炎症、高脂状态下Api6对小鼠巨噬细胞RAW264.7胆固醇转运的影响
发布时间：2018-12-15 12:31
【摘要】：目的 凋亡抑制因子6(Apoptosis Inhibitor 6,Api6),又称作AIM和Spα,是清道夫受体富含半胱氨酸残基家族新成员。Api6在免疫调节和肿瘤发生中发挥了重要作用,其抑制巨噬细胞凋亡、从而参与AS的病变发展的作用已得到人们的认识。但是作为清道夫受体,Api6的在脂质代谢中起到的作用还不为人们所熟知。本研究通过建立过表达Api6的RAW264.7小鼠巨噬细胞株,探讨在炎症和高脂状态下Api6对小鼠巨噬细胞胆固醇转运及相关基因表达的影响。 材料和方法 将体外扩增的Api6 cDNA全长片段插入载体PEGFP-N1中,构建表达Api6基因的质粒PEGFP-N1/Api6,酶切及测序鉴定。体外培养RAW264.7小鼠巨噬细胞,瞬时转染PEGFP-N1/Api6质粒,然后采用LPS和LDL刺激细胞建立炎症和高脂模型,油红O染色观察细胞内脂质蓄积的情况;RT-PCR检测在不同处理条件下细胞内胆固醇转运相关基因表达的情况。 结果 1.成功构建质粒PEGFP-N1/Api6;Western Blot和RT-PCR实验证实:与空质粒转染组相比, RAW264.7细胞瞬时转染质粒PEGFP-N1/Api6后,细胞内Api6蛋白表达增加,mRNA水平表达上调13倍。 2.油红O实验结果显示:单纯高脂以及炎症和高脂同时存在可以导致RAW264.7细胞内明显的脂质蓄积。但与空质粒转染组相比,瞬时转染PEGFP-N1/Api6的RAW264.7细胞内脂质蓄积更为明显。 3. RT-PCR实验结果显示:与空质粒转染组相比,过表达Api6对炎症和高脂状态下SRA和ABCG1的表达无明显影响,但过表达Api6会影响高脂负荷下LDLR的负反馈调节,清道夫受体CD36和胆固醇清除相关受体ABCA1的表达,从而导致细胞内胆固醇蓄积增加。 结论 炎症和高脂状态下,过表达Api6可以增加RAW264.7细胞内胆固醇的蓄积,其机制可能与Api6抑制LDLR的负反馈调节,上调清道夫受体CD36表达,以及下调胆固醇清除相关受体ABCA1的表达有关。
[Abstract]:Objective apoptosis inhibitor 6 (Apoptosis Inhibitor 6 (Api6), also known as AIM and Sp 伪, 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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
[Abstract]:Objective apoptosis inhibitor 6 (Apoptosis Inhibitor 6 (Api6), also known as AIM and Sp 伪, 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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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