PPARα基因多态性对妇科患者芬太尼静脉镇痛效应的影响

发布时间：2018-07-06 21:40

本文选题：过氧化物酶体增殖物激活受体α + 基因多态性　；参考：《郑州大学》2014年硕士论文

【摘要】：背景与目的芬太尼(fentanyl)是我国目前临床上应用最为广泛的强效麻醉性镇痛药，其起效快，维持时间短，无组胺释放作用，对呼吸、循环系统的影响轻微，但用于病人术后静脉自控镇痛术（PCIA）时，其镇痛效果存在较为显著的个体差异性。导致上述差异的原因是多方面的，其中遗传因素通过改变代谢酶的活性或者表达水平从而影响阿片类镇痛药的药物代谢动力学(pharmacokinetics)和药物效应动力学(pharmacodynamics）是主要原因之一。芬太尼主要经过肝脏代谢，肝脏细胞色素P450(cytochrome P450, CYP)3A4酶（CYP3A4）是其最重要的代谢酶，该酶可将芬太尼代谢为几乎无活性的去甲芬太尼。芬太尼药代动力学的个体差异提示CYP3A4与芬太尼静脉镇痛效应的个体差异密切相关。课题组前期通过体内药代动力学试验证实，CYP3A4*1G基因多态性可改变芬太尼在人体内的药代动力学参数，从而对其术后静脉镇痛效应产生影响，但具体机制尚不清楚。目前，国内外有关CYP3A4*1G多态性对CYP3A4的影响的研究结果存在争议，且近期研究提示绝大多数CYP3A4单核苷酸的突变频率很低，对表型的影响微弱，故推测存在由反式作用基因编码的转录调控因子或蛋白质通过其他途径调控酶活性，最终导致了CYP3A4的变异。过氧化物酶体增殖物激活受体α（PPARα）是核激素受体超家族中的配体激活受体，作为一种转录调控因子，可调控多种核内靶基因的表达。近期研究表明，PPARα影响CYP3A4酶活性，其中rs4253728单核苷酸多态性是对该酶活性最具影响力的因素之一。那么PPARα rs4253728(GA)基因多态性能否引起代谢酶活性发生改变，进而导致妇科患者芬太尼PCIA需求量的个体差异，亟待进一步探究。本课题旨在通过综合分析PPARα rs4253728(GA)基因多态性与芬太尼PCIA药物效应及其代谢酶活性的关系，探讨导致芬太尼PCIA个体差异的遗传学因素，力争为临床制订术后镇痛个体化的用药方案提供相应的理论依据。试验方案 1受试对象及试验分组选择2012年9月～2013年12月于我院诊断为“子宫肌瘤”拟于全身麻醉下行“腹式子宫全切术”或“腹式子宫肌瘤剔除术”的汉族妇科患者共168例，年龄20岁～50岁， ASAⅠ级～Ⅱ级，体重指数(body mass index, BMI)≤30kg/m2(1±20%)，所有受试患者术毕神志清醒，拔除气管导管后行病人静脉自控镇痛术(patient-controlled intravenous analgesia, PCIA)。排除标准：术前存在长期的酗酒史和（或）吸烟史；合并心血管系统疾病、肾脏疾病、肝脏疾病、精神神经系统疾病、糖尿病等内分泌系统疾病；患者术前存在慢性疼痛病史；长期的非甾体类镇痛药物服用史；患者在术前1个月内服用过CYP3A4酶抑制剂或者诱导剂；处于孕期或哺乳期的患者等。根据PPARα rs4253728GA的DNA测序结果将患者分为三组：野生型纯合子组（G/G）、突变型杂合子组(G/A)和突变型纯合子组(A/A)。 2麻醉与PCIA 本试验经郑州大学第一附属医院伦理委员会讨论批准，受试患者及其家属签署知情同意书。所有受试患者不给予任何术前用药。患者入室后常规监测心电图（electrocardiogram，ECG）、心率（heart rate，HR）、无创血压（noninvasiveblood pressure，NIBP）、呼吸（respiratory rate，RR）及脉搏氧饱和度（saturationof pulse oxygen，SpO2）。术中采取统一的全凭静脉麻醉（total intravenousanesthesia，，TIVA），具体如下：麻醉诱导：静脉注射咪达唑仑（midazolam，MDZ）0.01mg/kg、瑞芬太尼（remifentanil）2μg/kg、丙泊酚（propofol）0.5mg/kg和琥珀酰胆碱（succinylcholine）1.5mg/kg。麻醉维持：微量泵持续静脉输注丙泊酚6～8mg·kg-1·h-1和瑞芬太尼0.1～0.2μg·kg-1·min-1，阿曲库铵首次剂量0.6mg/kg，之后以0.1～0.2mg/kg间断静脉注射。术毕停止泵注静脉麻醉药物，吸痰，待患者自主呼吸、咳嗽反射等恢复、神志清楚、呼之能应、肌力恢复后拔除气管导管。拔除气管导管即刻使用痛觉视觉模拟评分（visual analogue scale, VAS）对患者的疼痛程度进行评估。若VAS＞3分，静脉注射芬太尼20μg/5min,直至VAS≤3分，记录期间芬太尼的滴定用量。所有患者术后VAS≤3分时行PCIA。PCIA装置：6300型CADD-Legacy电子镇痛泵。PCIA药物配方：氟哌利多5mg，芬太尼1.0mg，加入生理盐水稀释至100ml后注入镇痛泵。PCIA设置：芬太尼背景剂量0.5ml/h，追加剂量2ml/h，锁定时间为5min，最大限量145μg/h。镇痛有效的评价标准：患者活动时的VAS小于或等于3分。若芬太尼消耗量达到最大限量（145μg/h）时仍无法满足镇痛需求（VAS＞3分），可辅以其他非甾体类镇痛药物以确保患者围术期的舒适与安全，但要将该病例从本次试验中剔除。 3芬太尼PCIA效应的评价观察并记录患者在拔管即刻、术后的第1个24h、术后的第2个24h其VAS评分情况、芬太尼PCIA的消耗量。记录术后随访过程中各组患者术后头晕、恶心呕吐（PONV）、轻度镇静、皮肤瘙痒等不良反应的发生率。 4PPARα rs4253728GA多态性位点检测患者入室后肘静脉放置留置针，抽取外周静脉血2~3ml，采用酚-氯仿法抽提DNA，采用聚合酶链式反应（polymerase chain reaction, PCR）对所需的目的基因片段进行体外扩增，琼脂糖凝胶电泳验证扩增产物。采用DNA直接测序法检测PPARα rs4253728GA多态性位点并在此基础上进行分型。 5CYP3A4酶活性测定选择MDZ作为检测CYP3A4酶活性的探针药物，以MDZ代谢产物1-羟基咪达唑仑（1’-OH MDZ）与MDZ的比值作为衡量CYP3A4酶活性的指标。检测方法：麻醉诱导时静脉注射MDZ0.1mg/kg，60min后采集静脉血5ml，离心后取上层血浆，采用液相色谱-质谱法（liquid chromatography mass spectrometry,LC-MS）测定血浆1’-OH MDZ及血浆MDZ的浓度。 6统计学分析采用SPSS17.0统计学软件进行数据分析。计量资料以均数±标准差（x s）形式表示。等级资料采用秩和检验（wilcoxon rank sum test）。使用χ2检验检测等位基因和基因型是否符合Hard-Weinberg平衡。多组间的数据比较采用单因素方差分析（ANOVA）。组间比较采用最小显著差法（LSD）。为排除混杂因素的影响，不同组间患者芬太尼PCIA消耗量的比较采用协方差分析（covarianceanalysis）。突变的等位基因的数量与芬太尼PCIA消耗量间的相关性采用等级相关分析。各计量资料变量间的关系采用直线相关性分析。比较各组患者术后不良反应的发生情况采用χ2检验或者Fisher’s精确概率法。检验水准：α=0.05。结果 1一般资料纳入的168例女性患者在拔管即刻、术后24h、48h平均VAS评分分别为(5.9±1.3)，（2.3±0.6），（1.1±0.5），所有患者术后均达到有效镇痛标准。术后第1个24h、第2个24h芬太尼平均消耗量分别为（379.5±213.6）μg，（182.3±51.7）μg。3例患者术后出现头晕，47例患者出现恶心呕吐，3例患者出现轻度镇静，1例患者出现瘙痒，其不良反应的发生率分别为1.79%，27.98%，1.79%，0.60%，术后随访未见其他不良反应。 2中国汉族妇科患者PPARα（rs4253728GA）的等位基因频率 PPARα rs4253728GA等位基因在中国籍汉族妇科手术患者中的变异频率为20.4%。经检验，该等位基因及基因型的分布符合Hardy-Weinberg平衡(P0.05)，其等位基因频率高于非裔美国人(7.2%)，低于高加索人(27.3%)，与文献报道相符(P0.05)。 3PPARα（rs4253728GA）基因多态性对CYP3A4酶活性及芬太尼PCIA效应的影响按照直接测序的结果将患者分为野生型纯合子组（GG），突变型杂合子组（GA）和突变型纯合子组（AA）。三组患者一般资料的比较，差异无统计学意义(P0.05)。三组患者在拔管即刻及术后第1个、第2个24h的平均VAS评分组间比较差异无统计学意义(P0.05)。三组患者术后第1个24h芬太尼PCIA的消耗量组间比较差异无统计学意义(P0.05)。术后第2个24h芬太尼PCIA的消耗量组间比较，AA组患者显著低于GA组和GG组(P0.05)。AA组患者CYP3A4酶活性较GA组和GG组有所降低(P0.05)。3组患者术后不良反应的发生情况比较差异无统计学意义(P0.05)。结论 1．PPARα rs4253728GA等位基因在中国汉族妇科手术患者中的变异频率为20.4%； 2．PPARα rs4253728GA是一个可能有功能意义的突变，其对妇科患者CYP3A4酶活性及芬太尼PCIA效应可能产生影响
[Abstract]:Background and purpose
Fentanyl (fentanyl) is the most widely used strong anesthetic analgesic in China at present. It has quick effect, short duration, no histamine release and slight influence on respiratory and circulatory system, but the analgesic effect of postoperative intravenous analgesia (PCIA) has a significant individual difference. The reasons are multifaceted, among which genetic factors affect the pharmacokinetics (pharmacokinetics) and drug effect dynamics (pharmacodynamics) of opioid analgesics by altering the activity or expression of metabolic enzymes. The main causes are liver metabolism and liver cytochrome P450 (cytochrome P). 450, CYP) 3A4 enzyme (CYP3A4) is the most important metabolic enzyme. The enzyme can metabolize fentanyl to almost inactive, the individual differences in the pharmacokinetics of fentanyl suggest that the individual difference between CYP3A4 and fentanyl is closely related to the individual differences in the effect of fentanyl on intravenous analgesia. The state of state can change the pharmacokinetic parameters of fentanyl in human body, which can affect the effect of postoperative intravenous analgesia, but the specific mechanism is not clear. At present, the research results about the effect of CYP3A4*1G polymorphism on CYP3A4 at home and abroad are controversial, and recent studies suggest that the mutation frequency of the overwhelming majority of CYP3A4 mononucleotide is very low. The effect on the phenotype is weak, so it is speculated that the transcriptional regulator or protein encoded by the trans acting gene can regulate the activity of the enzyme through other pathways, and eventually leads to the variation of CYP3A4.
Peroxisome proliferator activated receptor alpha (PPAR alpha) is a ligand activated receptor in the nuclear hormone receptor superfamily. As a transcriptional regulator, the expression of a variety of target genes can be regulated. Recent studies have shown that PPAR alpha affects the activity of CYP3A4 enzymes, and the rs4253728 mono nucleoside polymorphism is the most influential factor in the activity of the enzyme. One of them is whether the PPAR alpha rs4253728 (GA) gene polymorphism can cause the metabolic enzyme activity to change, which will lead to the individual difference in the demand for fentanyl PCIA in gynecologic patients. The aim of this study is to analyze the relationship between the PPAR alpha rs4253728 (GA) gene polymorphism and the fentanyl PCIA drug effect and its metabolic enzyme activity. Objective to explore the genetic factors leading to individual differences in fentanyl PCIA, and to provide a theoretical basis for formulating individualized medication regimens for postoperative analgesia.
Test scheme
1 subjects and experimental groups
168 cases of Han gynecologic patients, aged from 20 to 50 years old, ASA grade I to grade II, and body mass index (body mass index, BMI) less than 30kg/m2 (1 + 20%), were selected from September 2012 to December 2013 in our hospital for "hysteromyoma" or "abdominal myomectomy". At the end of the operation, patient-controlled intravenous analgesia (PCIA) was removed after the tracheal catheter was removed. The exclusion criteria: a long history of alcohol abuse and / or smoking history before the operation; combined with cardiovascular diseases, renal diseases, liver diseases, mental and neurologic diseases, diabetes and other endocrine diseases; Patients have a history of chronic pain before surgery; a history of long-term nonsteroidal analgesics; patients who have taken CYP3A4 enzyme inhibitors or inducers within 1 months before operation; patients in pregnancy or lactation period, etc.. The patients are divided into three groups according to the DNA sequencing results of PPAR alpha rs4253728GA: wild type homozygote group (G/G), mutant heterozygosity The subgroup (G/A) and the mutant homozygote group (A/A).
2 anaesthesia and PCIA
The test was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University. The subjects and their families signed the informed consent. All the patients were not given any preoperatively. The patients received routine monitoring of electrocardiogram (electrocardiogram, ECG), heart rate (heart rate, HR), non invasive blood pressure (noninvasiveblood pressure, NIBP), and respiratory (RES). Piratory rate, RR) and pulse oxygen saturation (saturationof pulse oxygen, SpO2). A unified total intravenous anesthesia (total intravenousanesthesia, TIVA) was taken during the operation, as follows: anesthesia induction: intravenous midazolam (midazolam, MDZ), 2 micronutrium, propofol and succinyl Choline (succinylcholine) 1.5mg/kg. anesthesia maintenance: micropump continuous infusion of propofol 6 ~ 8mg. Kg-1. H-1 and remifentanil 0.1 ~ 0.2 g. Kg-1. Min-1, atracurium for the first time dose 0.6mg/kg, then intravenous injection of 0.1 ~ 0.2mg/kg. Recovery, clear mind, call it should be, after the recovery of muscle strength after removal of endotracheal tube.
The pain degree of the patients was assessed by visual analogue scale (VAS). If VAS > 3, intravenous fentanyl 20 u g/5min, until VAS < 3, and the titration amount of fentanyl during the recording period. All patients underwent VAS < 3 minutes as PCIA.PCIA device: type 6300 CADD-Legacy electronic town. .PCIA drug formula for pain pump: droperidol 5mg, fentanyl 1.0mg, adding saline to 100ml and injected to the analgesic pump.PCIA setting: fentanyl background dose 0.5ml/h, additional dose 2ml/h, locking time 5min, the maximum limit of 145 micron analgesia effectiveness evaluation criteria: Patients' activity VAS less than or equal to 3 points. If fentanyl consumption The maximum limit (145 u g/h) is still unable to meet the needs of analgesia (VAS > 3), supplemented with other non steroidal analgesics to ensure the comfort and safety of the patient during the perioperative period, but the case should be removed from this test.
Evaluation of the PCIA effect of 3 fentanyl
The patients were observed and recorded immediately after extubation, first 24h postoperatively, second 24h after the operation and the VAS score of the fentanyl, and the consumption of fentanyl PCIA. The incidence of adverse reactions such as postoperative dizziness, nausea and vomiting (PONV), mild sedation, and itching of the skin were recorded during the follow-up.
Detection of 4PPAR alpha rs4253728GA polymorphic loci
The patients were placed in the elbow vein after entering the room, extracting the peripheral venous blood 2~3ml, extracting DNA by the phenol chloroform method and using the polymerase chain reaction (polymerase chain reaction, PCR) to amplify the desired target gene fragment in vitro and the agarose gel electrophoresis to verify the increase of the product. The DNA direct sequencing method was used to detect the rs4253728GA polymorphism of PPAR. The sex loci are typed on this basis.
Determination of 5CYP3A4 enzyme activity
MDZ was selected as a probe drug to detect the activity of CYP3A4 enzyme. The ratio of MDZ metabolite 1- hydroxymidazolam (1 '-OH MDZ) to MDZ was used as an index to measure the activity of CYP3A4 enzyme. Romatography mass spectrometry (LC-MS) was used to determine plasma concentration of 1 '-OH MDZ and plasma MDZ.
6 statistical analysis
SPSS17.0 statistical software was used to analyze the data. The measurement data were expressed in the form of mean mean deviation (x s). Rank sum test (Wilcoxon rank sum test) was used for rank data. X 2 test was used to test whether alleles and genotypes were in accordance with Hard-Weinberg balance. The number of data among groups was compared by single factor analysis of variance (ANOVA). The minimum significant difference method (LSD) was used. In order to exclude the influence of confounding factors, the PCIA consumption of fentanyl in different groups was compared with covariance analysis (covarianceanalysis). The correlation between the number of mutation alleles and the PCIA consumption of fentanyl was analyzed by grade correlation analysis. The relationship between the variables of each measurement was linear. The incidence of adverse reactions in each group was compared by chi square test or Fisher 's exact probability method. The test level was =0.05. =0.05..
Result
1 general information
168 cases of female patients were removed immediately after extubation. The average VAS score of 24h and 48h after operation was (5.9 + 1.3), (2.3 + 0.6), (1.1 + 0.5). All patients achieved effective analgesia after operation. First 24h after operation, and second 24h fentanyl (379.5 + 213.6) mu g respectively, (182.3 + 51.7) Mu G.3 patients were dizzy after operation. There were 3 cases of mild sedation and 1 patients with itching. The incidence of adverse reactions was 1.79%, 27.98%, 1.79%, 0.60% respectively. No other adverse reactions were observed after the postoperative follow-up.
2 allele frequencies of PPAR alpha (rs4253728GA) in Chinese Han patients with gynecologic diseases
The mutation frequency of PPAR alpha rs4253728GA allele in Chinese gynecologic surgery patients is 20.4%., and the distribution of allele and genotype conforms to Hardy-Weinberg balance (P0.05). The allele frequency of the allele is higher than that of African Americans (7.2%), lower than the Caucasian (27.3%), and is consistent with the literature (P0.05).
Effects of 3PPAR alpha (rs4253728GA) gene polymorphism on CYP3A4 enzyme activity and fentanyl PCIA effect
According to the results of direct sequencing, the patients were divided into wild type homozygote group (GG), mutant heterozygote group (GA) and mutant homozygote group (AA). The comparison of the general data of the three groups was not statistically significant (P0.05). The three groups were extubated immediately and first after the operation, and there was no statistical difference between the average VAS scores of second 24h groups (P0 .05). There was no significant difference between the first 24h fentanyl PCIA consumption groups in three groups of patients (P0.05). After the second 24h fentanyl PCIA consumption group, the AA group was significantly lower than the GA group and the GG group (P0.05).AA group. The difference was not statistically significant (P0.05).
conclusion
The mutation frequency of 1.PPAR alpha rs4253728GA allele in Chinese Han patients undergoing gynecologic surgery was 20.4%.
2.PPAR alpha rs4253728GA is a potentially functional mutation, which may have an effect on CYP3A4 enzyme activity and fentanyl PCIA effect in gynecologic patients.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

【参考文献】