泡沫細胞內膽固醇流出。背景二級預防藥物的使用能夠為血運重建患者帶來臨床獲益,比較經皮冠狀動脈介入和冠狀動脈搭橋患者術后二級預防藥物的使用、血脂、血壓及血糖達標率及終點事件發(fā)生率情況的研究鮮見報道。目的通過對PCI和CABG術后患者二級預防藥物使用、血脂、血壓及血糖達標率及終點事件發(fā)生率的分析比較,探討PCI和CABG術后患者心血管相關危險因素控制情況是否存在差異及對預后的影響。方法回顧性分析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,從臨床電子病歷數(shù)據庫中提取資料,排除基線資料、用藥情況缺失患者7707例,最終有6523例(PCI=4728,CABG=1795)患者納入統(tǒng)計學分析。結果1.在未進行匹配的患者中,與CABG組相比,PCI組LDL-C1.8 mmol/L,LDL-C2.07 mmol/L及BP140/90mm Hg達標率更高(P均0.01),FBG和Hb A1C達標率在兩組之間無顯著性差異(P0.05)。傾向性評分匹配患者中LDL-C1.8 mmol/L、LDL-C2.07 mmol/L、BP140/90mm Hg、FBG及Hb A1C達標率與未匹配患者結果一致。2.在未匹配的PCI患者中,與年齡≥60歲的患者相比,年齡60歲的患者BP140/90mm Hg達標率顯著升高(P0.01),但LDL-C2.07mmol/L達標率顯著降低(P0.01);在未匹配CABG患者中,與年齡≥60歲的患者相比,年齡60歲的患者FBG7 mmol/L,Hb A1c7%及BP140/90mm Hg達標率顯著升高(P均0.01)(P0.05)。3.在未匹配的PCI患者中,與女性患者相比,男性患者LDL-C1.8 mmol/L,FBG7 mmol/L及Hb A1c7%達標率顯著升高(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%達標率均無顯著性差異(P均0.05)。4.在傾向性評分匹配后患者中,與CABG組相比,PCI組患者出院時使用他汀、ACEI/ARB、β受體阻滯劑的比例更高(P均0.01),合用依折麥布、貝特類降脂藥物的患者比例更高(P均0.01),兩組患者中阿司匹林使用率無顯著性差異(P0.05)。5.在未匹配患者,PCI組復合終點事件發(fā)生率顯著高于CABG組(P0.01);在傾向性評分匹配患者,PCI和CABG患者復合終點事件發(fā)生率無顯著性差異(P0.05)。多變量COX回歸分析發(fā)現(xiàn)LDL-C1.8 mmol/L和HBA1C7%是影響PCI和CABG患者復合終點事件發(fā)生的獨立預測因子,患者LDL-C1.8mmol/L和HBA1C7%達標與復合終點事件發(fā)生風險降低顯著相關。結論1.在總體和傾向性評分匹配的患者,PCI和CABG患者的血脂、血壓達標率均存在差異,兩組患者血脂、血糖及血壓達標率都不高;2.在PCI和CABG術后患者中,年齡60歲和年齡≥60歲患者血脂、血壓及血糖達標率存在差異,女性和男性患者血脂、血糖達標率存在差異;3.LDL-C1.8 mmol/L和HBA1C7%達標與復合終點事件發(fā)生風險降低顯著相關。
[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.
【學位授予單位】：首都醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R543.5

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7 孟華;;基于PCI總線的PC機再入遙測站平臺設計[A];中國工程物理研究院科技年報（2003）[C];2003年

8 陳燕;劉志虹;曾彩虹;黎磊石;;腎小球疾病患者腎間質泡沫細胞及其臨床意義[A];中華醫(yī)學會腎臟病學分會2006年學術年會論文集[C];2006年

9 王偉鈞;唐小我;楊晉浩;;滿足AR(1)需求過程的零售商庫存決策和牛鞭效應[A];第四屆中國智能計算大會論文集[C];2010年

10 施鴻毓;張紅旗;孫愛軍;姚康;黃榕，

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