炎癥、高脂狀態(tài)下Api6對小鼠巨噬細胞RAW264.7膽固醇轉(zhuǎn)運的影響
發(fā)布時間：2018-12-15 12:31
【摘要】：目的 凋亡抑制因子6(Apoptosis Inhibitor 6,Api6),又稱作AIM和Spα,是清道夫受體富含半胱氨酸殘基家族新成員。Api6在免疫調(diào)節(jié)和腫瘤發(fā)生中發(fā)揮了重要作用,其抑制巨噬細胞凋亡、從而參與AS的病變發(fā)展的作用已得到人們的認識。但是作為清道夫受體,Api6的在脂質(zhì)代謝中起到的作用還不為人們所熟知。本研究通過建立過表達Api6的RAW264.7小鼠巨噬細胞株,探討在炎癥和高脂狀態(tài)下Api6對小鼠巨噬細胞膽固醇轉(zhuǎn)運及相關基因表達的影響。 材料和方法 將體外擴增的Api6 cDNA全長片段插入載體PEGFP-N1中,構(gòu)建表達Api6基因的質(zhì)粒PEGFP-N1/Api6,酶切及測序鑒定。體外培養(yǎng)RAW264.7小鼠巨噬細胞,瞬時轉(zhuǎn)染PEGFP-N1/Api6質(zhì)粒,然后采用LPS和LDL刺激細胞建立炎癥和高脂模型,油紅O染色觀察細胞內(nèi)脂質(zhì)蓄積的情況;RT-PCR檢測在不同處理條件下細胞內(nèi)膽固醇轉(zhuǎn)運相關基因表達的情況。 結(jié)果 1.成功構(gòu)建質(zhì)粒PEGFP-N1/Api6;Western Blot和RT-PCR實驗證實:與空質(zhì)粒轉(zhuǎn)染組相比, RAW264.7細胞瞬時轉(zhuǎn)染質(zhì)粒PEGFP-N1/Api6后,細胞內(nèi)Api6蛋白表達增加,mRNA水平表達上調(diào)13倍。 2.油紅O實驗結(jié)果顯示:單純高脂以及炎癥和高脂同時存在可以導致RAW264.7細胞內(nèi)明顯的脂質(zhì)蓄積。但與空質(zhì)粒轉(zhuǎn)染組相比,瞬時轉(zhuǎn)染PEGFP-N1/Api6的RAW264.7細胞內(nèi)脂質(zhì)蓄積更為明顯。 3. RT-PCR實驗結(jié)果顯示:與空質(zhì)粒轉(zhuǎn)染組相比,過表達Api6對炎癥和高脂狀態(tài)下SRA和ABCG1的表達無明顯影響,但過表達Api6會影響高脂負荷下LDLR的負反饋調(diào)節(jié),清道夫受體CD36和膽固醇清除相關受體ABCA1的表達,從而導致細胞內(nèi)膽固醇蓄積增加。 結(jié)論 炎癥和高脂狀態(tài)下,過表達Api6可以增加RAW264.7細胞內(nèi)膽固醇的蓄積,其機制可能與Api6抑制LDLR的負反饋調(diào)節(jié),上調(diào)清道夫受體CD36表達,以及下調(diào)膽固醇清除相關受體ABCA1的表達有關。
[Abstract]:Objective apoptosis inhibitor 6 (Apoptosis Inhibitor 6 (Api6), also known as AIM and Sp 偽, is a new member of the scavenger receptor rich cysteine residue family. Api6 plays an important role in immunomodulation and tumorigenesis. Its role in inhibiting macrophage apoptosis and participating in the development of AS has been recognized. But as a scavenger receptor, the role of Api6 in lipid metabolism is not well known. The aim of this study was to investigate the effects of Api6 on cholesterol transport and related gene expression in murine macrophages under inflammatory and hyperlipidemic conditions by establishing RAW264.7 mouse macrophage cell lines with overexpression of Api6. Materials and methods the full-length Api6 cDNA fragment amplified in vitro was inserted into the vector PEGFP-N1 and the plasmid PEGFP-N1/Api6, expressing Api6 gene was digested and sequenced. The macrophages of RAW264.7 mice were cultured in vitro and transfected with PEGFP-N1/Api6 plasmid. Then the inflammatory and hyperlipidemic models were established by LPS and LDL stimulation cells. The accumulation of lipid in the cells was observed by oil red O staining. RT-PCR was used to detect the expression of cholesterol transporter related genes under different treatment conditions. Result 1. The successful construction of plasmid PEGFP-N1/Api6;Western Blot and RT-PCR confirmed that the expression of Api6 protein increased and the expression of mRNA increased 13 times after transient transfection of plasmid PEGFP-N1/Api6 in RAW264.7 cells compared with empty plasmid transfection group. 2. The results of oil red O experiment showed that hyperlipidemia and the presence of inflammation and hyperlipidemia could result in obvious lipid accumulation in RAW264.7 cells. However, compared with the empty plasmid transfection group, the accumulation of lipid in the RAW264.7 cells was more obvious than that in the transient transfection group. 3. RT-PCR results showed that overexpression of Api6 had no significant effect on the expression of SRA and ABCG1 in inflammation and hyperlipidemia, but overexpression of Api6 affected the negative feedback regulation of LDLR under high fat load. Scavenger receptor CD36 and cholesterol clearance associated receptor ABCA1 expression, resulting in increased intracellular cholesterol accumulation. Conclusion overexpression of Api6 can increase the accumulation of cholesterol in RAW264.7 cells under the condition of inflammation and hyperlipidemia. The mechanism may be related to the inhibition of Api6 on the negative feedback regulation of LDLR and the up-regulation of the expression of scavenger receptor CD36. And down-regulating the expression of cholesterol clearance related receptor ABCA1.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363
[Abstract]:Objective apoptosis inhibitor 6 (Apoptosis Inhibitor 6 (Api6), also known as AIM and Sp 偽, is a new member of the scavenger receptor rich cysteine residue family. Api6 plays an important role in immunomodulation and tumorigenesis. Its role in inhibiting macrophage apoptosis and participating in the development of AS has been recognized. But as a scavenger receptor, the role of Api6 in lipid metabolism is not well known. The aim of this study was to investigate the effects of Api6 on cholesterol transport and related gene expression in murine macrophages under inflammatory and hyperlipidemic conditions by establishing RAW264.7 mouse macrophage cell lines with overexpression of Api6. Materials and methods the full-length Api6 cDNA fragment amplified in vitro was inserted into the vector PEGFP-N1 and the plasmid PEGFP-N1/Api6, expressing Api6 gene was digested and sequenced. The macrophages of RAW264.7 mice were cultured in vitro and transfected with PEGFP-N1/Api6 plasmid. Then the inflammatory and hyperlipidemic models were established by LPS and LDL stimulation cells. The accumulation of lipid in the cells was observed by oil red O staining. RT-PCR was used to detect the expression of cholesterol transporter related genes under different treatment conditions. Result 1. The successful construction of plasmid PEGFP-N1/Api6;Western Blot and RT-PCR confirmed that the expression of Api6 protein increased and the expression of mRNA increased 13 times after transient transfection of plasmid PEGFP-N1/Api6 in RAW264.7 cells compared with empty plasmid transfection group. 2. The results of oil red O experiment showed that hyperlipidemia and the presence of inflammation and hyperlipidemia could result in obvious lipid accumulation in RAW264.7 cells. However, compared with the empty plasmid transfection group, the accumulation of lipid in the RAW264.7 cells was more obvious than that in the transient transfection group. 3. RT-PCR results showed that overexpression of Api6 had no significant effect on the expression of SRA and ABCG1 in inflammation and hyperlipidemia, but overexpression of Api6 affected the negative feedback regulation of LDLR under high fat load. Scavenger receptor CD36 and cholesterol clearance associated receptor ABCA1 expression, resulting in increased intracellular cholesterol accumulation. Conclusion overexpression of Api6 can increase the accumulation of cholesterol in RAW264.7 cells under the condition of inflammation and hyperlipidemia. The mechanism may be related to the inhibition of Api6 on the negative feedback regulation of LDLR and the up-regulation of the expression of scavenger receptor CD36. And down-regulating the expression of cholesterol clearance related receptor ABCA1.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363
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