【作者】 常琰子；

【导师】 张卫；

【作者基本信息】 郑州大学 ， 麻醉学， 2009， 硕士

【摘要】 【背景与目的】在我国芬太尼是常用的阿片类术后静脉镇痛药物,临床资料表明不同个体间术后镇痛芬太尼的需求量和不良反应存在显著差异。研究证实芬太尼静脉给药后主要在肝脏内通过细胞色素P450(cytochrome P450,CYP)3A4酶代谢成为无活性产物,CYP3A4酶活性在不同个体存在明显差异,且编码CYP3A4酶的基因存在多态性。研究提示CYP3A4基因多态性可能是造成不同个体药物代谢差异的基础。CYP3A4~*1G是目前已发现的中国人所有的CYP3A4单核苷酸多态性中发生频率最高的一个位点。研究提示该基因多态性可能影响其表型即CYP3A4酶活性,但结论不一。CYP3A4~*1G多态性是否会引起酶活性的改变,间接造成术后芬太尼静脉镇痛需要量的差异,尚不可知。本研究旨在通过分析CYP3A4~*1G多态性和CYP3A4酶活性,探讨妇科患者全麻术后芬太尼静脉自控镇痛效应个体差异的遗传因素,为临床制订芬太尼术后镇痛的个体化用药方案提供理论依据。【材料与方法】1.研究对象与分组190例妇科择期全麻下腹式子宫肌瘤剔除术或子宫全切术患者,ASAⅠ或Ⅱ级,年龄20-50岁,体重指数在正常范围(1±20%),拟行术后静脉镇痛者。排除有严重心血管疾病、糖尿病、严重肝脏病、肾脏病史,吸烟、酗酒史,慢性疼痛史、长期使用镇痛药物的患者,处于孕期或哺乳期的患者,并排除术前1个月内服用过对肝脏CYP3A4酶有诱导或抑制作用的药物或食物的患者。根据CYP3A4~*1G基因型检测结果将患者分为CYP3A4~*1/~*1(野生型纯合子)、CYP3A4~*1/~*1G(突变型杂合子)和CYP3A4~*1G/~*1G(突变型纯合子)三组。2.麻醉与镇痛研究设计得到医院伦理委员会同意,所有患者签署知情同意书后进入试验。术中采用统一的全凭静脉麻醉方法,麻醉诱导静脉注射咪达哗仑(MDZ)、异丙酚、瑞芬太尼和琥珀酰胆碱;静脉输注异丙酚和瑞芬太尼,间断静脉注射阿曲库铵维持麻醉;手术结束时停用所有麻醉药物,待患者自主呼吸恢复满意,神志清醒后拔除气管导管,采用视觉模拟评分(visual analog scale,VAS)评定术后即刻疼痛程度并记录,接电子镇痛泵行静脉芬太尼病人自控镇痛(patient-controlled intravenous analgesia,PCIA)。镇痛泵内药物配方为芬太尼1.0 mg、氟哌利多5 mg,加生理盐水至100 ml。PCIA设置:芬太尼背景剂量5μg/h、追加剂量20μg/次、锁定时间5 min、最大限量145μg/h。活动时疼痛VAS评分≤3分定义为有效镇痛,若已达到每小时最大剂量VAS评分仍＞3分,则辅用其他镇痛药物。观察并记录术后即刻和每个24h内芬太尼消耗量、平均VAS评分以及不良反应发生情况。3.基因多态性检测采用酚-氯仿法提取外周静脉血DNA,聚合酶链反应-限制性片段长度多态性分析(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)技术检测CYP3A4~*1G基因型,并通过PCR扩增产物基因测序以验证基因分型方法的可靠性。4.CYP3A4酶活性检测采用MDZ作为探针药物,麻醉诱导静脉注射MDZ1 h时抽取外周静脉血5 ml,液相色谱-质谱检测法(LC-MS)测定血浆中MDZ及其代谢产物1′-OH MDZ浓度。以1′-OH MDZ与MDZ比值作为CYP3A4酶活性的指标。5.统计学分析数据采用SPSS11.0软件进行分析,计量资料以(?)±s表示,以x~2检验检测等位基因和基因型分布是否符合Hardy-Weinberg平衡;x~2检验或Fisher’s精确概率检验比较不同种族间等位基因的发生频率;多组间数据进行单因素方差分析(ANOVA),各组间比较采用LSD法。组间芬太尼消耗量的比较采用协方差分析以排除混杂因素影响。不良反应发生率的比较采用x~2检验或Fisher’s精确概率法。突变等位基因数量与芬太尼消耗量之间的相关性采用等级相关分析,计量资料变量之间的关系采用直线相关分析。检验水准为α=0.05。【结果】1.一般情况190例女性患者中,术后即刻VAS评分较高(5.7±1.4),术后第1个24 h平均VAS评分为2.2±0.8分;术后第2个24 h平均VAS评分为1.3±0.4,均达到有效镇痛。术后第1个24h芬太尼消耗量为391.5±201.7μg;第2个24h芬太尼消耗量为191.2±43.5μg。使用芬太尼镇痛时术后恶心呕吐的发生率为28.9%;轻度镇静的发生率为1.6%;瘙痒的发生率为0.5%;未见其他不良反应。2.妇科患者中CYP3A4~*1G等位基因频率CYP3A4~*1G等位基因在中国汉族妇科手术患者中的变异频率为29.7%,等位基因和基因型分布符合Hardy-Weinberg平衡(P＞0.05)。CYP3A4~*1G等位基因频率与日本人(24.9%)和中国高脂血症患者(27.6%)相近(P＞0.05)。3.CYP3A4~*1G多态性对妇科患者CYP3A酶活性和术后芬太尼镇痛效应的影响CYP3A4~*1/~*1、CYP3A4~*1/~*1G和CYP3A4~*1G/~*1G三组间一般情况比较差异无统计学意义(P＞0.05);术后即刻及术后24 h平均VAS评分组间差异无统计学意义(P＞0.05)。术后24 h芬太尼消耗量三组间差异有统计学意义(P＜0.05),CYP3A4~*1G/~*1G组(260.0±101.1μg)低于CYP3A4~*1/~*1组(406.7±186.6μg)和CYP3A4~*1/~*1G组(396.8±222.6μg);术后24 h芬太尼消耗量与患者携带CYP3A4~*1G等位基因数量呈负相关(r=-0.14,P＜0.05)。三组间CYP3A4酶活性的比较差异有统计学意义(P＜0.05),CYP3A4~*1G/~*1G组(0.34±0.15)低于CYP3A4~*1/~*1G组(0.46±0.12)和CYP3A4~*1/~*1组(0.46±0.14)。三组间术后镇痛期间不良反应发生率的比较差异无统计学意义(P＞0.05)。【结论】1.中国汉族妇科手术患者中CYP3A4~*1G等位基因的变异频率为29.7%;2.CYP3A4~*1G是一个具有功能意义的突变,与CYP3A4酶活性和术后24 h芬太尼静脉镇痛消耗量降低有关。更多还原

【Abstract】 Background and ObjectiveFentanyl is a synthetic opioid that has been widely used in clinical practice and it is especially effective for induction and maintenance of anesthesia or control of analgesia.However,the effective dose of fentanyl for pain control varies greatly among individuals.Fentanyl is metabolized in the liver predominantly by the cytochrome P450 3A4(CYP3A4).CYP3A4 protein expression in the liver may vary up to 40-fold,leading to variations in drug metabolism and contributing to differences in individual response to the drug.Genetic variation within the CYP3A4 gene may contribute to interindividual variability in drug metabolism.Single nucleotide polymorphisms(SNPs) are the most common form of genetic variation in the CYP3A4.CYP3A4~*1G is a high-frequency allele in Chinese,the alteration of function remains unclear in vitro and in vivo.The influence of CYP3A4~*1G on fentanyl analgesic effect has not been reported.Due to the importance of CYP3A4 in the metabolism of fentanyl,we conducted this study to observe the impact of CYP3A4~*1G polymorphism on fentanyl effect for intravenous analgesia.The present study provides an important foundation and theoretical evidence for individualization of medication in the pain treatment.Materials and Methods1.SubjectsTotal one hundred and ninety patients,having an American Society of Anesthesiologists(ASA) physical status ofⅠorⅡ,aged 20-50 yr,within±20%of ideal body weight,who were admitted into our medical institution for elective abdominal total hysterectomy or myomectomy,were enrolled in the current study. Patients with a known history of significant cardiovascular disease,diabetes mellitus, alcohol or drug abuse,hepatic or renal dysfunction,pregnancy or nursing,and chronic analgesic use were excluded from the study.Patients who had consumed drugs known to inhibit or induce the expression of CYP3A4 enzymes one month prior to surgery were also excluded.The patients were divided into three groups according to the genotypes.2.Anesthetic Technique and AnalgesiaAll patients gave written informed consent for participating in the study and the study protocol was approved by the Institution Review Board at Zhengzhou University.A standardized general anesthesia protocol was used for all patients.We administered 0.1mg/kg midazolam,0.5 mg/kg propofol,2μg/kg remifentanil and 1 mg/kg succinylcholine for induction of anesthesia.Atracurium 0.6 mg/kg was administered intravenously as an initial dosage immediately after tracheal intubation was confirmed and then 0.1 to 0.2 mg/kg was administered by repeated boluses and 0.1 to 0.2μg/kg/min remifentanil and 6 to 8 mg/kg/h propofol were infused for maintenance of anesthesia.Postoperative PCA(1 mg fentanyl and 5 mg droperidol in 100 ml normal saline) was administered using a computer controlled infusion pump (CADD-Legacy 6300) which was programmed to give a 2 ml bolus of fentanyl solution with a 5 min lockout time,0.5 ml/h background infusion and a maximum of 145μg per hour.All patients received intravenous PCA with fentanyl once they were stable and awake.The delivered dose of fentanyl was recorded.VAS was used for assessing pain at rest during PCA.Successful analgesia was defined as a VAS score≤3.The VAS score and incidence of any adverse effects such as nausea,vomiting, respiratory depression and sedation were recorded.3.Genotyping assaysVenous blood samples were collected from all patients.DNA was extracted using a standard phenol/chloroform procedure.Genotyping of CYP3A4~*1G allele was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).The results for each genotype were confirmed in randomly selected individuals by direct sequence analysis.4.Evaluation of CYP3A4 activityPeripheral blood sample(5 ml) was collected from each patient.CYP3A4 activity was measured by determining the plasma ratio of 1’-hydroxymidazolam to midazolam 1 h after intravenous administration of 0.1 mg/kg midazolam for induction of anesthesia.Midazolam and 1’-hydroxymidazolam concentrations were determined using a liquid chromatography-mass spectrometry.5.Statistical analysisSPSS 11.0 software was used for statistical analyses.Values were reported as (?)+s.The allele frequencies were estimated from the observed numbers of each specific allele.Chi-square test was used to verify Hardy-Weinberg equilibrium. One-way analysis of variance was used to assess whether significant differences exist between the three genotypes.Data for the fentanyl consumption were compared using one-way analysis of variance with post hoc Bonferroni correction,multiple comparisons was performed before and after adjusted for age,weight of the patients and remifentanil consumption in the operation.The incidences of any adverse effects were analyzed using Chi-square test or Fisher exact test.A two tailed P-value of＜0.05 was considered statistically significant.Results1.General informationAmong the 190 patients,the VAS pain score immediately postoperatively was 5.7±1.4.At 24 h after surgery,it was 2.2±0.8.No one needed rescue management for inadequate pain control.The fentanyl consumption was 391.5±201.7μg and 191.2±43.5μg in the first and second 24 h postoperatively,respectively.The incidence of postoperative nausea and vomiting was 28.9%.The incidence of mild sedation and pruritus in our study was 1.6%and 0.5%,respectively.2.Frequency of CYP3A4~*1G alleleThe frequency of CYP3A4~*1G allele in gynecologic patients was 29.7%.The allele frequency was in Hardy-Weinberg equilibrium(P＞0.05).The allelic frequency of CYP3A4~*1G in our study was similar to that reported in Japanese(24.9%) and Chinese hyperlipidemic patients(27.6%)(P＞0.05).3.Association of CYP3A4~*1G gene polymorphism with CYP3A4 activity and fentanyl analgesic effectThere were no significant differences in general information among the three genotype groups(P＞0.05).No statistical difference in postoperative VAS pain scores was detected across genotypes.The subjects with the CYP3A4~*1G/~*1G genotype (260.0±101.1μg) need less fentanyl to achieve pain control than subjects carrying the CYP3A4~*1/*1(406.7±186.6μg) and CYP3A4~*1/~*1G(396.8±222.6μg) genotypes (P＜0.05).Fentanyl consumption increased in accordance with the number of ~*1G alleles(r=-0.14,P＜0.05 for linear trend).The activity of CYP3A4 in ~*1G/~*1G group(0.34±0.15) was lower than in ~*1/~*1(0.46±0.14) and ~*1/~*1G(0.46±0.12) groups(P＜0.05).There was no significant difference in incidences of adverse events among the different genotype groups(P＞0.05).Conclusions1.The frequency of CYP3A4~*1G allele in Chinese gynecologic patients is 29.7%.2.Carrying CYP3A4~*1G decrease the activity of CYP3A4 and patient-controlled intravenous fentanyl consumption.更多还原