基于CYP2C19、CYP2C9、CYP3A4、ABCB1基因多态性的伏立康唑个体化用药研究

发布时间：2018-06-20 16:34

  本文选题：伏立康唑 + CYP2C19　； 参考：《郑州大学》2017年硕士论文

【摘要】：背景近几十年来,侵袭性真菌感染的情况日益严峻,并伴随着较高的病死率,是院内感染的重要死亡原因之一。目前抗真菌药物仍是控制侵袭性真菌感染的主要手段;伏立康唑作为第二代三唑类抗真菌药,具有更强的靶点亲和力和抗真菌活性,已被多个指南推荐为治疗侵袭性曲霉菌感染的一线治疗药物。伏立康唑主要由肝脏CYP450酶系代谢,CYP2C19、CYP3A4、CYP2C9系其主要代谢酶,ABCB1是体内主要的转运蛋白,且CYP2C19、CYP3A4、CYP2C9、ABCB1存在的基因多态性能够不同程度的影响酶代谢或转运蛋白活性;稳态谷浓度是伏立康唑体内暴露剂量的评价指标,与治疗效果及不良反应密切相关;目前在伏立康唑的临床抗真菌治疗中仍存在着一些问题:1.伏立康唑治疗窗窄;2.伏立康唑个体差异显著;3.伏立康唑有较严重的不良反应;4.伏立康唑治疗费用高。伏立康唑的临床治疗受多种遗传因素及非遗传因素的影响,截止现在,尚不能完全解释伏立康唑个体差异的原因。当前亟需解决的就是探究伏立康唑显著个体差异的影响因素,从而实现伏立康唑的个体化用药。目的1、监测中国汉族血液病患者伏立康唑血清初始稳态谷浓度[(Css)min],探讨伏立康唑药代动力学特点;2、通过对中国汉族血液病患者CYP2C19,CYP3A4,CYP2C9以及ABCB1基因多态性的检测,初步了解河南省人民医院中国汉族血液病患者伏立康唑代谢酶及及ABCB1基因多态性的分布特点;3、探讨CYP2C19、CYP3A4、CYP2C9等主要代谢酶以及转运蛋白ABCB1的基因多态性对河南省人民医院中国汉族血液病患者(Css)min的影响;4、探讨河南省人民医院中国汉族血液病患者临床非遗传因素对(Css)min的影响。方法1、筛选于2015年3月至2016年9月收住河南省人民医院,符合2013年《血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则》血液病侵袭性真菌病的诊断标准,使用伏立康唑治疗或预防真菌感染并接受伏立康唑血药浓度监测的中国汉族血液病患者进行回顾性研究;2、采用高效液相色谱法(HPLC)监测伏立康唑血清谷浓度,通过全血DNA提取试剂盒提取DNA,紫外/可见光分光光度计对提取DNA进行浓度和质量的检测,并将DNA样本保存于-40℃冰箱中;3、采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术对21个CYP2C19、CYP3A4、CYP2C9、ABCB1等的单核苷酸多态性位点(SNPs)进行基因分型。PCR引物和单碱基延伸引物由Assay Designer软件包设计,检测过程采用Mass ARRAY系统完成。最终结果由Mass ARRAY RT软件系统实时读取,Mass ARRAY Typer软件系统完成基因分型;4、检索河南省人民医院电子病历系统,记录入选患者人口统计学资料、伏立康唑用药途径、基础疾病、合并用药、实验室检查以及SNPs分型结果和(Css)min等数据建立数据库;5、采用SPSS19.0统计学软件对数据进行统计学分析,采用χ~2检验对研究中21个位点的基因分型结果进行Hardy-Weinberg平衡检验;采用SHEsis在线软件平台进行连锁不平衡分析。单因素分析采用非参数检验(两组之间采用Mann-Whitney U检验,三组间采用Kruskal-Wallis检验),定量数据之间的相关性采用Spearman秩相关检验;最优尺度回归用于确定导致(Css)min变异性的影响因素,P0.05有统计学意义。结果1、本研究共纳入86名中国汉族血液病患者,106例次(Css)min,其中确诊侵袭性真菌病(Proven IFD)3例,临床诊断侵袭性真菌病(Probable IFD)11例,拟诊断侵袭性真菌病(Possible IFD)32例以及未确定侵袭性真菌病(Undetermined IFD)40例;(Css)min变异系数达65.8%。2、本研究中21个SNPs的次等位基因频率(Minor allele frequency,MAF)与全球MAF存在一定程度的差异性,其中rs17882687、rs12248560、rs1799853、rs2740574、rs35599367、rs10264272和rs41303343次等位基因频率(Minor allele frequency,MAF)非常低或为0;3、CYP2C19基因多态性是(Css)min变异性的重要影响因素,其中相对于快代谢型患者,(Css)min/D在存在rs4244285或rs4986893突变的(中间代谢型或慢代谢型)患者中更高,同时(Css)min/D与年龄呈正相关;4、在中间代谢型或慢代谢型患者中,年龄及rs4646437(CYP3A4)基因型是造成(Css)min/D个体间差异的影响因素;其中rs4646437的突变(CT或TT)及高年龄与较高的(Css)min/D具有相关性;在快代谢型患者中,BMI与(Css)min/D呈正相关。结论1、伏立康唑个体差异较大,(Css)min变异性显著;2、CYP2C19基因多态性是导致(Css)min变异性的重要影响因素;3、除CYP2C19基因型外,rs4646437(CYP3A4)基因型、年龄、BMI等因素的影响也需要考虑进去,特别是对于那些CYP2C19为中间代谢型或慢代谢型的汉族血液病患者。
[Abstract]:In the past few decades, invasive fungal infections have become increasingly severe, accompanied by a high mortality rate, which is one of the most important causes of death in hospital infection. Antifungal agents are still the main means of controlling invasive fungal infections. Voriconazole, as the second generation of three azole antifungal agents, has a stronger target affinity and resistance to truth. Bacterial activity has been recommended by several guidelines as a first-line treatment for invasive Aspergillus infection. Volconazole is mainly metabolized by the liver CYP450 enzyme system, CYP2C19, CYP3A4, and CYP2C9 are the main metabolic enzymes. ABCB1 is the main transporter in the body, and CYP2C19, CYP3A4, CYP2C9, and ABCB1 existing gene polymorphisms can affect enzymes in varying degrees. The activity of metabolic or transporter protein; the steady-state Valley concentration is the evaluation index of the exposure dose of voriconazole in vivo, which is closely related to the therapeutic effect and adverse reactions. There are still some problems in the clinical antifungal treatment of voriconazole: 1. the window of voriconazole is narrow, and 2. volitazol is significantly different; 3. volantiazole is more serious. 4. voriconazole is expensive. The clinical treatment of voriconazole is affected by a variety of genetic factors and non genetic factors. By now, the reasons for the individual differences in voriconazole can not be fully explained. The current urgent need to be solved is to explore the influence factors of the difference of voriconazole, so as to realize the individuals of voriconazole Objective 1 to monitor the initial steady-state Valley concentration of voriconazole in Chinese Han blood disease patients [(Css) min], to explore the pharmacokinetic characteristics of voriconazole; 2, through the detection of CYP2C19, CYP3A4, CYP2C9 and ABCB1 gene polymorphisms in Chinese Han hemopathy, a preliminary understanding of the volt of patients with hematological diseases in the Han nationality in Henan Province People's Hospital of China. The distribution characteristics of the polymorphism of the metabolic enzyme and ABCB1 gene of rirozazole; 3, to explore the effect of CYP2C19, CYP3A4, CYP2C9 and other major metabolic enzymes and transporter ABCB1 gene polymorphisms on the min of the Chinese Han hematological patients (Css) in Henan Province People's Hospital; 4, to explore the clinical non genetic factors of hematological diseases in the Han nationality of the Chinese Han nationality (C). SS) effect of min. Method 1, screening in Henan Province People's Hospital from March 2015 to September 2016, conforming to diagnostic criteria for invasive fungal disease of patients with hematological diseases / malignancies in 2013, diagnostic criteria for invasive fungal disease of hematopathy, the use of voriconazole to treat or prevent fungal infection and to receive the concentration of voriconazole in blood. The Chinese Han blood disease patients were monitored retrospectively. 2, the serum concentration of voriconazole was monitored by high performance liquid chromatography (HPLC), DNA was extracted from the whole blood DNA extraction kit and UV / visible photometer was used to detect the concentration and quality of the extracted DNA, and the DNA samples were stored in the -40 refrigerator, and the matrix supplemented by matrix supplemented. The single nucleotide polymorphic loci (SNPs) of 21 CYP2C19, CYP3A4, CYP2C9, ABCB1, and other single nucleotide polymorphic loci (SNPs) were designed by the aid of laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) technology, and the single base extension primers were designed by the Assay Designer software package. The detection process was completed by Mass ARRAY system. Real-time reading, Mass ARRAY Typer software system completed genotyping; 4, retrieving the Henan Province People's Hospital electronic medical record system, recording selected patient demographic data, voriconazole use, basic disease, combined medication, laboratory examination and SNPs typing results and (Css) Min data set up database; 5, using SPSS19.0 statistics. Statistical analysis of the data was carried out by the software. The Hardy-Weinberg balance test was carried out on the results of the genotyping of 21 loci in the study by the chi square ~2 test; the linkage disequilibrium analysis was carried out by the SHEsis online software platform. The single factor analysis used the non parametric test (two groups using Mann-Whitney U test and three groups using Kruskal-Wallis test. Spearman rank correlation test was used to determine the correlation between quantitative data. The optimal scaling regression was used to determine the factors affecting (Css) min variability, and P0.05 had statistical significance. Results 1, 86 Chinese Han hematological patients were included in this study, 106 cases (Css) min, among which 3 cases of invasive fungal disease (Proven IFD) were diagnosed, and the clinical diagnosis was invasive. 11 cases of Probable IFD, 32 cases of invasive fungal disease (Possible IFD) and 40 cases of undetermined invasive mycosis (Undetermined IFD); (Css) the coefficient of variation of Min is 65.8%.2. The secondary allele frequency of 21 SNPs (Minor allele) in this study has a certain degree of difference from that in the world. Rs12248560, rs1799853, rs2740574, rs35599367, rs10264272, and rs41303343 secondary alleles (Minor allele frequency, MAF) are very low or 0; 3, CYP2C19 gene polymorphism is an important factor in the variability of (Css). The patients with slow metabolic type were higher, and (Css) min/D was positively correlated with age; 4 in intermediate or slow metabolic patients, age and rs4646437 (CYP3A4) genotypes were the factors contributing to the difference among individuals of (Css) min/D; the mutation of rs4646437 (CT or TT) and higher age and higher (Css) min/D were related; in fast metabolic patients Among those, BMI was positively correlated with (Css) min/D. Conclusion 1, the individual differences in voriconazole, (Css) min variability, are significant; 2, CYP2C19 gene polymorphism is an important factor leading to (Css) min variability; 3, besides the CYP2C19 genotype, the influence of rs4646437 (CYP3A4) genotypes, age, BMI and other factors should also be taken into consideration, especially for those The 19 is the middle metabolism or slow metabolism Han blood disease patients.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
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本文编号：2044915