| 张欢,吴科锋,田银,等.基于HIF-1α活性的高通量药物筛选细胞模型的建立及应用[J].中国海洋药物,2025,(2):-. |
| 基于HIF-1α活性的高通量药物筛选细胞模型的建立及应用 |
| Establishment and application of high-throughput drug screening cell model based on HIF-1α activity |
| 投稿时间：2023-11-16 修订日期：2024-01-13 |
| DOI： |
| 中文关键词: 低氧诱导因子1α 低氧反应元件 海洋微生物 药物筛选模型 |
| English Keywords:hypoxia-inducible factor-1α hypoxia response element marine microorganisms drug screening model |
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| 中文摘要: |
| 目的 构建基于HIF-1α活性的高通量药物筛选细胞模型，筛选出能够抑制HIF-1α活性的海洋来源微生物次级代谢产物粗提物。方法 将缺氧反应元件HRE核苷酸序列插入萤火虫荧光素酶报告基因载体pGL6-TA的多克隆位点上，构建重组质粒pHIF1-TA-Luc，并与内参质粒pRL-SV40-C瞬时共转染人源肝癌细胞HepG2，低氧培养并以HIF-1α抑制剂PX-478作为阳性对照，建立双荧光素酶报告基因筛选系统。对231份海洋来源微生物发酵粗提物样品进行活性筛选；CCK-8法检测粗提物对HepG2细胞活力的影响。结果 通过基因测序鉴定，重组质粒构建成功；通过荧光素酶检测发现，与常氧对照组相比，低氧实验组中重组质粒的相对荧光素酶活性明显上升，差异有统计学意义（P＜0.05），提示细胞低氧模型构建成功；与空载对照组pGL6-TA相比，插入HRE序列的重组质粒实验组相对荧光素酶活性明显上升，差异有统计学意义（P＜0.05）；PX-478作用24 h后，与对照组（0 μmol/L）相比，其相对荧光素酶活性显著降低（P＜0.05），提示基于HIF-1α活性的高通量药物筛选细胞模型构建成功；粗提物H768、H1458、Z-160-4和Z-29-4作用在筛选模型上可显著降低相对荧光素酶活性；CCK-8法检测结果显示粗提物H768、H1458、Z-160-4和Z-29-4对细胞活力无明显影响，差异无统计学意义（P＞0.05）。结论 建立了基于HIF-1α活性的高通量药物筛选细胞模型，筛选结果发现海洋来源微生物发酵粗提物H768、H1458、Z-160-4和Z-29-4能显著抑制HIF-1α的转录水平（优化表述方式），其活性成分值得开展进一步药物化学研究。 |
| English Summary: |
| Objective To construct a high-throughput drug screening cell model based on HIF-1α activity and screen out the crude extracts of marine-derived microbial Secondary metabolite that can inhibit HIF-1α activity. Methods Recombinant plasmid pHIF1-TA-Luc was constructed by inserting the nucleotide sequence of hypoxia response element HRE into the multiple cloning site of firefly luciferase reporter vector pGL6-TA, a dual luciferase reporter gene screening system was established by transient co-transfection of human hepatoma cell line HepG2 with plasmid pRL-SV40-C and HIF-1α inhibitor PX-478 under hypoxia. The activity of 231 marine-derived microbial fermentation extracts was screened and the effect of the extracts on the viability of HepG2 cells was detected by CCK-8 method. Results The recombinant plasmid was successfully constructed by gene sequencing;Compared with the normoxia control group, the relative luciferase activity of the recombinant plasmid in the hypoxia experimental group increased significantly (P＜0.05) , suggesting that the hypoxia model was successfully constructed; The relative luciferase activity of the recombinant plasmid pGL6-TA was significantly higher than that of the control group (P＜0.05); Compared with the control group (0 μmol/L) , the relative luciferase activity of PX-478 decreased significantly (P＜0.05) after 24 hours of treatment, suggesting that a high-throughput drug screening cell model based on HIF-1α activity was successfully constructed; Crude extracts H768, H1458, Z-160-4 and Z-29-4 could significantly reduce relative luciferase activity in the screening model; The results of CCK-8 assay showed that the crude extracts H768, H1458, Z-160-4 and Z-29-4 had no significant effect on cell viability (P＞0.05). Conclusion A high-throughput drug screening cell model based on HIF-1α activity was established and the screening results showed that marine-derived microbial fermentation extracts H768, H1458, Z-160-4 and Z-29-4 could significantly inhibit HIF-1α transcription, its active components are worthy of further medicinal chemistry research. |
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