脂肪分化相关蛋白通过抑制中性胆固醇酯水解酶表达促进RAW264.7细胞内脂质积蓄
作者:
作者单位:

(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.湘南学院附属医院,湖南省郴州市 423000)

作者简介:

袁旭,硕士研究生,研究方向为动脉粥样硬化发病机制及防治,E-mail为178024852@qq.com。

基金项目:

国家自然科学基金资助项目(30971268)


Adipophilin enhances intracellular lipid accumulation by inhibiting neutral cholesterol ester hydrolase in RAW264.7 macrophages
Author:
Affiliation:

1.Institute of Cardiovascular Disease, University of South China, Hengyang 421001, Hunan, China;2.Affiliated Hospital of Xiangnan University, Chenzhou 423000, Hunan, China)

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    摘要:

    目的 探讨RAW264.7巨噬细胞中脂肪分化相关蛋白(adipophilin)调控中性胆固醇酯水解酶(nCEH)表达进而介导脂质积蓄的作用机制。 方法 用已成功构建的沉默与高表达Adipophilin的逆转录病毒质粒载体转染包装细胞PA317,继而收集病毒液感染RAW264.7巨噬细胞,筛选出Adipophilin沉默细胞株和高表达细胞株。逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹分析技术(Western blot)分别检测nCEH的mRNA和蛋白表达水平,液体闪烁计数仪、高效液相色谱法检测细胞内胆固醇流出及胆固醇酯含量。 结果 ①高表达Adipophilin的细胞组胆固醇、胆固醇酯含量明显高于空白对照组(P<0.01),而沉默Adipophilin表达的细胞组则显著低于空白对照组(P<0.01),Adipophilin可抑制nCEH mRNA和蛋白的表达。②用100 nmol/L蛋白激酶Cδ(PKCδ)激动剂佛波酯(PMA)孵育高表达Adipophilin的RAW264.7细胞30 min后,nCEH的mRNA与蛋白表达水平明显降低(P<0.05),而用100 nmol/L PKCδ抑制剂卡马拉素(Rottlerin)同样孵育30 min后,nCEH的表达水平较对照组增高(P<0.05)。 结论 Adipophilin促进巨噬细胞内脂质积蓄可能通过抑制nCEH表达来实现,在这一调控机制中PKCδ发挥了重要作用。

    Abstract:

    Aim To explore the mechanism of adipophilin regulating neutral cholesterol ester hydrolase (nCEH) expression thus mediating lipid accumulation in RAW264.7 macrophages. Methods The constructed retroviral plasmid vectors with adipophilin of no expression and over expression were transfected into package cell PA317, then collected virus solution was used to infect RAW264.7 macrophages by screening and identifying to obtain RAW264.7 cell lines with adipophilin stably silenced and highly expressed. The mRNA and protein expression levels of nCEH were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. High performance liquid chromatography (HPLC) was taken to detect the contents of intracellular cholesterol ester. Results The cholesterol and cholesterol ester levels were significantly higher in adipophilin over-expression group than in the control group (P<0.01), while dramatically lower than that in the control group when adipophilin was supressed (P<0.01). In addition, adipophilin inhibited nCEH expression. Furthermore, in RAW264.7 macrophages with high adipophilin expression, treatment with 100 nmol/L protein kinase Cδ agonist PMA for 30 min resulted in remarkable reductions of nCEH mRNA and protein expression(P<0.05), on the contrary, treatment with 100 nmol/L PKCδ inhibitor Rottlerin for 30 min increased the expression of nCEH(P<0.05). Conclusion Adipophilin may exert influence over lipid accumulation by inhibiting the expression of nCEH in RAW264.7 macrophages, and PKCδ played an important role in this regulatory mechanism.

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袁旭,孟磊,陈雁斌,张瑞,袁中华.脂肪分化相关蛋白通过抑制中性胆固醇酯水解酶表达促进RAW264.7细胞内脂质积蓄[J].中国动脉硬化杂志,2018,26(2):115~121.

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  • 收稿日期:2017-09-01
  • 最后修改日期:2017-11-18
  • 在线发布日期: 2018-03-07