Calphostin C抑制脂肪分化相关蛋白诱导的ACAT1表达  

Calphostin C Inhibit Adipophilin-induced Expression of ACAT 1

作　　者：袁中华[1] 陶媛[1] 刘清南 黄谙非 刘晓辉[4] 田国平[5] 唐朝克[1] 易光辉[1] 王佐[1] 

机构地区：[1]南华大学心血管疾病研究所动脉硬化学湖南省重点实验室,湖南省衡阳市421001 [2]益阳医学高等专科学校护理系基础护理学教研室,湖南省益阳市413000 [3]深圳市西丽医院病理科,广东省深圳市518050 [4]苏州大学唐仲英血液学研究中心,江苏省苏州市215400 [5]南华大学附属第二医院心内科,湖南省衡阳市421001

出　　处：《中国动脉硬化杂志》2012年第12期1057-1063,共7页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金(30971268);教育部留学回国人员科研启动基金(教外司留[2008]890号)

摘　　要：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。Aim To investigate the signaling pathways involved in the process which adipophilin induced the in- tracellular lipid accumulation. Methods We obtained the RAW264. 7 cells of high or low expression of adipophilin by constructing retroviral vector. Then the ceils were incubated with 300 nmol/L of Calphostin C （ PKC inhibitor） for 16 h or incubated with 50 mg/L ox-LDL simultaneously. The expression of ACAT1 was detected by RT-PCR and Western blot.Results The results showed that RAW264. 7 cells with high expression of adipophilin increased the expression of ACAT1, and decreased in cells of low expression of adipophilin. Calphostin C inhibited the expression of ACAT1 in the cells with high or low expression of adipophilin. Compared with the cells incubated without Calphostin C, the difference was signifi- cant. Calphostin C also inhibited the expression of ACAT1 when incubated the cells of high expression of adipophilin with ox-LDL for lipid-loading. Both mRNA and protein were significantly decreased after incubated for 16 h and the difference was significant compared with the control. In the condition of lipid-loaded, Calphostin C could inhibit ACAT1 expression time-dependently in cells of low expression of adipophilin. Conclusion The results indicated that PKC signaling mol- ecules affected adipophilin expression and further affected the expression of ACAT1. It suggests that PKC signal could be related to adipophilin causing intracellular lipid accumulation.

关 键 词：脂肪分化相关蛋白 乙酰辅酶A 胆固醇酰基转移酶1 Calphostin C 

分 类 号：R363[医药卫生—病理学]