高脂、炎症状态下Api6在巨噬细胞中的表达研究

发布时间：2018-07-26 13:04

【摘要】： 目的凋亡抑制因子6(Apoptosis Inhibitor 6,Api6)也叫作AIM或Spα,是清道夫受体富含半胱氨酸残基家族新成员,在免疫反应、肿瘤发生中发挥着重要的作用;但作为清道夫受体,Api6在脂质代谢中的作用尚未得到深入研究。本研究选用人THP-1单核/巨噬细胞和小鼠RAW264.7巨噬细胞探讨①高脂、炎症状态下,两种巨噬细胞脂质蓄积情况;②高脂和/或炎症状态下,清道夫受体SR-A、CD36和Api6在两种巨噬细胞中的表达;③高脂和/或炎症状态下,核受体LXRalpha、LXRbeta表达与Api6表达的相关关系。材料和方法 1.从人新鲜血浆中提取LDL,氧化制备成oxLDL;采用oxLDL刺激细胞建立高脂模型,采用脂多糖(LPS)刺激细胞建立炎症模型,油红O染色观察巨噬细胞脂质蓄积情况。 2.提取Balb/c小鼠腹腔巨噬细胞mRNA,逆转录扩增获得Api6cDNA全长,构建入pCDNA3.1质粒中,获得可以表达Api6基因的质粒pCDNA3.1/Api6。 3.实时荧光定量PCR技术(real-time quantitative PCR)检测Api6、CD36、SR-A、LXRalpha、LXRbeta、ABCA1、ABCG1等基因在各种处理条件下,两种巨噬细胞中mRNA的表达水平。结果 1.研究发现200 ng/ml的LPS和100μg/ml的oxLDL处理可以导致体外培养的两种巨噬细胞出现异常的脂质蓄积。 2.成功构建了pCDNA3.1/Api6质粒,并对RAW264.7巨噬细胞进行瞬时转染,使Api6表达上调了12500倍。 3.高脂情况下,清道夫受体SRA在RAW264.7细胞和THP-1细胞中的表达分别上调了6倍、2.5倍和6倍、44倍。但同样处理条件下,Api6在RAW264.7细胞的表达没有明显上调;而炎症和高脂同时存在时,Api6在THP-1细胞中的表达却上调了2.7倍。 4. LXR受体激动剂T0901317可以使RAW264.7细胞中LXR的靶基因ABCA1和ABCG1基因表达分别上调22倍和7倍;THP-1巨噬细胞中ABCA1和ABCG1基因表达均上调9倍;但LXR受体激动剂T0901317没有改变Api6在RAW264.7细胞中的表达,Api6在THP-1细胞中的表达则上调了2倍。Api6在各种处理条件下表达水平的变化趋势与LXRα的表达水平相关。结论 200 ng/ml的LPS和100μg/ml的oxLDL处理可以导致体外培养的RAW264.7和THP-1细胞出现异常的脂质蓄积。炎症、高脂状态下,Api6的表达水平与核受体LXR受体α亚型的表达水平明显相关;由于缺乏LXRα受体,体外培养的小鼠巨噬细胞株RAW264.7细胞Api6基础表达量较低,因此可以作为载体细胞,用以建立过表达Api6的细胞模型深入研究Api6在脂质代谢中的作用。
[Abstract]:Objective apoptosis inhibitor 6 (Apoptosis Inhibitor 6 (Api6), also known as AIM or Sp 伪, is a new member of scavenger receptor rich cysteine residue family, which plays an important role in immune response and tumorigenesis. However, the role of Api6 as a scavenger receptor in lipid metabolism has not been further studied. In this study, human THP-1 mononuclear / macrophages and mouse RAW264.7 macrophages were used to investigate 1 hyperlipidemia and 2 hyperlipidemia and / or inflammation. The expression of scavenger receptor SR-AG CD36 and Api6 in macrophages was found to be related to the expression of LXR beta and Api6 under hyperlipidemia and / or inflammation. Materials and methods 1. LDLs were extracted from human fresh plasma and oxidized to form oxLDL.The hyperlipidemia model was established by oxLDL stimulation cells, inflammatory model was established by lipopolysaccharide (LPS) stimulation cells, and lipid accumulation of macrophages was observed by oil red O staining. 2. The Balb/c mouse peritoneal macrophage mRNAs were extracted, the full length of Api6cDNA was obtained by reverse transcription amplification, and the plasmid pCDNA3.1 / Api6.3 was constructed into the pCDNA3.1 plasmid. Real-time fluorescence quantitative PCR (real-time quantitative PCR) was used to detect the expression of mRNA in two kinds of macrophages. Result 1. It was found that 200 ng/ml LPS and 100 渭 g/ml oxLDL could induce abnormal lipid accumulation in two kinds of macrophages cultured in vitro. PCDNA3.1/Api6 plasmid was successfully constructed and transient transfection of RAW264.7 macrophages was carried out. The expression of Api6 was up-regulated by 12500 times. Under hyperlipidemia, the expression of scavenger receptor SRA in RAW264.7 cells and THP-1 cells was increased by 6 times, 2.5 times and 6 times, respectively. However, the expression of Api6 in RAW264.7 cells was not significantly up-regulated under the same treatment condition, but the expression of Api6 in THP-1 cells was increased by 2.7 times when inflammation and hyperlipidemia were present at the same time. LXR receptor agonist T0901317 could up-regulate the expression of ABCA1 and ABCG1 genes of LXR target genes in RAW264.7 cells by 22 times and 7 times respectively. The expression of ABCA1 and ABCG1 genes in THP-1 macrophages were all up-regulated by 9 times. However, LXR receptor agonist T0901317 did not change the expression of Api6 in RAW264.7 cells. However, the expression of Api6 in THP-1 cells increased by 2 times. The change trend of Api6 expression level was related to the expression of LXR 伪 under various conditions. Conclusion 200 ng/ml LPS and 100 渭 g/ml oxLDL could induce abnormal lipid accumulation in cultured RAW264.7 and THP-1 cells. The expression level of Api6 was significantly related to the expression of nuclear receptor LXR receptor 伪 subtype under the condition of inflammation and hyperlipidemia. Due to the lack of LXR 伪 receptor, the basic expression of Api6 in murine macrophage cell line RAW264.7 in vitro was low, so it could be used as carrier cell. To establish a cell model of overexpression of Api6 to study the role of Api6 in lipid metabolism.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R364.2

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