高表达和敲减adipophilin对细胞内ERK1/2活性、PPARγ表达和细胞内脂质蓄积的影响(英文)

The Effects of Highexpression and Knockdown Adipophilin in The Activity of ERK1/2 and Expression of PPARγ and Lipid Accumulation in Cells

本课题组以前的研究表明,adipophilin通过ERK1/2-PPARγ信号转导通路促进细胞内的脂质蓄积.为了研究高表达和敲减adipophilin是否影响RAW264.7细胞内ERK1/2的活性、PPARγ的表达以及细胞内的脂质蓄积,从而进一步证实这一通路,阐明adipophilin促进泡沫细胞形成的机制.重组pQCXIP-HA-Adipophilin和pSuper-retro-adipophilin siRNA逆转录病毒载体经酶切检测证实,并用SofastTM介导转染到包装细胞PA317中,经培养后释放逆转录病毒.将收集的逆转录病毒感染RAW264.7细胞,经嘌呤霉素筛选后获得稳定高表达和敲减adipophilin的细胞系.用50 mg/L的氧化低密度脂蛋白处理细胞24 h后,用油红O染色法和高效液相色谱法测定细胞内的脂质蓄积情况,用半定量RT-PCR和蛋白质印迹分别检测adipophilin和PPARγ的mRNA和蛋白质的表达,用蛋白质印迹对与动脉粥样硬化发病有关的ERK1/2及其磷酸化进行检测.酶切结果表明,pQCXIP-HA-Adipophilin和pSuper-retro-adipophilin siRNA重组逆转录病毒载体构建成功.在荷脂情况下,pQCXIP-HA-Adipophilin转染的细胞能明显增加细胞内的脂质蓄积,但使PPARγ的表达和ERK1/2的磷酸化下调,这些作用可被adipophilin siRNA逆转.结果表明,adipophilin与泡沫细胞的形成有关,adipophilin可能是通过ERK1/2-PPARγ途径促进细胞内的脂质蓄积.

Our previous studies have showed that adipophilin promoted intracellular lipids accumulation through ERK1/2-PPARγ signaling pathway.In the study we explored that whether highexpression and knockdown adipophilin affects the activity of ERK1/2 and the expression of PPARγ and lipid accumulation in RAW264.7 cells,and further certified that adipophilin promoted intracellular lipids accumulation through this pathway.The recombinant retroviral vetors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA were verified by the methods of enzyme-digesting.The recombinant retroviral vetors were transfected into PA317 cell by mediating SofastTM,which can induce retroviruses release.Then we used the collected retroviruses to infect RAW264.7 cells and achieved adipophilin gene highexpression and knockdown RAW264.7 cell lines applying puromycin screening.After the infected RAW264.7 cells incubated with 50 mg/L Ox-LDL for 24 h,the lipids accumulation were measured by Oil red O staining and HPLC,the expression of mRNA and proteins of adipophilin and PPARγ were detected by semi-quantitative RT-PCR and Western blot respectively,and phosphorylation of ERK1/2 was analyzed by Western blot too.The results of enzyme-digesting confirmed the recombinant retroviral vectors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA as expected.In the situation of Dutch fat,transfected cells with pQCXIP-HA-Adipophilin significantly increased the accumulation of lipids,but reduced the expression of PPARγ and the phosphorylation of ERK1/2,which were reversed in cells with pSuper-retro-adipophilin siRNA transfection.Our results showed that ERK1/2 and PPARγ may be related with lipids accumulation caused by adipophilin expression in macrophages incubated with modified LDL.Therefore,adipophilin might contribute,in vivo,to lipid accumulation in the intima of the arterial wall through the ERK1/2-PPARγ signaling pathway.