本文選題：過氧化物酶體增殖物激活受體α + 基因多態(tài)性��； 參考：《鄭州大學》2014年碩士論文

【摘要】：背景與目的 芬太尼(fentanyl)是我國目前臨床上應用最為廣泛的強效麻醉性鎮(zhèn)痛藥，其起效快，維持時間短，無組胺釋放作用，對呼吸、循環(huán)系統(tǒng)的影響輕微，但用于病人術(shù)后靜脈自控鎮(zhèn)痛術(shù)（PCIA）時，其鎮(zhèn)痛效果存在較為顯著的個體差異性。導致上述差異的原因是多方面的，其中遺傳因素通過改變代謝酶的活性或者表達水平從而影響阿片類鎮(zhèn)痛藥的藥物代謝動力學(pharmacokinetics)和藥物效應動力學(pharmacodynamics）是主要原因之一。芬太尼主要經(jīng)過肝臟代謝，肝臟細胞色素P450(cytochrome P450, CYP)3A4酶（CYP3A4）是其最重要的代謝酶，該酶可將芬太尼代謝為幾乎無活性的去甲芬太尼。芬太尼藥代動力學的個體差異提示CYP3A4與芬太尼靜脈鎮(zhèn)痛效應的個體差異密切相關(guān)。課題組前期通過體內(nèi)藥代動力學試驗證實，CYP3A4*1G基因多態(tài)性可改變芬太尼在人體內(nèi)的藥代動力學參數(shù)，從而對其術(shù)后靜脈鎮(zhèn)痛效應產(chǎn)生影響，但具體機制尚不清楚。目前，國內(nèi)外有關(guān)CYP3A4*1G多態(tài)性對CYP3A4的影響的研究結(jié)果存在爭議，且近期研究提示絕大多數(shù)CYP3A4單核苷酸的突變頻率很低，對表型的影響微弱，故推測存在由反式作用基因編碼的轉(zhuǎn)錄調(diào)控因子或蛋白質(zhì)通過其他途徑調(diào)控酶活性，最終導致了CYP3A4的變異。 過氧化物酶體增殖物激活受體α（PPARα）是核激素受體超家族中的配體激活受體，作為一種轉(zhuǎn)錄調(diào)控因子，可調(diào)控多種核內(nèi)靶基因的表達。近期研究表明，PPARα影響CYP3A4酶活性，其中rs4253728單核苷酸多態(tài)性是對該酶活性最具影響力的因素之一。那么PPARα rs4253728(GA)基因多態(tài)性能否引起代謝酶活性發(fā)生改變，進而導致婦科患者芬太尼PCIA需求量的個體差異，亟待進一步探究。本課題旨在通過綜合分析PPARα rs4253728(GA)基因多態(tài)性與芬太尼PCIA藥物效應及其代謝酶活性的關(guān)系，探討導致芬太尼PCIA個體差異的遺傳學因素，力爭為臨床制訂術(shù)后鎮(zhèn)痛個體化的用藥方案提供相應的理論依據(jù)。 試驗方案 1受試對象及試驗分組 選擇2012年9月～2013年12月于我院診斷為“子宮肌瘤”擬于全身麻醉下行“腹式子宮全切術(shù)”或“腹式子宮肌瘤剔除術(shù)”的漢族婦科患者共168例，年齡20歲～50歲， ASAⅠ級～Ⅱ級，體重指數(shù)(body mass index, BMI)≤30kg/m2(1±20%)，所有受試患者術(shù)畢神志清醒，拔除氣管導管后行病人靜脈自控鎮(zhèn)痛術(shù)(patient-controlled intravenous analgesia, PCIA)。排除標準：術(shù)前存在長期的酗酒史和（或）吸煙史；合并心血管系統(tǒng)疾病、腎臟疾病、肝臟疾病、精神神經(jīng)系統(tǒng)疾病、糖尿病等內(nèi)分泌系統(tǒng)疾病；患者術(shù)前存在慢性疼痛病史；長期的非甾體類鎮(zhèn)痛藥物服用史；患者在術(shù)前1個月內(nèi)服用過CYP3A4酶抑制劑或者誘導劑；處于孕期或哺乳期的患者等。根據(jù)PPARα rs4253728GA的DNA測序結(jié)果將患者分為三組：野生型純合子組（G/G）、突變型雜合子組(G/A)和突變型純合子組(A/A)。 2麻醉與PCIA 本試驗經(jīng)鄭州大學第一附屬醫(yī)院倫理委員會討論批準，受試患者及其家屬簽署知情同意書。所有受試患者不給予任何術(shù)前用藥。患者入室后常規(guī)監(jiān)測心電圖（electrocardiogram，ECG）、心率（heart rate，HR）、無創(chuàng)血壓（noninvasiveblood pressure，NIBP）、呼吸（respiratory rate，RR）及脈搏氧飽和度（saturationof pulse oxygen，SpO2）。術(shù)中采取統(tǒng)一的全憑靜脈麻醉（total intravenousanesthesia，，TIVA），具體如下：麻醉誘導：靜脈注射咪達唑侖（midazolam，MDZ）0.01mg/kg、瑞芬太尼（remifentanil）2μg/kg、丙泊酚（propofol）0.5mg/kg和琥珀酰膽堿（succinylcholine）1.5mg/kg。麻醉維持：微量泵持續(xù)靜脈輸注丙泊酚6～8mg·kg-1·h-1和瑞芬太尼0.1～0.2μg·kg-1·min-1，阿曲庫銨首次劑量0.6mg/kg，之后以0.1～0.2mg/kg間斷靜脈注射。術(shù)畢停止泵注靜脈麻醉藥物，吸痰，待患者自主呼吸、咳嗽反射等恢復、神志清楚、呼之能應、肌力恢復后拔除氣管導管。 拔除氣管導管即刻使用痛覺視覺模擬評分（visual analogue scale, VAS）對患者的疼痛程度進行評估。若VAS＞3分，靜脈注射芬太尼20μg/5min,直至VAS≤3分，記錄期間芬太尼的滴定用量。所有患者術(shù)后VAS≤3分時行PCIA。PCIA裝置：6300型CADD-Legacy電子鎮(zhèn)痛泵。PCIA藥物配方：氟哌利多5mg，芬太尼1.0mg，加入生理鹽水稀釋至100ml后注入鎮(zhèn)痛泵。PCIA設(shè)置：芬太尼背景劑量0.5ml/h，追加劑量2ml/h，鎖定時間為5min，最大限量145μg/h。鎮(zhèn)痛有效的評價標準：患者活動時的VAS小于或等于3分。若芬太尼消耗量達到最大限量（145μg/h）時仍無法滿足鎮(zhèn)痛需求（VAS＞3分），可輔以其他非甾體類鎮(zhèn)痛藥物以確�；颊邍g(shù)期的舒適與安全，但要將該病例從本次試驗中剔除。 3芬太尼PCIA效應的評價 觀察并記錄患者在拔管即刻、術(shù)后的第1個24h、術(shù)后的第2個24h其VAS評分情況、芬太尼PCIA的消耗量。記錄術(shù)后隨訪過程中各組患者術(shù)后頭暈、惡心嘔吐（PONV）、輕度鎮(zhèn)靜、皮膚瘙癢等不良反應的發(fā)生率。 4PPARα rs4253728GA多態(tài)性位點檢測 患者入室后肘靜脈放置留置針，抽取外周靜脈血2~3ml，采用酚-氯仿法抽提DNA，采用聚合酶鏈式反應（polymerase chain reaction, PCR）對所需的目的基因片段進行體外擴增，瓊脂糖凝膠電泳驗證擴增產(chǎn)物。采用DNA直接測序法檢測PPARα rs4253728GA多態(tài)性位點并在此基礎(chǔ)上進行分型。 5CYP3A4酶活性測定 選擇MDZ作為檢測CYP3A4酶活性的探針藥物，以MDZ代謝產(chǎn)物1-羥基咪達唑侖（1’-OH MDZ）與MDZ的比值作為衡量CYP3A4酶活性的指標。檢測方法：麻醉誘導時靜脈注射MDZ0.1mg/kg，60min后采集靜脈血5ml，離心后取上層血漿，采用液相色譜-質(zhì)譜法（liquid chromatography mass spectrometry,LC-MS）測定血漿1’-OH MDZ及血漿MDZ的濃度。 6統(tǒng)計學分析 采用SPSS17.0統(tǒng)計學軟件進行數(shù)據(jù)分析。計量資料以均數(shù)±標準差（x s）形式表示。等級資料采用秩和檢驗（wilcoxon rank sum test）。使用χ2檢驗檢測等位基因和基因型是否符合Hard-Weinberg平衡。多組間的數(shù)據(jù)比較采用單因素方差分析（ANOVA）。組間比較采用最小顯著差法（LSD）。為排除混雜因素的影響，不同組間患者芬太尼PCIA消耗量的比較采用協(xié)方差分析（covarianceanalysis）。突變的等位基因的數(shù)量與芬太尼PCIA消耗量間的相關(guān)性采用等級相關(guān)分析。各計量資料變量間的關(guān)系采用直線相關(guān)性分析。比較各組患者術(shù)后不良反應的發(fā)生情況采用χ2檢驗或者Fisher’s精確概率法。檢驗水準：α=0.05。 結(jié)果 1一般資料 納入的168例女性患者在拔管即刻、術(shù)后24h、48h平均VAS評分分別為(5.9±1.3)，（2.3±0.6），（1.1±0.5），所有患者術(shù)后均達到有效鎮(zhèn)痛標準。術(shù)后第1個24h、第2個24h芬太尼平均消耗量分別為（379.5±213.6）μg，（182.3±51.7）μg。3例患者術(shù)后出現(xiàn)頭暈，47例患者出現(xiàn)惡心嘔吐，3例患者出現(xiàn)輕度鎮(zhèn)靜，1例患者出現(xiàn)瘙癢，其不良反應的發(fā)生率分別為1.79%，27.98%，1.79%，0.60%，術(shù)后隨訪未見其他不良反應。 2中國漢族婦科患者PPARα（rs4253728GA）的等位基因頻率 PPARα rs4253728GA等位基因在中國籍漢族婦科手術(shù)患者中的變異頻率為20.4%。經(jīng)檢驗，該等位基因及基因型的分布符合Hardy-Weinberg平衡(P0.05)，其等位基因頻率高于非裔美國人(7.2%)，低于高加索人(27.3%)，與文獻報道相符(P0.05)。 3PPARα（rs4253728GA）基因多態(tài)性對CYP3A4酶活性及芬太尼PCIA效應的影響 按照直接測序的結(jié)果將患者分為野生型純合子組（GG），突變型雜合子組（GA）和突變型純合子組（AA）。三組患者一般資料的比較，差異無統(tǒng)計學意義(P0.05)。三組患者在拔管即刻及術(shù)后第1個、第2個24h的平均VAS評分組間比較差異無統(tǒng)計學意義(P0.05)。三組患者術(shù)后第1個24h芬太尼PCIA的消耗量組間比較差異無統(tǒng)計學意義(P0.05)。術(shù)后第2個24h芬太尼PCIA的消耗量組間比較，AA組患者顯著低于GA組和GG組(P0.05)。AA組患者CYP3A4酶活性較GA組和GG組有所降低(P0.05)。3組患者術(shù)后不良反應的發(fā)生情況比較差異無統(tǒng)計學意義(P0.05)。 結(jié)論 1．PPARα rs4253728GA等位基因在中國漢族婦科手術(shù)患者中的變異頻率為20.4%； 2．PPARα rs4253728GA是一個可能有功能意義的突變，其對婦科患者CYP3A4酶活性及芬太尼PCIA效應可能產(chǎn)生影響
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R614

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