氧化型低密度脂蛋白诱导RAW264.7细胞表达V型和X型分泌型磷脂酶A2
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Expression of GroupⅤand GroupⅩSecretory Phospholipase A2 on RAW264.7 Cell Induced by Oxidized Low Density Lipoprotein
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    摘要:

    目的通过研究不同氧化程度的氧化型低密度脂蛋白对鼠巨噬细胞系RAW264.7细胞表达Ⅳ型胞浆型磷脂酶A2、V型和X型分泌型磷脂酶A2的调节作用,探讨氧化型低密度脂蛋白在促动脉粥样硬化过程中的炎症机制。方法利用不同的氧化方法对低密度脂蛋白进行氧化修饰,分别制备轻微修饰低密度脂蛋白、轻度氧化低密度脂蛋白和重度氧化低密度脂蛋白,将此三型氧化型低密度脂蛋白用于刺激RAW264.7细胞,分别孵育4 h及8 h后收集细胞裂解液和细胞培养上清液,分别用逆转录聚合酶链反应法检测胞浆型磷脂酶A2、V型和X型分泌型磷脂酶A2的mRNA表达,Western blotting法检测胞内和细胞培养上清液中V型和X型分泌型磷脂酶A2蛋白的表达。结果与PBS对照组及低密度脂蛋白组相比,RAW264.7细胞经各型氧化型低密度脂蛋白刺激后胞浆型磷脂酶A2的mRNA表达差异无显著性(P>0.05),但V型分泌型磷脂酶A2和X型分泌型磷脂酶A2的mRNA表达水平和胞内及细胞培养液中蛋白的表达水平均明显升高(P<0.05)。与轻微修饰低密度脂蛋白组和重度氧化低密度脂蛋白组相比,轻度氧化低密度脂蛋白组诱导V型分泌型磷脂酶A2和X型分泌型磷脂酶A2的mRNA和蛋白表达作用更强(P<0.05),而轻微修饰低密度脂蛋白组的作用又要强于重度氧化低密度脂蛋白组(P<0.05)。结论氧化型低密度脂蛋白可诱导RAW264.7细胞表达V型和X型分泌型磷脂酶A2,而两型轻度氧化的氧化型低密度脂蛋白诱导V型和X型分泌型磷脂酶A2表达的能力明显强于重度氧化程度的低密度脂蛋白,说明轻度氧化程度的氧化型低密度脂蛋白具有较强的诱导巨噬细胞产生炎症反应的能力。

    Abstract:

    Aim To investigate whether the expression of groupⅣcytosolic phospholipase A2(cytosolic phospholipase A2,cPLA2),groupⅤsecretory phospholipase A2(sPLA2-Ⅴ) and groupⅩsecretory phospholipase A2 (sPLA2-Ⅹ) could be regulated by oxidized low density lipoprotein(ox-LDL) of different oxidation levels in the mouse macrophage cell line RAW264.7 cells,and to explore the inflammatory mechanisms of the interactions between ox-LDL and macrophages in the process of promoting atherosclerosis.Methods 3 types of ox-LDL(minimally modified low density lipoprotein or MM-LDL,lightly ox-LDL or ox_(4h)-LDL,heavily ox-LDL or ox_(48h)-LDL) were prepared by different oxidation ways.3 types of ox-LDL were used to incubate with RAW264.7 cells for 4 h and 8 h,then the cell lysate and cell culture supernatant were collected.The mRNA expression and protein expression of cPLA2,sPLA2-Ⅴand sPLA2-Ⅹwere determined respectively by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting analysis. Results The mRNA expression and protein expression of sPLA2-Ⅴand sPLA2-Ⅹwere undetectable in untreated RAW264.7 cell.Compared with the PBS control group and the LDL group,the mRNA expression of cPLA2 was not affected by all types of ox-LDL(P>0.05).But the mRNA expression and protein expression of sPLA2-Ⅴand sPLA2-Ⅹwere significantly enhanced by the stimulation of MM-LDL and ox_(4h)-LDL in RAW264.7 cell(P<0.05),while ox_(48h)-LDL had a slight ability to induce the expression of sPLA2-Ⅴand sPLA2-Ⅹ(P<0.05).However,PBS control group and native LDL failed to induce the expression of sPLA2-Ⅴand sPLA2-Ⅹ.Conclusion The exposure of ox-LDL to RAW264.7 cells did not affect the mRNA expression of cPLA2,but could significantly induce RAW264.7 cells to express sPLA2-Ⅴand sPLA2-Ⅹ.Two types of mildly oxidized LDL(MM-LDL and ox_(4h)-LDL)-induced expression of sPLA2-Ⅴand sPLA2-Ⅹwas stronger than that of heavily oxidized LDL(ox_(48h)-LDL),indicating that mildly oxidized LDL was capable of inducing a strong inflammatory response on macrophages.

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韩冠平,敖彩卉,薛林,赵春玉,齐丽彤.氧化型低密度脂蛋白诱导RAW264.7细胞表达V型和X型分泌型磷脂酶A2[J].中国动脉硬化杂志,2009,17(6):453~458.

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  • 收稿日期:2009-04-01
  • 最后修改日期:2009-06-06
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