同型半胱氨酸对THP-1单核细胞源性泡沫细胞形成中ABCA1、ACAT1表达的影响

摘要

目的 探讨同型半胱氨酸(Hcy)对THP-1单核细胞源性泡沫细胞形成中三磷酸腺苷-结合转运子A1( ABCA1)和酰基辅酶A:胆固醇酰基转移酶(ACAT1)表达的影响.方法 将THP-1单核细胞与佛波酯(PMA)、氧化低密度脂蛋白(ox-LDL)共同培养,复制泡沫细胞模型,并分别用50、100、200、500 μmol/L Hcy和100μnol/L Hcy+叶酸+维生素B12( Vit B12)干预72 h,并设对照组(不加入Hcy).采用油红0染色检测泡沫细胞的形成.采用酶终点法测定细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)含量的变化,观察Hcy对泡沫细胞CE流出的影响.采用荧光定量逆转录-聚合酶链反应(RT-PCR)测定ABCA1、ACAT1 mRNA表达；免疫印迹法检测ABCA1、ACAT1蛋白表达.结果 油红O染色显示Hcy加剧了泡沫细胞的形成,但不呈量效关系.实验组(加入50、100、200、500 μmol/L Hcy和100 μmol/L Hcy+叶酸+Vit B12)泡沫细胞阳性百分率均高于对照组(P＜0.05、P＜0.01),以100 μmol/L Hcy组效应最为明显(P＜0.01).在Hcy的干预下,泡沫细胞胞内TC、FC、CE流出减少,与对照组比较差异有统计学意义(P＜0.05),以100 μmol/L Hcy组效应最为明显(P＜0.01).100μmol/L Hcy+叶酸+VitB12组与100.μmol/L Hcy组比较,前者泡沫细胞形成减少,胆固醇流出增多.RTPCR结果显示ABCA1 mRNA表达下调,ACAT1 mRNA表达上调,均以100 μmol/L Hcy组效应最为明显(P＜0.01)；免疫印迹法检测ABCA1、ACAT1蛋白的表达与其mRNA表达一致.结论 Hcy下调了ABCA1的表达,上调了ACAT1的表达,促使了泡沫细胞形成.%Objective To investigate the influence of homocysteine (Hcy)on the expressions of ATP-binding cassette transporter Al(ABCAl)and acylcoenzyme A: cholesterol acyltransferase-1 (ACAT1)in the formation of THP-1 monocyte-derived foam cells. Methods THP-1 monocytes were cultured with phorbol myristate acetate (PMA)and oxidized low density lipoprotein(ox-LDL)so as to copy foam cell model, and then intervened by 50, 100, 200 and 500 μmol/L Hcy and 100 μmol/L Hey +folic acid + vitamin B12(Vit B12)for 72 h. The control group (not intervened by Hey)was set. The formation of foam cells was analyzed by the oil red 0 staining. The intracellular total cholesterol (TC), free cholesterol (FC)and cholesterol ester(CE)of foam cells were measured by enzymatic end point determination in order to observe the effects of Hcy on foam cell CE efflux. Fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR)detected the expressions of ABCA1 mRNA and ACAT1 mRNA. Western blotting tested the expressions of ABCA1 protein and ACAT1 protein. Results After the intervention of Hey, oil red 0 staining showed that the formation of foam cells was increased significantly, but not in dose-effect relationship. The positive percentage of experimental group (50, 100, 200 and 500 μmol/L Hey and 100 μmol/L Hey + folic acid + Vit B12)was higher than that of control group (P <0.05 ,P <0.01). The 100 μmol/L Hey group was the most significant one(P <0.01). The outflow of foam intracellular TC, FC and CE was reduced significantly compared with control group (P < 0.05), and the 100 μmol/L Hey group was the most significant one (P < 0.01). The 100 μmol/L Hey + folic acid + Vit B12 group showed that the formation of foam cells was decreased, whereas thecholesterol efflux was increased compared with 100 μmol/L Hey group. RT-PCR showed that the expression of ABCA1 mRNA was decreased, but ACAT1 mRNA was increased, and the 100 μmol/L Hey group was the most significant effect one(P < 0.01). Western blotting showed that the expressions of ABCA1 protein and ACAT1 protein were consistent with mRNA expression. Conclusions Hey reduces the expression of ABCA1, in the contrast, the expression of ACAT1 is increased. Both of them promote the formation of foam cells.