血栓性疾病个体化用药指导基因芯片试剂盒的制备和效果评价

doi:10.13481/j.1671-587x.20190613

吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (06): 1267-1274.doi: 10.13481/j.1671-587x.20190613

血栓性疾病个体化用药指导基因芯片试剂盒的制备和效果评价

袁语泽¹, 马明星², 王国强¹, 关雪娃¹, 王紫嫣¹, 郭英俏³, 郑敬彤¹, 章宏⁴, 陈光⁵, 王放¹

1. 吉林大学基础医学院病原生物学教研室, 吉林长春 130021;
2. 吉林寰基生物科技有限公司, 吉林长春 130012;
3. 吉林大学基础医学院遗传学教研室, 吉林长春 130021;
4. 吉林大学基础医学院生理学教研室, 吉林长春 130021;
5. 吉林大学第一医院血管外科, 吉林长春 130021

收稿日期:2019-02-18 出版日期:2019-12-05 发布日期:2019-12-05
通讯作者: 王放,教授,博士研究生导师(Tel:0431-85619509,E-mail:wf@jlu.edu.cn) E-mail:wf@jlu.edu.cn
作者简介:袁语泽(1994-),女,吉林省吉林市人,在读医学博士,主要从事血栓性疾病早期诊断和机制方面的研究。
基金资助:
吉林省发改委产业创新专项资金项目资助课题（2016C050-3）；吉林省科技厅中药办科技成果转化计划项目资助课题（20170311040YY）；吉林省发改委产业技术研究与开发项目资助课题（2017c058-1）

Preparation of gene chip detection kit for individualized treatment of thrombotic diseases and evaluation on its effectiveness

YUAN Yuze¹, MA Mingxing², WANG Guoqiang¹, GUAN Xuewa¹, WANG Ziyan¹, GUO Yingqiao³, ZHENG Jingtong¹, ZHANG Hong⁴, CHEN Guang⁵, WANG Fang¹

1. Department of Pathogen Biology, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
2. Jilin Huanji Biotechnology Co. Ltd., Changchun 130012, China;
3. Department of Genetics, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
4. Department of Physiology, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
5. Department of Vascular Surgery, First Hospital, Jilin University, Changchun 130021, China

Received:2019-02-18 Online:2019-12-05 Published:2019-12-05

摘要/Abstract

摘要： 目的：探讨血栓性疾病（TD）个体化用药指导基因芯片的制备过程，阐明一种快速、高通量检测氯吡格雷和华法林药物代谢基因的方法。方法：根据氯吡格雷药物代谢基因位点（CYP3A4、CYP2C19*17、CYP2C19*2和CYP2C19*3）和华法林药物代谢基因位点（CYP4F2*3、GGCX、VKORC1-2、VKORC1-1、CYP2C9*2和CYP2C9*3）设计相应的探针和引物，制备TD个体化用药指导的基因芯片检测试剂盒。经过DNA提取、PCR扩增、杂交、洗脱和扫描等步骤对基因芯片的灵敏性、重复性和稳定性进行评价。收集3个地区的150例TD受试者样本并进行药物代谢基因检测，以受试者在临床中接受的用药指导及6个月后患者的疾病恢复情况为依据，评价基因芯片试剂盒的有效率。结果：基因芯片检测2种药物的代谢基因均出现良好的杂交信号；芯片检测PCR扩增产物的最低检测浓度为10³ copies·mL^-1；对不同批次的3张芯片及同一批次的10张芯片进行平行测定，符合率为100%；芯片具有稳定性，有效期长达6个月；来源于3个地区的150例TD样本验证基因芯片检测试剂盒的有效率在90%以上。结论：成功制备了高通量检测氯吡格雷和华法林药物代谢基因的TD个体化用药指导基因芯片试剂盒，该方法灵敏度高，重复性好，稳定性强，制备效果优良。

关键词: 血栓性疾病, 基因芯片, 氯吡格雷, 华法林

Abstract: Objective: To investigate the preparation process of gene chips for individualized treatment of thrombotic diseases (TD),and to elucidate a rapid and high-throughput method for detecting the drug metabolism genes of clopidogrel and warfarin. Methods: Different kinds of probes and primers were designed according to the clopidogrel drug metabolism gene sites (CYP3A4,CYP2C19*17,CYP2C19*2, and CYP2C19*3) and warfarin drug metabolism gene sites (CYP4F2*3,GGCX,VKORC1-2,VKORC1-1,CYP2C9*2, and CYP2C9*3) to prepare the gene chip detection kit for individualized treatment of TD.The steps of microarray detection were DNA extraction,PCR amplification,hybridization,elution and scanning.The sensitivity,repeatability and stability of the gene chips were evaluated. The samples of 150 subjects with TD from three regions were selected,and the drug metabolism genes were detected.Based on the medical guidances in the clinic and the disease recovery status after 6 months,the effectiveness of gene chip detection kit was determined. Results: The effective hybridization signals were observed in the metabolism genes of two kinds of drugs detected by gene chips;the minimum detection concentration of PCR amplification products was 10³ copies·mL^-1 detected by gene chip;the coincidence rate of simultaneous determination of three chips in different batches and parallel measurement of 10 chips in the same batch was 100%;the chip was stable and could be stored for at least 6 months;the effective rate of gene chip detection kit in the 150 TD subjects from three regions was more than 90%. Conclusion: The high-throughput gene chip detection kit of individualized treatment of TD to detect the drug metabolism genes of clopidogrel and warfarin are successfully established,and the method has high sensitivity,satisfactory repeatability,strong stability and excellent preparation effect.

Key words: thrombotic disease, gene chip, clopidogrel, warfarin

中图分类号:

R540.4

袁语泽, 马明星, 王国强, 关雪娃, 王紫嫣, 郭英俏, 郑敬彤, 章宏, 陈光, 王放. 血栓性疾病个体化用药指导基因芯片试剂盒的制备和效果评价[J]. 吉林大学学报(医学版), 2019, 45(06): 1267-1274.

YUAN Yuze, MA Mingxing, WANG Guoqiang, GUAN Xuewa, WANG Ziyan, GUO Yingqiao, ZHENG Jingtong, ZHANG Hong, CHEN Guang, WANG Fang. Preparation of gene chip detection kit for individualized treatment of thrombotic diseases and evaluation on its effectiveness[J]. Journal of Jilin University(Medicine Edition), 2019, 45(06): 1267-1274.

参考文献

[1] 刘丽,季婷婷,戴淑娟,等.血栓性疾病的药物治疗研究进展[J].齐齐哈尔医学院学报,2018,39(23): 2807-2808.
[2] FURIE B,FLAUMENHAFT R.Thiol isomerases in thrombus formation [J].Circ Res,2014,114(7): 1162-1173.
[3] 刘莅欣,胡桃红.血栓性疾病抗栓治疗的研究进展[J].中国临床医生,2013,41(5): 15-17.
[4] 徐可.冠心病患者基因多态性、氯吡格雷反应性及临床预后的相关性研究[D].南京:南京医科大学,2018.
[5] 梅丹,都丽萍,刘昌伟.基因多态性与华法林个体化用药[J].中华外科杂志,2016,54(2): 144-147.
[6] GRAHAM B R,MENON B K.Clopidogrel load reduces emboli in carotid artery stenosis with free-floating thrombus [J].Can J Neurol Sci,2017,44(5): 594-596.
[7] 张琳,王丹.氯吡格雷基因检测的临床应用分析[J].世界最新医学信息文摘,2018,18(76): 175.
[8] TAN S S N,FONG A Y Y,MEJIN M,et al.Association of CYP2C19*2 polymorphism with clopidogrel response and 1-year major adverse cardiovascular events in a multiethnic population with drug-eluting stents [J].Pharmacogenomics,2017,18(13): 1225-1239.
[9] LIU R,ZHOU Z Y,CHEN Y B,et al.Associations of CYP3A4,NR1I2,CYP2C19 and P2RY12 polymorphisms with clopidogrel resistance in Chinese patients with ischemic stroke [J].Acta Phamaco Sin,2016,37(7): 882-888.
[10] FLORA D R,RETTIE A E,BRUNDAGE R C,et al.CYP2C9 genotype-dependent warfarin pharmacokinetics: impact of CYP2C9 genotype on R- and S-warfarin and their oxidative metabolites [J].J Clin Pharmacol,2017,57(3): 382-393.
[11] SHENDRE A,BROWN T M,LIU N,et al.Race-specific influence of CYP4F2 on dose and risk of hemorrhage among warfarin users [J].Pharmacotherapy,2016,36(3): 263-272.
[12] GEMMATI D,BURINI F,TALARICO A,et al.The active metabolite of warfarin (3'-Hydroxywarfarin) and correlation with INR,warfarin and drug weekly dosage in patients under oral anticoagulant therapy: A pharmacogenetics study [J].PLoS One,2016,11(9): e0162084.
[13] KRISHNA KUMAR D,SHEWADE D G,LORIOT M A,et al.Effect of CYP2C9,VKORC1,CYP4F2 and GGCX genetic variants on warfarin maintenance dose and explicating a new pharmacogenetic algorithm in South Indian population [J].Eur J Clin Pharmacol,2014,70(1): 47-56.
[14] WANG J.From DNA biosensors to gene chips [J].Nucleic Acids Res,2000,28(16): 3011-3016.
[15] ORTEL T L,ERKAN D,KITCHENS C S.How I treat catastrophic thrombotic syndromes [J].Blood,2015,126(11): 1285-1293.
[16] KAZUI M,NISHIYA Y,ISHIZUKA T,et al.Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite [J].Drug Metab Dispos,2010,38(1): 92-99.
[17] SAIZ-RODRIGUEZ M,ROMERO-PALACIAN D,VILLALOBOS-VILDA C,et al.Influence of CYP2C19 phenotype on the effect of clopidogrel in patients undergoing a percutaneous neurointervention procedure [J].Clin Pharmacol Ther,2019,105(3): 661-671.
[18] ZHUO Z L,XIAN H P,LONG Y,et al.Association between CYP2C19 and ABCB1 polymorphisms and clopidogrel resistance in clopidogrel-treated Chinese patients [J].Anatol J Cardiol,2018,19(2): 123-129.
[19] HOLMES M V,PEREL P,SHAH T,et al.CYP2C19 genotype,clopidogrel metabolism,platelet function,and cardiovascular events: a systematic review and meta-analysis [J].JAMA,2011,306(24): 2704-2714.
[20] DEAN L.Warfarin therapy and the genotypes CYP2C9 and VKORC1[J].Med Gene Summ,2012,2: 257-263.
[21] KULMYRZAEVA N,TATARUNAS V,SKIPSKIS V,et al. Gene polymorphism of CYP2C19*2,*3 and CYP3A4* 1B and early stent thrombosis: case reports and literature review [J].Pers Med,2016,13(5): 423-428.
[22] GONZALEZ DELLA VALLE A,KHAKHARIA S,GLUECK C J,et al.VKORC1 variant genotypes influence warfarin response in patients undergoing total joint arthroplasty: a pilot study [J].Clin Orthop Relat R,2009,467(7): 1773-1780.
[23] 徐晓丽,林娟,鄢仁祥.基因芯片与高通量测序技术的原理与应用的比较[J].中国生物化学与分子生物学报,2018,34(11): 1166-1174.
[24] SCHNEIDER A K,NIEMEYER C M.DNA Surface Technology: from gene sensors to integrated systems for life and materials sciences [J].Angew Chem Int Ed Engl,2018,57(52): 16959-16967.
[25] 周宏伟.Verigene基因芯片法检测与氯吡格雷代谢相关基因CYP2C19多态性[C].中华医学会(Chinese Medical Association),中华医学会微生物学与免疫学分会.中华医学会第十二次全国临床微生物学术年会暨第十一次全球华人临床微生物学与感染症学术年会论文汇编.北京:中华医学会(Chinese Medical Association),中华医学会微生物学与免疫学分会,2015:1.
[26] FEREIDOUNI M,MOOSSAVI M,KAZEMI T,et al.Association between polymorphisms of VKORC1 and CYP2C9 genes with warfarin maintenance dose in a group of warfarin users in Birjand city,Iran [J].J Cell Biochem,2019,120(6): 9588-9593.
[27] 石佳,贾佳,李国福.危重症患者静脉血栓栓塞症风险评估及预防研究进展[J].中国实用内科杂志,2019,39(2):185-188.

血栓性疾病个体化用药指导基因芯片试剂盒的制备和效果评价

Preparation of gene chip detection kit for individualized treatment of thrombotic diseases and evaluation on its effectiveness

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