建立以HIF-1α为靶标的高通量筛选防治动脉粥样硬化先导化合物的细胞模型

A cell model for high-throughput screening lead compounds targeting HIF-1α for atherosclerosis treatment

目的建立一个高通量筛选防治动脉粥样硬化先导化合物的体外细胞模型。方法克隆HIF-1α低氧反应元件(hypoxia response element,HRE)至荧光素酶报告基因表达载体pGL3-Enhancer,构建荧光素酶表达载体pGL3-HIF-1α-HRE,转染人单核细胞THP-1并筛选稳定表达细胞株THP-1-HIF-1α-HRE。结果 Real Time-PCR检测表明低氧培养可以有效上调THP-1-HIF-1α-HRE细胞HIF-1α蛋白表达和荧光素酶活性,而洛伐他汀和姜黄素预处理可以有效抑制低氧引起的THP-1-HIF-1α-HRE细胞内HIF-1α蛋白表达及荧光素酶活性。结论成功建立了筛选抗动脉粥样硬化的先导化合物体外高通量细胞模型THP1-HIF-1α-HRE。

Objective To establish a high-throughput in-vitro screening cell model for anti-atherosclerosis leading compounds.Methods Hypoxia response element(HRE)was cloned into a luciferase reporter vector,pGL3-Enhancer,to construct pGL3-HIF-1α-HRE.The THP-1 human monocyte cell line was infected with the pGL3-HIF-1α-HRE and a stable cell line,THP-1-HIF-1α-HRE,was screened.Results Real-time PCR assay showed that HIF-1αexpression and luciferase activity in THP-1-HIF-1α-HRE cells was effectively upregulated by hypoxia.The increase of HIF-1αexpression and luciferase activity induced by hypoxia was significantly inhibited by lovastatin or curcumin.Conclusion THP1-HIF-1α-HRE,an in-vitro cell model for high-throughput screening lead compounds for anti-atherosclerosis(AS)was successfully established.