靶向γ-谷氨酰羧化酶的出/凝血药物筛选体系的建立及初步应用
Development and Preliminary Application of a Screening System for Bleeding or Clotting Drugs Targeting γ-glutamyl Carboxylase
刘迅婕 1洪敏雯 1潘杨 1李世新 1李清峰 2任方哲 1郝振宇3
作者信息
- 1. 扬州大学生物科学与技术学院,扬州,225009
- 2. 扬州大学附属医院,扬州,225009
- 3. 扬州大学生物科学与技术学院,扬州,225009;扬州大学附属医院,扬州,225009
- 折叠
摘要
维生素K依赖性凝血缺陷Ⅰ型疾病(vitamin K-dependent coagulation factors deficiency 1,VKCFD1)是一种罕见的常染色体隐性遗传出血性疾病,由γ-谷氨酰羧化酶(gamma-glutamyl carboxylase,GGCX)基因突变引起.目前,VKCFD1的主要治疗药物是维生素K,,但由于其并非直接靶向GGCX,临床效果存在巨大的个体差异.因此,筛选直接针对GGCX的靶向药物十分必要,而关键是构建靶向GGCX的药物评价体系.本研究基于维生素K环氧化物还原酶(vitamin K epoxide reductase,VKOR)基因敲除的细胞系(DKO HEK293),利用CRISPR/Cas9技术将内源性GGCX进一步敲除(GCKO细胞系).随后,将致病突变的GGCX(I532T)与凝血因子X(coagulation factor X,FX)或抗凝因子C(protein C,PC)稳定双表达形成报告细胞系.基于该细胞系,通过检测FX和PC的羧化效率,进行GGCX靶向药物的初步筛选.结果发现,血根碱和高三尖杉酯碱抑制细胞增殖,几乎完全抑制FX和PC的羧化活性.原阿片碱、尿囊素和顺式乌头酸同时显著提高FX和PC的羧化活性,且与细胞增殖无关.此外,京尼平、地奥司明、番泻苷A和(+)-儿茶素水合物对FX羧化活性具有显著的促进作用,而苯乙酰胺和硝酸硫胺素则对PC羧化水平具有显著的促进作用,表明本研究所建立的以GGCX为靶点的出/凝血药物筛选体系可以高效、及时反馈药物针对FX和PC的羧化水平的影响,为筛选治疗VKCFD1的药物提供有力的工具和参考.
Abstract
Vitamin K-dependent coagulation factors deficiency 1(VKCFD1)is a rare autosomal recessive bleeding disorder caused by mutations in the gamma-glutamyl carboxylase(GGCX)gene.Currently,the main treatment for VKCFD1 is vitamin K1,but there are significant variations in clinical outcomes due to its indirect targeting of GGCX.It is essential to identify drugs that target GGCX directly,and a crucial step in this process is to establish a drug evaluation system for GGCX.In this study,we utilized a cell line(DKO HEK293)with the vitamin K epoxide reductase(VKOR)gene knocked out and further knocked out endogenous GGCX using CRISPR/Cas9 technology(GCKO cell line).We then created a reporter cell line by stably expressing both the pathogenic GGCX mutant(I532T)and either coagulation factor X(FX)or anticoagulant protein C(PC).Based on this cell line,a preliminary screening of drugs targeting GGCX was conducted by measuring the carboxylation efficiency of FX and PC.The results showed that sanguinarine and homoharringtonine hindered cell proliferation and nearly completely blocked the carboxylation of FX and PC.Protopine,allantoin,and cis-aconitate significantly increased it and without affecting cell proliferation.In addition,genipin,diosmin,sennoside A,and(+)-catechin hydrate significantly enhanced FX carboxylation activity,while 2-phenylacetamide and thiamine nitrate notably boosted PC carboxylation levels.It demonstrates that the screening system for bleeding or clotting drugs targeting GGCX established in this study can provide efficient and timely feedback on the effects of the carboxylation of FX and PC,providing powerful tool and reference for screening drugs for the treatment of VKCFD1.
关键词
VKCFD1/γ-谷氨酰羧化酶/药物筛选/ELISA/出/凝血疾病Key words
VKCFD1/γ-glutamyl carboxylase/Drug screening/ELISA/Bleeding or clotting disorders引用本文复制引用
基金项目
国家自然科学基金项目(82370136)
江苏省自然科学基金项目(BK20231333)
江苏省大学生创新创业训练计划项目(202211117057Z)
出版年
2024
NETL
NSTL
万方数据