血清高密度脂蛋白亚类1对人单核源性巨噬细胞泡沫化的影响

目的 探讨血清高密度脂蛋白(HDL)亚类HDL1对人外周血单核源性巨噬细胞泡沫化的影响,认识其在HDL抗动脉粥样硬化(As)中所发挥的作用.方法 采用分段浓度聚丙烯酰胺凝胶电泳(sd-PAGE)法同步分离和定最制备血清HDL1.采用密度梯度离心法和塑料吸附法从人外周血中分离单核细胞,以50 nmol/L佛波酯(PMA)刺激48 h使之转化为巨噬细胞.细胞分为对照组与实验组,以HDL1干预后分别测定细胞内总胆固醇(TC)、游离胆固醇(FC)与蛋白质的含量,观察HDL1对细胞内TC/蛋白比值影响的量效关系和时效关系.结果 成功地建立了人单核源性泡沫细胞的模型,采用sd-PAGE法有效地从血清中分离出HDL1.经不同浓度(0、0.1、1.0和10.0 ms/ L)的HDL1作用后,细胞中TC/蛋白的比值随HDL1浓度的增加而旱剂茸依赖性降低(由36.9±1.1下降至6.2±0.4,P<0.01).细胞的泡沫化程度也随着HDL1浓度的升高而降低,且在浓度(10.0 mg/ L)一定的情况下,细胞中TC/ 蛋白比值随HDL1处理时间(6～24 h)的延长而呈时间依赖性降低(6 h:16.9±0.9,24 h:6.4±0.6,P<0.01).结论 HDL1通过降低细胞内TC的含量,一定程度上减轻了巨噬细胞的泡沫化程度,从而在HDL抗As的过程中发挥作用.     

不对称二甲基精氨酸对THP-1巨噬细胞源性泡沫细胞内胆固醇含量的影响

目的 观察不对称二甲基精氨酸（ADMA）对THP-1巨噬细胞源性泡沫细胞内胆固醇含量的影响.方法 THP-1单核细胞给予160nmol/L佛波酯（PMA）孵育48h,诱导分化成巨噬细胞,与80mg/L氧化型低密度脂蛋白（ox-LDL）孵育24h,使巨噬细胞转化为泡沫细胞,用油红O染色在光镜下鉴定泡沫细胞形态及变化.再以不同浓度ADMA（1μmol/L、5μmol/L、10μmol/L、20μmol/L、30μmol/L）作用泡沫细胞24h,以20μmol/L ADMA作用泡沫细胞不同时间（0h,6h,12h,24h,48h）,酶比色法检测泡沫细胞内胆固醇的含量.结果 ①单核细胞加人PMA诱导48h后,分化为巨噬细胞,再经ox-LDL诱导24h后转化为泡沫细胞.②与对照组相比,5μmol/L、10μmol/L、20μmol/L,30μmol/L的ADMA作用于,THP-1巨噬细胞源性泡沫细胞24h后,细胞内胆固醇含量均明显增加（P＜0.01）,③与对照组相比,20μmol/L的ADMA作用于THP-1巨噬细胞源性泡沫细胞12h,24h,48h,细胞内胆固醇含量均明显增加（P＜0.01）.结论 ①单核细胞株THP-1经PMA诱导后,可分化为巨噬细胞,再经ox-LDL诱导后,可转化为泡沫细胞.②ADMA可以增加THP-1巨噬细胞源性泡沫细胞内胆固醇的含量.     

罗格列酮对THP-1源性泡沫细胞内胆固醇和氧化型低密度脂蛋白及丙二醛水平的影响

目的 观察罗格列酮干预THP-1源性泡沫细胞内胆固醇和氧化型低密度脂蛋白(ox-LDL)及丙二醛(MDA)水平的变化及其防治动脉粥样硬化的可能机制.方法 培养THP-1细胞,佛波脂(PAM)使之巨噬化,ox-LDL使巨噬细胞形成泡沫细胞,罗格列酮和泡沫细胞孵育48 h.用荧光分光光度法检测细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)的含量;ELISA法测定ox-LDL;TBARS法检量MDA.结果 THP-1细胞经PAM和ox-LDL孵育后,有大量泡沫细胞形成.细胞内TC、FC、CE、ox-LDL、MDA均增多(P＜0.05＝;罗格列酮干预后,细胞内TC、FC、CE、ox-LDLD、MDA均显著减少(P＜0.05＝.结论 罗格列酮可通过降低细胞内胆固醇的聚积和减轻细胞的氧化抗动脉粥样硬化.     

正常人两种极低密度脂蛋白亚组分对小鼠腹腔巨噬细胞脂质堆积的影响

本文研究了正常人极低密度脂蛋白两种亚组分，即贫含载脂蛋白Ｅ的ＶＬＤＬ和富含载脂蛋白Ｅ的ＶＬＤＬ对小鼠腹腔巨旨质堆积的影响。     

正常人两种极低密度脂蛋白亚组分对小鼠腹腔巨噬细胞脂质堆积的影响

本文研究了正常人极低密度脂蛋白（ｖｅｒｙｌｏｗｄｅｎｓｉｔｙｌｉｐｏｐｒｏｔｅｉｎ，ＶＬＤＬ）两种亚组分，即贫含载脂蛋白Ｅ的ＶＬＤＬ和富含载脂蛋白Ｅ的ＶＬＤＬ对小鼠腹腔巨噬细胞（ｍｏｕｓｅｐｅｒｉｔｏｎｅａｌｍａｃｒｏｐｈａｇｅ，ＭＰＭ）脂质堆积的影响。将贫含载脂蛋白Ｅ的ＶＬＤＬ和富含载脂蛋白Ｅ的ＶＬＤＬ分别与ＭＰＭ温育２４ｈ后，细胞内甘油三酯（ｔｒｉｇｌｙｃｅｒｉｄｅ，ＴＧ）和胆固醇酯（ｃｈｏｌｅｓｔｅｒｙｌｅｓｔｅｒ，ＣＥ）随ＶＬＤＬ浓度的增加而增加（方差分析，Ｐ＜０．０５），且对ＭＰＭ内ＴＧ的增加量以贫含载脂蛋白Ｅ的ＶＬＤＬ居多（Ｐ＜０．０１），而对ＭＰＭ内ＣＥ的增加量两者无显著性差异（Ｐ＞０．０５）。这提示，贫含载脂蛋白Ｅ的ＶＬＤＬ和富合载脂蛋白Ｅ的ＶＬＤＬ均能促进ＭＰＭ内ＴＧ、ＣＥ堆积，从而使巨噬细胞（ｍａｃｒｏｐｈａｇｅ，ＭＰ）转变为泡沫细胞。     

同型半胱氨酸对人THP-1巨噬细胞白细胞介素-8蛋白表达的影响

目的:探讨同型半胱氨酸(Hcy)对人THP-1巨噬细胞白细胞介素(IL)-8蛋白表达及分泌的影响。方法:在由0.1μmol/L佛波脂(PMA)诱导分化的人THP-1巨噬细胞中加入不同浓度的Hcy,并孵育不同时间。用酶联免疫吸附法检测培养上清液中IL-8蛋白的含量。结果:经PMA诱导后,THP-1细胞贴壁生长,分化成巨噬细胞。在一定浓度范围之内,Hcy呈剂量依赖性刺激THP-1巨噬细胞分泌IL-8蛋白,0.05mmol/L即可促进其分泌,增加为阴性对照组的1.28倍(P<0.01);0.2mmol/LHcy使IL-8的产量增加到阴性对照组的1.55倍(P<0.01)。0.1mmol/LHcy干预后,IL-8蛋白在3h即高于阴性对照组,到48h仍明显高于阴性对照组。Hcy刺激IL-8分泌呈剂量和时间依赖关系。结论:Hcy能促进人THP-1巨噬细胞IL-8的表达和分泌,可能为其诱导动脉粥样硬化的重要机制之一。     

酰基辅酶A胆固醇酰基转移酶1基因过表达对泡沫细胞形成的影响

目的研究在三种不同细胞中过表达酰基辅酶A胆固醇酰基转移酶1基因对泡沫细胞形成的影响。方法构建携带酰基辅酶A胆固醇酰基转移酶1全长cDNA的pCDNA3.1质粒载体并稳定转染体外培养的人THP-1单核细胞、小鼠RAW264.7单核巨噬细胞和人胚肾293上皮细胞,以油红O染色法检测在乙酰化低密度脂蛋白作用下转染前后三种细胞形成泡沫细胞的情况。结果在相同的脂质负荷条件下,转染酰基辅酶A胆固醇酰基转移酶1基因的THP-1单核细胞和RAW264.7巨噬细胞同未转染的细胞相比泡沫细胞的形成数量增加,而人胚肾293上皮细胞无论是否转染酰基辅酶A胆固醇酰基转移酶1基因均不易形成泡沫细胞。结论单核巨噬细胞中过表达酰基辅酶A胆固醇酰基转移酶1基因可促进泡沫细胞的形成。     

Phenolic-extract from argan oil (Argania spinosa L.) inhibits human low-density lipoprotein (LDL) oxidation and enhances cholesterol efflux from human THP-1 macrophages

Argan oil is rich in unsaturated fatty acids, tocopherol and phenolic compounds. These protective molecules make further study of its cardiovascular diseases (CVDs) action interesting. Furthermore, no previous study has explored the antioxidant activity of argan oil in comparison with olive oil. The present study was conducted to evaluate the beneficial properties of Virgin argan oil phenolic extracts (VAO-PE) towards CVD by: (A) protecting human (low-density lipoprotein, LDL) against lipid peroxidation and (B) promoting high-density lipoprotein (HDL)-mediated cholesterol efflux. Human LDLs were oxidized by incubation with CuSO(4) in the presence of different concentrations of VAO-PE (0-320mug/ml). LDL lipid peroxidation was evaluated by conjugated diene and MDA formation as well as Vitamin E disappearance. Incubation of LDL with VAO-PE significantly prolonged the lag-phase and lowered the progression rate of lipid peroxidation (P<0.01) and reduced the disappearance of Vitamin E in a concentration-dependent manner. Incubation of HDL with VAO-PE significantly increased the fluidity of the HDL phospholipidic bilayer (P=0.0004) and HDL-mediated cholesterol efflux from THP-1 macrophages. These results suggest that Virgin argan oil provides a source of dietary phenolic antioxidants, which prevent cardiovascular diseases by inhibiting LDL-oxidation and enhancing reverse cholesterol transport. These properties increase the anti-atherogenic potential of HDL.     

乙酰化低密度脂蛋白促进胆固醇酰基转移酶活性升高的机制研究

目的 :研究乙酰化低密度脂蛋白 (Ac LDL)对人THP 1单核细胞 (MC)分化的巨噬细胞 (MP)酰基辅酶A、胆固醇酰基转移酶 1(ACAT 1)活性的影响及其机制。方法 :体外培养人THP 1单核细胞系 ,由佛波酯 (PMA)作用使其分化为MP ,后者再由乙酰化低密度脂蛋白Ac LDL进行脂质负荷转变为泡沫细胞。该过程中以放射性同位素标记底物法检测ACAT 1酶活性的变化 ,并用Westernblot法检测ACAT 1酶蛋白的表达及RT PCR法检测A CAT 1mRNA的水平。结果 :在MC分化为MP的过程中ACAT 1酶活性升高了 2倍 ,差异有统计学意义 (P <0 .0 5 ) ,酶蛋白及mRNA水平呈现类似变化趋势 ;Ac LDL作用于巨噬细胞 ,使ACAT1酶活性、酶蛋白及mRNA进一步升高 ,差异有统计学意义 (P <0 .0 5 )。结论 :MC分化为MP的过程中ACAT 1表达上调 ,使ACAT 1活性增强 ,而Ac LDL可进一步促进ACAT 1基因表达增加 ,升高ACAT 1活性。     

肺炎衣原体通过上调酰基辅酶A:胆固醇酰基转移酶1诱导人单核细胞株THP-1源性泡沫细胞形成的实验研究

目的 观察酰基辅酶A:胆固醇酰基转移酶1(ACAT1)在肺炎衣原体(C.pn)诱导泡沫细胞形成中的作用,初步探讨C.pn诱导泡沫细胞形成的机制.方法 人单核细胞株(THP-1)给予160 mmol/L佛波酯(PMA)孵育48 h,诱导分化为巨噬细胞后随机分为4组:阴性对照组、阳性对照组、C.pn感染组和ACAT抑制剂58-035+C.pn感染组.分别运用RT-PCR和Western blot检测各组ACAT1 mRNA和蛋白表达.运用油红O染色观察细胞浆内脂滴的变化,用酶荧光学法检测细胞内胆固醇酯含量的变化.结果 C.pn感染呈浓度和时间依赖性地上调ACAT1 mRNA和蛋白表达.高浓度的C.pn(5×105和1×106IFU)感染THP-1源性巨噬细胞48 h后,细胞浆内脂滴明显增多,胆固醇酯与总胆固醇的比值明显增加(＞50%).ACAT抑制剂58-035可以抑制C.pn诱导的细胞浆内脂滴的增多.随着58-035浓度的增加,逐渐抑制C.pn诱导的细胞内胆固醇酯含量的增加.结论 ACAT1表达上调是C.pn诱导泡沫细胞形成的机制之一,这可能为进一步阐明C.pn感染促进动脉粥样硬化发生发展提供一个新的理论依据.     

白细胞介素10对人THP-1源巨噬细胞泡沫化过程中SR-A表达的影响

目的:本研究旨在观察白细胞介素-10(IL-10)对人THP-1源巨噬细胞泡沫化过程中清道夫受体A(SR-A)表达的影响,探讨在动脉粥样硬化形成中IL-10对氧化型低密度脂蛋白(ox-LDL)诱导巨噬细胞泡沫化的干预作用。方法:体外建立泡沫细胞培养体系,将细胞分为5组即THP-1单核细胞组,巨噬细胞组,IL-10刺激巨噬细胞组,ox-LDL刺激巨噬细胞组(泡沫细胞组),ox-LDL和IL-10联合刺激巨噬细胞组。采用RT-PCR和Westernbloting分别检测SR-A的mRNA和蛋白表达变化,脂质油红O化学染色方法检测各组细胞脂质摄取量的情况。结果:当THP-1分化为巨噬细胞时,SR-A开始大量表达;IL-10刺激可显著抑制巨噬细胞组SR-A的表达,其mRNA和蛋白分别下降为未刺激前的0.6倍和0.7倍,泡沫细胞形成率也下降为未刺激前的0.6倍。巨噬细胞经ox-LDL刺激形成泡沫细胞时,SR-A的表达进一步升高,其mRNA和蛋白分别增加为巨噬细胞组的1.5倍和1.4倍,泡沫细胞形成率提高为巨噬细胞组的1.7倍。在此过程中加入IL-10联合刺激,观察到SR-A的表达量有显著降低,其mRNA和蛋白均下降为单用ox-LDL刺激巨噬细胞组的0.4倍,ox-LDL的摄取量也下降为单用ox-LDL刺激巨噬细胞组的0.3倍。结论:IL-10抑制巨噬细胞SR-A表达,IL-10对ox-LDL诱导巨噬细胞SR-A的表达具明显干预作用。IL-10通过抑制SR-A表达可能在抗动脉粥样硬化的发生中发挥重要作用。     

阿托伐他汀对THP-1巨噬细胞CD36表达的影响

目的研究阿托伐他汀(atorvastatin)对THP-1巨噬细胞B类清道夫受体CD36表达的影响。方法用5!mol/L阿托伐他汀与THP-1巨噬细胞孵育不同时间;用不同浓度的阿托伐他汀与THP-1巨噬细胞共同孵育24h,采用RT-PCR与Westernblotting分别检测THP-1巨噬细胞CD36mRNA与蛋白质的表达。结果阿托伐他汀使THP-1巨噬细胞CD36mRNA与蛋白质的表达下调(P<0.05),且时间与剂量呈依赖关系。结论阿托伐他汀能抑制THP-1巨噬细胞CD36的表达。     

Accumulation of cholesteryl esters in macrophages incubated with human lipoprotein-antibody autoimmune complex

The interaction of mouse peritoneal and human pericardial macrophages with lipoprotein (LP)-antibody (Ab) immune complex isolated from the serum of ischemic heart disease (IHD) patients has been studied. It is shown that the Ab of the autoimmune complex belongs to IgG class, and the antigen is the LP with d less than 1.063 g/ml. Incubation of mouse peritoneal macrophages with such complex led to the activation of [14C]oleic acid incorporation into cholesteryl esters by 2.5-2.8-fold, in comparison with the experiments where macrophages were incubated with free apoprotein (apo) B-containing LP isolated from the same serum. Incubation of human pericardial macrophages with autologous LP-Ab immune complex led to the transformation of macrophages into foam cells. These data lead to the conclusion that formation of LP-Ab autoimmune complexes may play an important role in the formation of atherosclerotic lesions of the arteries.     

葡萄糖对THP-1源性巨噬细胞酰基辅酶A:胆固醇酰基转移酶1表达的影响

研究葡萄糖对人THP-1单核分化巨噬细胞酰基辅酶A：胆固醇酰基转移酶1（ACAT-1）表达的影响。发现高糖作用下，巨噬细胞ACAT-1的mRNA及蛋白表达增加，这可能是糖尿病血管病变机制之一。     

载脂蛋白A1对胆固醇逆转运及三磷酸腺苷结合盒转运体A1表达的影响

目的 观察载脂蛋白A1(apoAl)对人急性单核细胞白血病细胞株(THP-1)源性泡沫细胞内胆固醇、胆固醇酯含量和三磷酸腺苷结合盒转运蛋白A1(ABCAl)表达的影响和机制.方法 以50 ng/ml的佛波酯诱导人THP-1,使其分化为巨噬细胞,再经50μg/ml氧化型低密度脂蛋白充分诱导使其转变为负脂的泡沫细胞;然后用不同浓度apoAl(5μg/ml、10μg/ml、15μg/ml、20 μg/ml)孵育泡沫细胞24 h,以apoAl(10μg/ml)孵育泡沫细胞6 h、12 h、24 h.采用油红O染色观察细胞内脂滴的变化,用氧化酶法检测细胞内总胆固醇、游离胆固醇和胆固醇酯的含量;用反转录聚合酶链反应检测不同浓度apoAl干预泡沫细胞后ABCA1 mRNA的表达;用免疫荧光法检测经不同浓度和不同时间apoAl和ABCAl多克隆抗体干预泡沫细胞后ABCA1蛋白的表达.结果 (1)THP-1经佛波酯(50 ng/ml)和氧化型低密度脂蛋白(50μg/ml)分别干预48 h后可成功诱导为富含脂质的泡沫细胞;(2)apoA1可促进THP-1巨噬细胞源性泡沫细胞内胆固醇逆向转运,减少细胞内胆固醇含量,呈浓度依赖性和时间依赖性;(3)随着apoA1干预浓度增加,THP-1巨噬细胞源性泡沫细胞内ABCA1 mRNA的表达变化不显著,但蛋白表达增加,0 μg/ml与5 μg/ml、10μg/ml、15μg/ml、20μg/ml apoAl干预浓度下油红O染色阳性细胞率分别为(55±4)%与(43±9)%、(33±4)%、(28±1)%、(26±2)%,差异有统计学意义(均P<0.05);(4)ABCAl抗体干预可增加THP-1巨噬细胞源性泡沫细胞ABCA1蛋白的表达.结论 apoA1-ABCA1通路参与泡沫细胞胆固醇逆向转运,apoA1起关键作用,apoA1增加ABCA1蛋白的表达可能与降低ABCA1蛋白的降解有关.     

The regulation of EN-RAGE (S100A12) gene expression in human THP-1 macrophages

EN-RAGE is a ligand for the receptor for advanced glycation end products (RAGE) and may be involved in the development of diabetic macro- and micro-angiopathy. This study is designed to investigate the regulation of EN-RAGE gene expression in human macrophages. The amounts of EN-RAGE mRNA were measured in cultured human THP-1 macrophages after treatment with various stimuli known to modulate atherosclerosis. First, interleukin-6 (IL-6), a proinflammatory cytokine, increased the level of EN-RAGE mRNA by approximately 2-fold in a time- and a dose-dependent fashion. EN-RAGE protein was detected in the cultured medium and increased significantly by the addition of IL-6. The induction was abolished by pretreatment with the JAK kinase inhibitor and cycloheximide, but not with the MEK kinase inhibitor. Second, pioglitazone (PIO), a thiazolidinedione, decreased the level of EN-RAGE mRNA by approximately 25% of the basal in a time- and a dose-dependent fashion. Pioglitazone also inhibited the induction of EN-RAGE mRNA by IL-6. These results indicate the production of EN-RAGE is induced by IL-6 through de novo protein synthesis via the JAK-STAT kinase pathway and inhibited by the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) in human macrophages.     

Enhanced removal of cholesterol from macrophage foam cells to serum from type IV hypertriglyceridemic subjects

Hypertriglyceridemia being an independent cardiovascular risk factor, we have compared the potential of sera from asymptomatic hypertriglyceridemic (HTG) type IIb, type IV or normolipidemic (NLP) subjects to promote both fractional cholesterol efflux and cellular cholesterol mass changes using macrophage foam cells. The J774 cells loaded with cholesterol by incubation with acetylated LDL were incubated in the absence or presence of cAMP to upregulate ABCA1 (ATP binding cassette transporter A1) and then incubated for 24 h with 1% serum. Compared with NLP, type IV sera exhibited a major increase in ABCA1-dependent efflux while type IIb sera exhibited a moderate but not significant increased ABCA1-mediated efflux. Moreover, positive correlations were established between ABCA1-dependent efflux and the serum preβ-HDL or TG concentrations. The major finding was that the sera from type IV induced higher total cholesterol and cholesteryl ester mass depletions from ABCA1-expressing cells compared with other groups. Moreover, negative correlations were obtained between total cholesterol or cholesteryl ester mass changes and serum preβ-HDL levels. In conclusion, we demonstrated for the first time that the serum preβ-HDL present in high proportions in type IV HTG subjects are not only responsible for higher cholesterol efflux potential but also for increased abilities to promote net removal of cholesterol from macrophage foam cells.     

氧化型脂蛋白(a)对鼠腹腔巨噬细胞增殖的影响

为探讨氧化型脂蛋白（ａ）对巨噬细胞增殖的影响,采用细胞计数和ＭＴＴ掺入观察比较天然型脂蛋白（ａ）、氧化型脂蛋白（ａ）和丙二醛修饰脂蛋白（ａ）对培养的鼠腹腔巨噬细胞增殖的影响。结果发现,氧化型脂蛋白（ａ）在蛋白浓度为２．５～４０ｍｇ／Ｌ时刺激巨噬细胞增殖,蛋白浓度为４０ｍｇ／Ｌ时巨噬细胞数为对照组的１．５倍（Ｐ〈０．０５）。而天然型脂蛋白（ａ）和丙二醛修饰脂蛋白（ａ）对巨噬细胞增殖无影响。结果提示,氧化型脂蛋白（ａ）可能通过促进巨噬细胞增殖加速动脉粥样硬化的发展。