本文選題：ALDH2基因多態(tài)性 + 人體藥代動(dòng)力學(xué)　； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：目的：1.采用DNA微陣列芯片法檢測(cè)從人外周血提取的DNA中線粒體乙醛脫氫酶2(aldehyde dehydrogenase2, ALDH2)基因多態(tài)性,并篩選符合臨床試驗(yàn)標(biāo)準(zhǔn)的受試者。2.進(jìn)行中國(guó)健康男性志愿者單次噴服硝酸異山梨酯噴霧劑后硝酸異山梨酯(ISDN)及其兩代謝物(2-單硝酸異山梨酯和5-單硝酸異山梨酯)藥代動(dòng)力學(xué)研究,并評(píng)價(jià)ALDH2基因多態(tài)性對(duì)三者藥代動(dòng)力學(xué)的影響。3.進(jìn)行中國(guó)健康男性志愿者單次噴服單硝酸異山梨酯噴霧劑后5-單硝酸異山梨酯(5-ISMN)藥代動(dòng)力學(xué)研究,并評(píng)價(jià)ALDH2基因多態(tài)性對(duì)其藥代動(dòng)力學(xué)的影響。4.運(yùn)用分子對(duì)接軟件模擬酶與底物的作用過(guò)程,以硝酸甘油(GTN)為參考藥,從分子水平探討ALDH2基因多態(tài)性對(duì)ISDN和5-ISMN藥代動(dòng)力學(xué)的影響。 方法：1.入組前,采集每位受試者肘靜脈血3mL, EDTA抗凝,采用血液DNA提取試劑盒,提取血樣DNA; ALDH2基因特異引物經(jīng)PCR擴(kuò)增后,將帶有生物素標(biāo)記的擴(kuò)增產(chǎn)物與固定在醛基基片上的ALDH2基因型檢測(cè)探針進(jìn)行特異性雜交反應(yīng),并通過(guò)酶促顯色反應(yīng),使特異性雜交信號(hào)顯色；對(duì)芯片進(jìn)行掃描,得到樣品DNA與ALDH2基因位點(diǎn)的野生型和突變型探針雜交形成的雜交圖像；經(jīng)BaiO BE-2.0生物芯片識(shí)讀儀分析該圖像,判斷待檢樣品的基因型。 經(jīng)查體和實(shí)驗(yàn)室檢查后,篩選出20名健康男性受試者,在了解試驗(yàn)內(nèi)容、權(quán)利、義務(wù)和風(fēng)險(xiǎn)的基礎(chǔ)上簽署知情同意書(shū)。 2.篩選出的20名健康男性受試者,按基因型分為野生組和突變雜合組,單次噴服硝酸異山梨酯噴霧劑3.75mg,于給藥前和給藥后2、4、6、8、10、12、15、20、30、45min和1、1.5、2、3、4、6、8、12、24h,肘靜脈取血5mL置于試管中(肝素抗凝),5000rpm、離心5min,取上層血漿,-20℃保存待測(cè)。GC-ECD法測(cè)定血漿中ISDN、5-ISMN和2-單硝酸異山梨酯(2-ISMN)的濃度,采用DAS2.0.1藥代動(dòng)力學(xué)軟件分別計(jì)算三者的藥代動(dòng)力學(xué)參數(shù)(t1/2、Tmax、Cmax、 AUC0-t和AUC0-∞),并對(duì)兩組藥代動(dòng)力學(xué)參數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析(代謝物Cmax、 AUC0-t和AUC0-t采用校正值比較。參數(shù)校正值是代謝物Cmax、AUC0-t、AUC0-∞與母藥相應(yīng)藥代動(dòng)力學(xué)參數(shù)的比值)。 3.上述受試者,單次噴服單硝酸異山梨酯噴霧劑5mg,于藥前和藥后8、15、20、30、45min、1、1.5、2、3、6、8、12、24h,肘靜脈取血5mL,置于試管中(肝素抗凝),5000rpm、離心5min,取上層血漿,-20℃保存待測(cè)。GC-ECD法測(cè)定血漿中5-ISMN的濃度,采用DAS2.0.1藥代動(dòng)力學(xué)軟件計(jì)算其藥代動(dòng)力學(xué)參數(shù)(t1/2、Cmax、AUC0-t和AUC0-∞),并對(duì)兩組藥代動(dòng)力學(xué)參數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 4.運(yùn)用SYBYL-X1.1軟件,分別模擬ALDH2野生酶、ALDH2突變酶與GTN、ISDN、5-ISMN的作用過(guò)程,以GTN為參照藥,分別比較兩酶與ISDN、5-ISMN的結(jié)合力。 結(jié)果：1.經(jīng)基因分型,本課題篩選出20名健康男性受試者,其中ALDH2基因野生型(ALDH2*1/*1)14名、突變雜合型(ALDH2*1/*2)6名。 2.20名受試者單次噴服硝酸異山梨酯噴霧劑3.75mg后,野生型和突變雜合型受試者體內(nèi)ISDN藥代動(dòng)力學(xué)參數(shù)如下：t1/2分別為(0.994±0.309)h和(1.342±0.086) h, Tmax分別為(0.105±0.018)h和(0.139±0.014)h,Cmax分別為(33.611±2.065)ng/mL和(42.615±3.003)ng/mL, AUC0-t分別為(18.999±3.798)ng/mL·h和(31.188±1.427)ng/mL·h, AUC0-∞分別為(20.915-4.383)ng/mL·h和(34.576±0.898)ng/mL·h。5-ISMN藥代動(dòng)力學(xué)參數(shù)如下：t1/2分別為(4.554±0.858)h和(5.849±0.781)h,Tmax分別為(0.857±0.128)h和(1.083±0.204)h, Cmax分別為(63.595±4.941)ng/mL和(83.962±5.512)ng/mL, AUC0-t分別為(286.433±72.682)ng/mL·h和(482.731±100.705)ng/mL·h, AUC0-∞分別為(300.173±68.283) ng/mL·h和(519.607±96.383)ng/mL-h;2-ISMN藥代動(dòng)力學(xué)參數(shù)如下：tl/2分別為(3.852±0.859)h和(3.940±0.887)h,Tmax分別為(0.470±0.148)h和(0.667±0.204)h,Cmax分別為(12.321±1.325)ng/mL和(16.647±2.530)ng/mL, AUC0-t分別為(36.501±4.564)ng/mL·h和(65.941±13.547)ng/mL·h, AUC0-∞分別為(42.407±5.402)ng/mL·h和(75.098±17.972)ng/mL·h。 突變雜合組與野生組相比,ISDN的t1/2和Tmax分別延長(zhǎng)42.16%(p＜0.001)和32.38%(p＜0.001),Cmax、AUC0-t和AUCo-∞分別增加26.79%(p＜0.01)、64.16%(p＜0.01)和65.32%(p＜0.01)；5-ISMN的t1/2和Tmax分別延長(zhǎng)28.44%(p＜0.05)和26.37%(p＜0.05),Cmax、AUC0-t和AUC0-∞的校正值分別增加4.13%(p＞0.05)、2.67%(p＞0.05)和4.71%(p＞0.05)；2-ISMN的t1/2和Tmax分別延長(zhǎng)2.28%(p＞0.05)和41.91%(p＞0.05),Cmax、AUC0-t和AUC0-∞的校正值分別增加6.56%(p＞0.05)、10.05%(p＞0.05)和7.12%(p＞0.05)。 3.20名受試者單次噴服單硝酸異山梨酯噴霧劑5mg后,野生型和突變雜合型受試者體內(nèi)5-ISMN藥代動(dòng)力學(xué)參數(shù)如下：t1/2分別為(4.981±1.248)h和(5.365±0.824) h, Tmax分別為(0.821±0.117)h和(0.833±0.119)h,Cmax分別為(109.332±5.668)ng/mL和(114.108±4.529) ng/mL, AUC0-t分別為(607.626±68.854) ng/mL·h和(658.459±89.621) ng/mL·h,AUC0-∞分別為(634.798±83.358) ng/mL·h和(690.937±105.191) ng/mL·h。 突變雜合組與野生組相比,t1/2和Tmax分別延長(zhǎng)了7.72%(p＞0.05)和1.45%(p0.05), Cmax、AUC0-t和AUC0-∞分別增加4.37%(p＞0.05),8.37%(p＞0.05)和8.84%(p＞0.05)。 4. SYBYL-X1.1軟件對(duì)ALDH2野生酶與GTN、ISDN、5-ISMN作用過(guò)程的打分分別為6.49、5.45和3.41,對(duì)ALDH2突變酶與GTN、ISDN、5-ISMN作用過(guò)程的打分分別為3.82、2.42和3.22。 結(jié)論：研究結(jié)果表明,ALDH2基因多態(tài)性影響ISDN的人體藥代動(dòng)力學(xué)。噴服硝酸異山梨酯噴霧劑后,與野生組相比,突變雜合組ISDN的t1/2、Tmax, Cmax、AUC0-t和AUC0-∞都增加,且差異具有統(tǒng)計(jì)學(xué)意義；與野生組相比,突變雜合組5-ISMN的t1/2和Tmax都增加,且差異有統(tǒng)計(jì)學(xué)意義,而其它藥動(dòng)學(xué)校正參數(shù)間的差異無(wú)統(tǒng)計(jì)學(xué)意義；但兩組2-ISMN校正藥代動(dòng)力學(xué)參數(shù)間差異無(wú)統(tǒng)計(jì)學(xué)意義。 ALDH2基因多態(tài)性不影響5-ISMN的人體藥代動(dòng)力學(xué)。噴服單硝酸異山梨酯噴霧劑后,兩組間5-ISMN藥代動(dòng)力學(xué)參數(shù)存在差異,但差異均無(wú)統(tǒng)計(jì)學(xué)意義。 ALDH2野生酶與GTN、ISDN的結(jié)合力顯著高于ALDH2突變酶與二者的結(jié)合力；但兩酶與5-ISMN結(jié)合力差異不明顯。
[Abstract]:Objective: 1. the polymorphisms of mitochondrial acetaldehyde dehydrogenase 2 (aldehyde dehydrogenase2, ALDH2) gene in DNA extracted from human peripheral blood were detected by DNA microarray, and.2. was selected for the single dose of Isosorbide Dinitrate Spray of Chinese healthy male volunteers after single injection of isosorbate nitrate (ISDN) in Chinese healthy male volunteers. Pharmacokinetics of its two metabolites (2- isosorbide mononitrate and 5- isosorbide mononitrate), and evaluation of the effect of ALDH2 gene polymorphism on the pharmacokinetics of the three,.3. pharmacokinetics of 5- mononitrate 5- mononitrate (5-ISMN) after single injection of Isosorbide 5-Mononitrate Spray in Chinese healthy male volunteers was studied and ALDH2 was evaluated for ALDH2 The effect of gene polymorphism on its pharmacokinetics.4. using molecular docking software to simulate the action process of enzyme and substrate, using nitroglycerin (GTN) as the reference drug, to investigate the effect of ALDH2 gene polymorphism on the pharmacokinetics of ISDN and 5-ISMN from the molecular level.
Methods: before 1., the blood samples of each recipient were collected from the elbow vein blood 3mL, EDTA anticoagulant, and blood DNA extraction kit was used to extract the blood sample DNA. The specific primers of ALDH2 gene were amplified by PCR, and the amplified products with biotin markers were specifically hybridized with the ALDH2 genotypic probe fixed on the aldehyde base. The color reaction made the specific hybridization signal color, and the chip was scanned and the hybrid images of the wild type and mutant probe of the DNA and ALDH2 gene loci were hybridized. The image was analyzed by the BaiO BE-2.0 biochip reader to determine the genotype of the samples to be tested.
After physical examination and laboratory examination, 20 healthy male subjects were screened and informed consent was obtained on the basis of knowing the contents, rights, duties and risks of the experiment.
2. selected 20 healthy male subjects were divided into the wild group and the mutant heterozygous group according to the genotype, the single injection of Isosorbide Dinitrate Spray 3.75mg, 2,4,6,8,10,12,15,20,30,45min and 1,1.5,2,3,4,6,8,12,24h before and after administration, and the 5mL in the elbow vein (heparin anticoagulant), 5000rpm, centrifugation 5min, the upper plasma, -20 The concentration of ISDN, 5-ISMN and isosorbide mononitrate (2-ISMN) in plasma was determined by.GC-ECD, and the pharmacokinetic parameters of the three groups were calculated by DAS2.0.1 pharmacokinetics software (t1/2, Tmax, Cmax, AUC0-t and AUC0-) respectively, and the pharmacokinetic parameters of the two groups were statistically analyzed. Comparison of calibration values. Parameter correction is the ratio of metabolites Cmax, AUC0-t, AUC0- infinity to the corresponding pharmacokinetic parameters of the parent drug.
3. the above subjects were given a single dose of Isosorbide 5-Mononitrate Spray 5mg, and 5mL was taken from 8,15,20,30,45min, 1,1.5,2,3,6,8,12,24h, and elbow vein before and after the drug. It was placed in a test tube (heparin anticoagulant), 5000rpm, centrifugation 5min, taking the upper plasma, -20 C and.GC-ECD method to determine the concentration of 5-ISMN in plasma, and the DAS2.0.1 pharmacokinetics software was used. The pharmacokinetic parameters (t1/2, Cmax, AUC0-t and AUC0- infinity) were calculated, and the pharmacokinetic parameters of the two groups were analyzed statistically.
4. SYBYL-X1.1 software was used to simulate the action process of ALDH2 wild enzyme, ALDH2 mutase and GTN, ISDN, 5-ISMN respectively. The binding force of the two enzyme to ISDN and 5-ISMN was compared with GTN as the reference.
Results: 1. according to genotyping, 20 healthy male subjects were screened out in this study. Among them, 14 were wild type (ALDH2*1/*1) of ALDH2 gene, 6 were mutant heterozygous (ALDH2*1/*2).
After a single dose of Isosorbide Dinitrate Spray 3.75mg, the ISDN pharmacokinetic parameters of wild and mutant heterozygous subjects were as follows: t1/2 was (0.994 + 0.309) H and (1.342 + 0.086) h respectively, Tmax was (0.105 + 0.018) H and (0.139 + 0.014) h respectively, Cmax was (33.611 + 2.065) ng/mL and (42.615 + 3.003) ng/mL, AUC0 -t (18.999 + 3.798) ng/mL / h and (31.188 + 1.427) ng/mL. H respectively, AUC0- infinity (20.915-4.383) ng/mL. H and (34.576 + 0.898) ng/mL. H.5-ISMN pharmacokinetic parameters are as follows: t1/2 is (4.554 + 0.858) and (5.849 + 0.781) respectively, respectively (0.857 + 0.128) and (1.083 +) 962 + 5.512) ng/mL, AUC0-t (286.433 + 72.682) ng/mL / h and (482.731 + 100.705) ng/mL. H respectively, AUC0- infinity is (300.173 + 68.283) ng/mL. H and (519.607 + 96.383) ng/mL-h, 2-ISMN pharmacokinetic parameters are as follows: tl/2 is respectively (3.852 + 0.859) and [3.940 +] respectively. Not (12.321 + 1.325) ng/mL and (16.647 + 2.530) ng/mL, AUC0-t (36.501 + 4.564) ng/mL. H and (65.941 + 13.547) ng/mL. H, AUC0- infinity respectively (42.407 + 5.402) ng/mL h and (75.098 + 17.972) ng/mL. H.
Compared with the wild group, the t1/2 and Tmax of ISDN were prolonged by 42.16% (P < 0.001) and 32.38% (P < 0.001), Cmax, AUC0-t and AUCo- infinity were increased by 26.79% (P < 0.01), 64.16% (P < 0.01) and 65.32% (P < 0.01), respectively. Increase 4.13% (P > 0.05), 2.67% (P > 0.05) and 4.71% (P > 0.05); t1/2 and Tmax of 2-ISMN extend 2.28% (P > 0.05) and 41.91% (P > 0.05), Cmax, AUC0-t and AUC0- infinity respectively increase 6.56% (P > 0.05), 10.05% (0.05 > 0.05).
After a single dose of Isosorbide 5-Mononitrate Spray 5mg, the 5-ISMN pharmacokinetics parameters of wild and mutant heterozygous subjects were as follows: t1/2 was (4.981 + 1.248) H and (5.365 + 0.824) h respectively, Tmax was (0.821 + 0.117) H and (0.833 + 0.119) h respectively, Cmax was (109.332 + 5.668) ng/mL and (114.108 + 4.529) ng/mL, AUC0-t were (607.626 + 68.854) ng/mL? H and (658.459 + 89.621) ng/mL? H, AUC0- AUC0- were (634.798 + 83.358) ng/mL? H and (690.937 + 105.191) ng/mL? H., respectively.
Compared with the wild group, the t1/2 and Tmax increased by 7.72% (P > 0.05) and 1.45% (P0.05), Cmax, AUC0-t and AUC0-, respectively 4.37% (P > 0.05), 8.37% (P > 0.05) and 8.84% (P > 0.05), respectively.
4. SYBYL-X1.1 software was divided into 6.49,5.45 and 3.41 for the action of ALDH2 wild enzyme and GTN, ISDN and 5-ISMN, respectively. The action process of ALDH2 mutase and GTN, ISDN and 5-ISMN were 3.82,2.42 and 3.22..
Conclusion: the results show that the polymorphism of ALDH2 gene affects the pharmacokinetics of ISDN in the human body. Compared with the wild group, the t1/2, Tmax, Cmax, AUC0-t and AUC0- infinity of the mutant heterozygous group are all increased, and the difference has statistical significance compared with the wild group. Compared with the wild group, the t1/2 and Tmax of 5-ISMN in the mutant heterozygous group are all increased. Plus, the difference was statistically significant, but there was no statistical difference between the positive parameters of other pharmacokinetic schools, but there was no statistical difference between the two groups of 2-ISMN correction pharmacokinetic parameters.
ALDH2 gene polymorphism did not affect the pharmacokinetics of 5-ISMN in human body. After spraying Isosorbide 5-Mononitrate Spray, there were differences in the pharmacokinetic parameters of 5-ISMN between the two groups, but the difference was not statistically significant.
The binding capacity of ALDH2 wild enzyme to GTN and ISDN was significantly higher than that of ALDH2 mutant enzyme, but the binding force between two enzyme and 5-ISMN was not obvious. Two
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R969.1

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