人载脂蛋白A-I表达上调剂的发现与活性研究  

Identification and characterization of novel upregulator of human Apolipoprotein A-I

作　　者：杜郁[1] 贾晓健[1] 王丽[1] 王丽非[1] 姜华军[1] 杨帆[1] 司书毅[2] 杨媛[1] 洪斌[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所卫生部抗生素生物工程重点实验室,北京100050 [2]中国医学科学院国家新药(微生物)筛选实验室,北京100050

出　　处：《中国医药生物技术》2014年第2期95-102,共8页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30801401);"重大新药创制"国家科技重大专项(2012ZX09301002-003;2012ZX09301002-001)

摘　　要：目的筛选上调人载脂蛋白A-I(ApoA-I)基因表达的新型活性化合物,并在表达和功能水平对其作用进行研究。方法利用人ApoA-I基因表达上调剂筛选模型对化合物库进行筛选;对发现的活性化合物在ApoA-I启动子水平进行量效关系研究;利用实时荧光定量RT-PCR检测ApoA-I mRNA的表达水平;利用Western blot、ELISA和流式细胞技术检测ApoA-I的蛋白表达;利用胆固醇流出试验检测ApoA-I介导的巨噬细胞胆固醇外流的情况。结果从20 000样次化合物筛选中得到活性化合物5238B-69,在一定浓度范围内存在转录调控活性的量-效关系,当化合物浓度为0.30μg/ml时,上调ApoA-I表达活性达到最高值(408%),EC50为0.01μg/ml。5238B-69能显著上调HepG2细胞中ApoA-I mRNA的水平;同时,Western blot结果显示5238B-69能增加HepG2细胞ApoA-I蛋白的表达。ELISA结果显示,与对照相比,5238B-69作用48 h时,胞外ApoA-I水平增加了48%,流式细胞检测表明,5238B-69作用24 h后,胞内ApoA-I蛋白水平增加了21.4%。功能分析试验显示,5238B-69作用HepG2细胞上调生成的ApoA-I,能促进巨噬细胞RAW264.7内[3H]标记的胆固醇的流出。结论得到一个具有上调ApoA-I表达的活性化合物,在提高ApoA-I表达水平和生物学功能方面均有明显的效果。Objective To find potential small molecules that increase endogenous synthesis of apolipoprotein A-I （ApoA-I）, and to identify its ability of increasing ApoA-I at expression and functional levels. Methods Compounds screening was performed with established screening model based on the ApoP-Luc HepG2 cell line. Dose-response relationship of the active compound was achieved in promoter activity. The expression of ApoA-I in HepG2 cells regulated by the compound was detected by real-time quantitative reverse transcription-polymerase chain reaction （RT-PCR）, Western blot, ELISA and flow cytometry analysis. The cholesterol efflux assay was applied to investigate the effect of promoted ApoA-I on mediating lipid transfer in RAW 264.7 cells. Results In the present study, compound 5238B-69 was found using the screening model. 5238B-69 increased ApoA-I promoter activity in a dose-dependent manner with EC50 of 0.01 μg/ml, and maximal activity of 408%at 0.30μg/ml. 5238B-69 significantly increased ApoA-I mRNA level and protein level in HepG2 cells. ELISA and flow cytometry analysis showed 5238B-69 could increase ApoA-I protein by 48%and 21.4%compared with control in the media and cells, respectively. The functional efflux assay further illustrated the role of ApoA-I induced by 5238B-69 with RAW 264.7 cells. Conclusion A potential small molecular upregulator was obtained, which could significantly increase the expression of human ApoA-I in liver cells and promote cholesterol efflux from mice macrophages.

关 键 词：载脂蛋白A-I 基因表达调控 动脉粥样硬化 转录水平 

分 类 号：R341[医药卫生—基础医学]