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  本文選題：脂肪分化相關(guān)蛋白 + 中性膽固醇酯水解酶�。� 參考：《南華大學(xué)》2015年碩士論文

【摘要】：[目的]獲得adipophilin通過n CEH促使細(xì)胞內(nèi)脂質(zhì)積聚的可靠證據(jù),闡明adipophilin通過n CEH促使細(xì)胞內(nèi)脂質(zhì)積聚的機(jī)制,為動脈粥樣硬化的防治尋找新靶點(diǎn)。[方法]將adipophilin沉默表達(dá)和穩(wěn)定高表達(dá)的逆轉(zhuǎn)錄病毒質(zhì)粒載體轉(zhuǎn)染PA317包裝細(xì)胞,收集病毒,感染RAW264.7細(xì)胞,篩選,制備高和沉默表達(dá)adipophilin的細(xì)胞。在高和沉默表達(dá)adipophilin的RAW264.7細(xì)胞中,分別用RT-PCR和Western blot檢測n CEH的m RNA和蛋白表達(dá)的變化;高效液相色譜法檢測細(xì)胞內(nèi)膽固醇酯的含量。在高表達(dá)adipophilin的RAW264.7細(xì)胞中,用PKCδ激動劑0.1μmol/L PMA預(yù)孵育30 min后,或者用PKCδ特異性抑制劑0.1μmol/L Rottlerin預(yù)孵育30 min后,分別用RT-PCR和Western blot檢測n CEH的m RNA水平和蛋白表達(dá)的改變;高效液相色譜法來檢測細(xì)胞內(nèi)膽固醇酯含量。[結(jié)果]在高表達(dá)adipophilin的RAW264.7細(xì)胞中,膽固醇酯的含量明顯增加,在沉默表達(dá)adipophilin的RAW264.7細(xì)胞中,膽固醇酯的含量顯著減少;同時,adipophilin能夠抑制n CEH m RNA和蛋白的表達(dá);在高表達(dá)adipophilin的RAW264.7細(xì)胞中,用PKCδ激動劑0.1μmol/L PMA預(yù)孵育30 min后,n CEH的m RNA與蛋白表達(dá)水平明顯降低,用PKCδ特異性抑制劑0.1μmol/L Rottlerin預(yù)孵育30 min后,n CEH的m RNA和蛋白表達(dá)水平顯著增高。[結(jié)論]高表達(dá)adipophilin促進(jìn)巨噬細(xì)胞內(nèi)膽固醇酯的蓄積,抑制n CEH m RNA和蛋的表達(dá);adipophilin通過PKCδ抑制n CEH促進(jìn)巨噬細(xì)胞內(nèi)脂質(zhì)蓄積。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R543.5

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