Adipophilin通过PKCδ抑制nCEH促进巨噬细胞内脂质蓄积

发布时间：2018-05-18 00:16

本文选题：脂肪分化相关蛋白 + 中性胆固醇酯水解酶　；参考：《南华大学》2015年硕士论文

【摘要】：[目的]获得adipophilin通过n CEH促使细胞内脂质积聚的可靠证据,阐明adipophilin通过n CEH促使细胞内脂质积聚的机制,为动脉粥样硬化的防治寻找新靶点。[方法]将adipophilin沉默表达和稳定高表达的逆转录病毒质粒载体转染PA317包装细胞,收集病毒,感染RAW264.7细胞,筛选,制备高和沉默表达adipophilin的细胞。在高和沉默表达adipophilin的RAW264.7细胞中,分别用RT-PCR和Western blot检测n CEH的m RNA和蛋白表达的变化;高效液相色谱法检测细胞内胆固醇酯的含量。在高表达adipophilin的RAW264.7细胞中,用PKCδ激动剂0.1μmol/L PMA预孵育30 min后,或者用PKCδ特异性抑制剂0.1μmol/L Rottlerin预孵育30 min后,分别用RT-PCR和Western blot检测n CEH的m RNA水平和蛋白表达的改变;高效液相色谱法来检测细胞内胆固醇酯含量。[结果]在高表达adipophilin的RAW264.7细胞中,胆固醇酯的含量明显增加,在沉默表达adipophilin的RAW264.7细胞中,胆固醇酯的含量显著减少;同时,adipophilin能够抑制n CEH m RNA和蛋白的表达;在高表达adipophilin的RAW264.7细胞中,用PKCδ激动剂0.1μmol/L PMA预孵育30 min后,n CEH的m RNA与蛋白表达水平明显降低,用PKCδ特异性抑制剂0.1μmol/L Rottlerin预孵育30 min后,n CEH的m RNA和蛋白表达水平显著增高。[结论]高表达adipophilin促进巨噬细胞内胆固醇酯的蓄积,抑制n CEH m RNA和蛋的表达;adipophilin通过PKCδ抑制n CEH促进巨噬细胞内脂质蓄积。
[Abstract]:[objective] to obtain the reliable evidence that adipophilin induces intracellular lipid accumulation through n CEH, and to elucidate the mechanism of adipophilin inducing intracellular lipid accumulation through n CEH, so as to find a new target for the prevention and treatment of atherosclerosis. [methods] the retroviral vector expressing adipophilin silenced and stably high expression was transfected into PA317 packaging cells, the virus was collected, RAW264.7 cells were infected, and the cells expressing adipophilin with high or silent expression were screened. The expression of m RNA and protein of n CEH was detected by RT-PCR and Western blot, and the content of cholesterol ester was detected by high performance liquid chromatography in RAW264.7 cells with high and silence expression of adipophilin. After 30 min preincubation with PKC 未 agonist 0.1 渭 mol/L PMA or 30 min preincubated with PKC 未 specific inhibitor 0.1 渭 mol/L Rottlerin, the changes of m RNA level and protein expression of n CEH were detected by RT-PCR and Western blot, respectively. High performance liquid chromatography (HPLC) was used to detect the content of cholesterol ester in cells. [results] in RAW264.7 cells with high expression of adipophilin, the content of cholesterol ester was significantly increased, the content of cholesterol ester was significantly decreased in RAW264.7 cells with silencing expression of adipophilin, and adipophilin could inhibit the expression of n CEH m RNA and protein. In RAW264.7 cells with high expression of adipophilin, the expression of m RNA and protein of n CEH was significantly decreased after preincubation with PKC 未 agonist 0.1 渭 mol/L PMA for 30 min, and the expression level of m RNA and protein of n CEH was significantly increased after incubation with 0 1 渭 mol/L Rottlerin, a specific inhibitor of PKC 未, for 30 min. [conclusion] overexpression of adipophilin can promote the accumulation of cholesterol ester in macrophages, and inhibit the expression of n CEH m RNA and egg by inhibiting the accumulation of lipid in macrophages by PKC 未.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R543.5

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