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目的我们已经发现adipophilin通过改变乙酰辅酶A∶胆固醇酰基转移酶1(ACAT1)的表达来促进细胞内脂质蓄积,病理状态下形成泡沫细胞,成为动脉粥样硬化的始动因素。本研究旨在探讨该过程所涉及的信号通路,阐明adipophilin引起细胞内脂质积聚的机制。方法通过克隆adipophilin基因,构建逆转录病毒载体pQCXIP-HA-Adi,使用siRNA技术构建pSuper-retro-adipophilin siRNA逆转录病毒载体,包装病毒后,感染RAW264.7细胞,筛选后获得高或低表达adipophilin的细胞。将该细胞分别与300 nmol/L PKC抑制剂Calphostin C孵育16 h或同时再加入50 mg/L氧化型低密度脂蛋白(ox-LDL)共孵育,应用RT-PCR和Western blot检测细胞内ACAT1的表达。当低表达adipophilin的细胞与Calphostin C共孵育时,在不同的时间点取样,RT-PCR和Westernblot检测各时间点细胞内ACAT1的表达。结果高表达adipophilin细胞中ACAT1表达增加,低表达adipophilin细胞中ACAT1表达减少。无论在高或低表达adipophilin的细胞中,Calphostin C能够抑制ACAT1的表达,与不孵育Calphostin C组相比差别有显著性。与ox-LDL孵育使高表达adipophilin的细胞荷脂,同样能够发现Calphostin C抑制ACAT1的表达。低表达adipophilin的细胞与Calphostin C共孵育1 h后,ACAT1 mRNA开始下降;孵育8 h后,ACAT1蛋白开始下降;孵育16 h后ACAT1 mRNA及蛋白表达均明显下降,与对照组相比差别有显著性。高及低表达adipophilin或负荷脂质状态下,Calphostin C能够时间依赖性的抑制ACAT1的表达。结论 adipophilin引起细胞内脂质积聚的机制可能是,PKC信号分子作用于adipophilin,并进一步影响ACAT1的表达,最终导致细胞内脂质积聚。
Objectives We have found that adipophilin promotes intracellular lipid accumulation by altering the expression of acetyl-CoA: cholesterol acyltransferase 1 (ACAT1), forming foam cells under pathological conditions and becoming the initiating factor of atherosclerosis. The purpose of this study was to investigate the signaling pathways involved in this process and to elucidate the mechanism by which adipophilin causes lipid accumulation in cells. Methods The recombinant retroviral vector pQCXIP-HA-Adi was constructed by cloning the adipophilin gene. The pSuper-retro-adipophilin siRNA retroviral vector was constructed by siRNA technique. After packaging the virus, RAW264.7 cells were infected and screened for high or low expression of adipophilin Cell. The cells were incubated with 300 nmol / L PKC inhibitor Calphostin C for 16 h or 50 mg / L ox-LDL respectively. RT-PCR and Western blot were used to detect the expression of ACAT1 expression. When adipophilin-deficient cells were co-incubated with Calphostin C, samples were taken at different time points and RT-PCR and Western blot were used to detect intracellular ACAT1 expression at each time point. Results The expression of ACAT1 in adipophilin cells was increased, and the expression of ACAT1 in adipophilin cells with low expression was decreased. Calphostin C inhibited the expression of ACAT1 in both high and low adipophilin-expressing cells, which was significantly different from that of non-incubated Calphostin C group. Incubation with ox-LDL by lipofectin-overexpressing cells also showed that Calphostin C inhibited ACAT1 expression. ACAT1 mRNA began to decline after co-incubated with Calphostin C for 1 h in adipophilin-deprived cells. The ACAT1 protein began to decline after 8 h of incubation. The mRNA and protein expression of ACAT1 decreased significantly after 16 h of incubation, which was significantly different from that of control group Sex. Calphostin C inhibited the expression of ACAT1 in a time-dependent manner under high and low expression of adipophilin or lipid-loaded state. Conclusions The mechanism by which adipophilin induces intracellular lipid accumulation may be that PKC signaling acts on adipophilin and further influences the expression of ACAT1, eventually leading to intracellular lipid accumulation.