细胞色素P450 2J2在细胞模型中具有抗动脉粥样硬化作用
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广东省自然科学基金资助项目(S2013010011957);广东省医学科学技术研究基金(WSTJJ20111108;广文字第036092号);广州市珠江科技新星资助项目(2013J2200027)


Cytochrome P450 2J2 Exhibits Anti-atherosclerosis Effects in Two Cultured Cell Models
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    摘要:

    目的 通过在离体培养的人脐静脉内皮细胞(HUVEC)以及人外周单核细胞来源的泡沫细胞模型上过表达细胞色素P450 2J2(CYP2J2),探讨CYP2J2对动脉粥样硬化的影响。方法 构建pLVX-IRES-ZsGreen1-CYP2J2慢病毒载体,感染HUVEC及人外周单核细胞来源的泡沫细胞,逆转录-聚合酶链反应(RT-PCR)和Western blot分别检测CYP2J2在细胞内的表达情况。离体培养HUVEC分为空白对照组、空载体组(pLV组)和CYP2J2基因过表达组(pLV-CYP2J2组),通过MTS法分别于转染后的12、24、48、72 h测定细胞的增殖情况,通过Transwell实验检测48 h后的细胞迁移能力。另外,采用密度梯度离心法从冠心病患者外周血中分离出单核细胞,以50 nmol/L佛波酯刺激48 h使之转化为巨噬细胞,然后加入终浓度为80 mg/L的氧化型低密度脂蛋白使之转化为泡沫细胞;细胞亦分为空白对照组、pLV组和pLV-CYP2J2组,培养48 h后收集细胞,然后通过油红O染色观察细胞内脂滴变化,通过总胆固醇测定试剂盒检测细胞内总胆固醇的含量。结果 RT-PCR、Western blot结果显示,pLV-CYP2J2病毒感染后,HUVEC以及泡沫细胞中CYP2J2的mRNA和蛋白表达均显著升高。与空白对照组、pLV组相比,pLV-CYP2J2处理显著增强了HUVEC在24、48、72 h的细胞增殖能力以及在48 h的细胞迁移能力,而对12 h的细胞增殖则无统计学差异。在人外周单核细胞来源的泡沫细胞模型中,pLV-CYP2J2处理显著降低了泡沫细胞的数量、泡沫细胞内油红脂滴的数量以及总胆固醇的含量。结论 CYP2J2基因过表达可促进HUVEC的细胞增殖和迁移,降低人外周单核细胞来源的泡沫细胞形成,提示CYP2J2具有抗动脉粥样硬化的作用。

    Abstract:

    Aim To investigate the effect of cytochrome P450 2J2 (CYP2J2) on atherosclerosis by overexpression of CYP2J2 in the cultured human umbilical vein endothelial cell (HUVEC) and human peripheral monocyte-derived foam cell model. Methods Recombinant pLVX-IRES-ZsGreen1-CYP2J2 lentiviral vectors were infected into HUVECs and human peripheral monocyte-derived foam cells. CYP2J2 expressions in cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cultured HUVECs were divided into control group, vehicle group (pLV group) and CYP2J2 overexpression group (pLV-CYP2J2 group). Cell proliferation and migration were determined by MTS and Transwell assay. In addition, monocytes were isolated from the peripheral blood of patients with coronary heart disease by Ficoll-Hypaque density gradient centrifugation and plastic adsorptive process. The isolated cells were induced and stimulated by phorbol-12-myristate-13-acetate (PMA) at concentration of 50 nmol/L for 48 h to transfer them into macrophages, then adding oxidized low density lipoprotein (ox-LDL) at a final concentration of 80 mg/L to make the cells transformed into foam cells. Cells were also divided into control group, pLV group and pLV-CYP2J2 group, all of them were collected in 48 h after the virus transfection. Intracellular lipid droplets and total cholesterol were detected by oil red O staining and the total cholesterol assay kit. Results The results of RT-PCR and Western blot demonstrated that CYP2J2 mRNA and protein expression were significantly increased in both HUVECs and foam cells after pLV-CYP2J2 virus infection. Compared with the control group and pLV group, pLV-CYP2J2 infection significantly enhanced the cell proliferation of HUVECs in 24, 48, 72 h, and the cell migration in 48 h, but there was no significant difference in cell proliferation in 12 h. In addition, in human peripheral monocyte-derived foam cell model, pLV-CYP2J2 infection significantly reduced the number of foam cells, oil red lipid droplets and total cholesterol content. Conclusion CYP2J2 can be overexpressed in HUVECs and human peripheral monocyte-derived foam cells, which promotes the proliferation and migration of HUVECs, and reduces the formation of foam cells, so it suggests that CYP2J2 has the function of anti-atherosclerosis.

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李 锐,邱 健,石 磊,严华成,张 园,李 晋,阮云军,肖 华.细胞色素P450 2J2在细胞模型中具有抗动脉粥样硬化作用[J].中国动脉硬化杂志,2015,23(12):1223~1228.

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  • 收稿日期:2015-06-10
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  • 在线发布日期: 2015-12-26