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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 study showed that adipophilin promotes intracellular lipid accumulation through the ERK1 / 2-PPARγ signaling pathway.In order to investigate whether high expression and knockdown of adipophilin affect ERK1 / 2 activity in RAW264.7 cells, Expression and intracellular lipid accumulation, further confirming this pathway, elucidate the mechanism by which adipophilin promotes the formation of foam cells.Recombinant pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA retroviral vectors were confirmed by restriction enzyme digestion and stained with SofastTM Mediated transfection into the packaging cells PA317, after the release of retroviruses.Transfection of RAW264.7 cells collected retrovirus acquired by puromycin screening obtained stable expression and adipophilin knockout cell line with 50 After 24 h treatment with oxidized low density lipoprotein (mg / L), the intracellular lipid accumulation was determined by oil red O staining and high performance liquid chromatography. The levels of adipophilin and PPARγ were detected by semi-quantitative RT-PCR and Western blot respectively The expression of ERK1 / 2 and phosphorylation of ERK1 / 2 in atherosclerosis were detected by Western blotting.Results of digestion showed that pQCXIP-HA-A dipophilin and pSuper-retro-adipophilin siRNA recombinant retroviral vector was constructed successfully.PQCXIP-HA-Adipophilin transfected cells can significantly increase intracellular lipid accumulation in the case of lipid-loaded, but the expression of PPARγ and ERK1 / 2 , And these effects can be reversed by adipophilin siRNA.The results show that adipophilin is involved in the formation of foam cells, and adipophilin may promote intracellular lipid accumulation through the ERK1 / 2-PPARγ pathway.