發(fā)布時(shí)間：2018-04-13 22:20

  本文選題：造血干細(xì)胞移植(HSCT) + 急性移植物抗宿主病(aGVHD)��； 參考：《蘇州大學(xué)》2015年碩士論文

【摘要】：目的基于氣象色譜質(zhì)譜聯(lián)用(GC-MS)的方法,探究造血干細(xì)胞移植后急性移植物抗宿主病(a GVHD)的血漿代謝標(biāo)志物。方法本文采用GC-MS的研究方法分別對(duì)移植后7天內(nèi)、移植后14天、28天及移植后29天至60天四個(gè)時(shí)間點(diǎn)的61位患者174例血漿樣本進(jìn)行處理,利用自動(dòng)質(zhì)譜退卷積定性系統(tǒng)(AMDIS)和美國(guó)國(guó)家標(biāo)準(zhǔn)與技術(shù)研究院(NIST)標(biāo)準(zhǔn)庫(kù)并結(jié)合保留指數(shù)對(duì)血漿中的化合物進(jìn)行鑒定,再應(yīng)用正交偏最小二乘判別分析(OPLS-DA)、t檢驗(yàn)、遺傳算法(GA)等多種統(tǒng)計(jì)學(xué)方法選取特征代謝物,并應(yīng)用Logistic回歸合并受試者工作特征曲線(ROC曲線)對(duì)代謝標(biāo)志物組合進(jìn)行最終篩選與評(píng)價(jià)。結(jié)果(1)建立了穩(wěn)定、可靠的適用于人血漿的GC-MS代謝組學(xué)分析方法。本研究對(duì)該分析方法的日內(nèi)穩(wěn)定性、日間穩(wěn)定性和預(yù)處理的重現(xiàn)性進(jìn)行考察。實(shí)驗(yàn)結(jié)果表明上述三項(xiàng)考察中共有峰面積相對(duì)標(biāo)準(zhǔn)偏差(RSD)15%的數(shù)目占到所有共有峰的95%以上,說(shuō)明該方法能能夠滿足血漿中代謝物相對(duì)定量的需要。(2)GC-MS掃描獲得的總離子圖(TIC)憑借AMDIS的解析合并NIST 11庫(kù)的檢索并聯(lián)合保留指數(shù)的比對(duì)共鑒定出54種化合物,包括氨基酸、脂肪酸和糖類等。(3)應(yīng)用OPLS-DA方法分別對(duì)不同時(shí)間點(diǎn)的所有化合物信息進(jìn)行分析,各個(gè)時(shí)間點(diǎn)陰性組陽(yáng)性組分界清晰,聯(lián)合OPLS-DA模型下的散點(diǎn)圖(S-Plot)和變異權(quán)重參數(shù)值(VIP)值選取出不同時(shí)間點(diǎn)的a GVHD血漿特征代謝物,分別是:移植后7天內(nèi):尿素;移植后14天:尿素,棕櫚酸;移植后28天:尿素;移植后29天至60天:乳酸,羥基乙酸,果糖,塔羅糖。(4)分別對(duì)4個(gè)時(shí)間點(diǎn)所有鑒定出的化合物行獨(dú)立樣本t檢驗(yàn),以p≤0.05為標(biāo)準(zhǔn)選取出相應(yīng)時(shí)間點(diǎn)的數(shù)個(gè)a GVHD血漿特征代謝物,分別為:移植后7天內(nèi):丙氨酸;移植后14天:山梨醇,甘油酸;移植后28天:甘氨酸,山梨醇;移植后29天至60天:乳酸,丙酸,纈氨酸,尿素,甘氨酸,絲氨酸,山梨醇,葡萄糖,肌醇,丙二醇,α-生育酚,羥基乙酸,2,3-二羥基丁酸乙酯。(5)應(yīng)用GA算法分別對(duì)不同時(shí)間點(diǎn)所有化合物信息進(jìn)行計(jì)算,每個(gè)時(shí)間點(diǎn)計(jì)算5次,取位次靠前的十種化合物,其結(jié)果分別是:移植后7天內(nèi):丙氨酸,3-羥丁酸,甘油,苯丙氨酸,山梨醇,酪氨酸,油酸,鳥氨酸,磷酸,甘油酸;移植后14天:丙氨酸,尿素,山梨醇,尿酸,棕櫚酸單甘油酯,門冬氨酸,甘油酸,甘露糖,十五酸,花生酸;移植后28天:甘油,脯氨酸,甘氨酸,絲氨酸,山梨醇,亞油酸,油酸,色氨酸,磷酸,甘油酸;移植后29天至60天:乳酸,草酸,纈氨酸,甘氨酸,蘇氨酸,山梨醇,肌醇,丙二醇,α-生育酚,甘油酸。(6)將4個(gè)時(shí)間點(diǎn)所有化合物分別用Logistic回歸方程進(jìn)行計(jì)算,以α入=0.05,α出=0.10為標(biāo)準(zhǔn)選取各個(gè)時(shí)間點(diǎn)的特征代謝物,其結(jié)果為:移植后7天內(nèi):丙氨酸,尿素,酪氨酸;移植后14天:山梨醇,甘油酸,十五酸;移植后28天:甘氨酸,山梨醇,甘油酸;移植后29天至60天:乳酸,草酸,蘇氨酸,丙二醇。(7)將上述每種方法選取的特征代謝物按照時(shí)間點(diǎn)進(jìn)行合并,每個(gè)時(shí)間點(diǎn)化合物以不同的組合引入Logistic回歸方程,計(jì)算相應(yīng)的預(yù)測(cè)值,并用其繪制ROC曲線,直到某個(gè)組合滿足:1)ROC曲線下組合的p值0.01;2)ROC曲線的AUC0.85;3)ROC曲線截點(diǎn)的靈敏度和特異性均70%為止,即將該組合作為這個(gè)時(shí)間點(diǎn)的代謝標(biāo)志物組合。最終的組合是:移植后7天內(nèi):丙氨酸+尿素+酪氨酸;移植后14天:丙氨酸+尿素+山梨醇+尿酸+棕櫚酸單甘油酯+門冬氨酸+甘油酸+正十五酸+花生酸;移植后28天:丙氨酸+絲氨酸+山梨醇+油酸+甘氨酸;移植后29天至60天:乳酸+草酸+蘇氨酸+丙二醇。其中移植后7天內(nèi)這個(gè)時(shí)間點(diǎn)篩選出的組合,其ROC曲線的AUC達(dá)到0.935,靈敏度和特異性分別達(dá)到90.5%和81.8%,是所有組合中對(duì)a GVHD的發(fā)生預(yù)測(cè)能力最強(qiáng)的。結(jié)論(1)基于GC-MS方法建立的代謝模型可以用于造血干細(xì)胞移植后a GVHD的預(yù)測(cè)。(2)造血干細(xì)胞移植后所選取的各個(gè)時(shí)間點(diǎn)均可篩選出預(yù)測(cè)和區(qū)分能力良好的a GVHD特異的代謝標(biāo)志物組合。
[Abstract]:Based on Meteorological chromatography-mass spectrometry (GC-MS) method, inquiry after hematopoietic stem cell transplantation for acute graft-versus-host disease (a GVHD) of the plasma metabolic markers. Methods this paper adopts the research methods of GC-MS respectively for 7 days after transplantation, 14 days after transplantation, 28 days and 29 days to 60 days after transplantation four time points in 61 patients, 174 cases of plasma samples were processed by automated mass spectral deconvolution & identification system (AMDIS) and the National Institute of standards and Technology (NIST) standard library and identification of compounds in plasma combined with retention index, using orthogonal partial least squares discriminant analysis (OPLS-DA), t test the genetic algorithm (GA), and other statistical methods of characteristic metabolites, and the application of Logistic regression with receiver operating characteristic curve (ROC curve) of the final screening and evaluation of metabolic markers. Results (1) to establish a stable and reliable. For GC-MS metabolic plasma analysis method. The research on the analysis method of stability in a day, the day the stability and reproducibility of pretreatment were investigated. The experimental results show that the three peaks of the relative standard deviation (RSD) to the number 15% account for all of the common peaks above 95%, indicating this method can meet the needs of the relative quantification of metabolites in plasma. (2) the total ion map scanned by GC-MS (TIC) by comparison with NIST AMDIS 11 retrieval analysis library and combined retention index of 54 compounds were identified, including amino acids, fatty acids and carbohydrates (3) were. Analysis and application of OPLS-DA method at different time points of all the compounds, each time point negative group, positive group clear demarcation, scattered united under the OPLS-DA model diagram (S-Plot) and the variation of weight parameter value (VIP) values extracted at different time points A GVHD features of plasma metabolites, respectively: 7 days after transplantation: 14 days after transplantation: urea; urea, palmitic acid; urea; 28 days after transplantation: 29 to 60 days after transplantation: lactic acid, glycolic acid, fructose, talose. (4) of all 4 time points identified compounds for independent samples t test, respectively with P lower than 0.05 for the standard selected a GVHD plasma characteristics of metabolites, the corresponding time: 7 days after transplantation: alanine; 14 days after transplantation: sorbitol, glycerol acid; 28 days after transplantation: glycine, sorbitol; 29 to 60 days after transplantation lactic acid, propionic acid, urea, glycine, valine, serine, sorbitol, glucose, inositol, propylene glycol, tocopherol, glycolic acid, 2,3- two hydroxy ethyl butyrate. (5) the application of GA algorithm at different time points of all the compounds information is calculated, each time point is calculated from 5 times, ranking ten compound front, the results Don't is: 7 days after transplantation: alanine, 3- hydroxybutyric acid, glycerol, sorbitol, phenylalanine, tyrosine, oleic acid, ornithine, phosphoric acid, glycerol acid; 14 days after transplantation: alanine, urea, sorbitol, uric acid, palmitic acid monoglyceride, aspartic acid, glycerol acid, mannose, fifteen acid, arachidic acid 28 days after transplantation; glycerol, proline, glycine, serine, sorbitol, linoleic acid, oleic acid, tryptophan, phosphoric acid, glycerol acid; 29 to 60 days after transplantation: lactic acid, oxalic acid, valine, glycine, threonine, sorbitol, inositol, propylene glycol, alpha tocopherol, glyceric acid (6). The 4 time point of all the compounds were used Logistic regression equation to calculate, alpha =0.05, alpha =0.10 as the standard for selecting characteristic metabolites each time point, the results are as follows: 7 days after transplantation: urea, alanine, tyrosine; 14 days after transplantation: sorbitol, glycerol acid, fifteen acid; 28 days after transplantation: glycine, Yamanashi Alcohol, glycerol acid; 29 to 60 days after transplantation: lactic acid, oxalic acid, threonine, propylene glycol. (7) the characteristic metabolites of each method are combined according to the selected time points, each time point compounds in different combinations using the Logistic regression equation, calculate the predicted value, and draw the curve with ROC which, until a combination meet: 1) the combination of P ROC under the curve value of 0.01; 2) ROC curve AUC0.85; 3) the sensitivity and specificity of the ROC curve cut point are 70% so far, is the combination as the time point of metabolic markers. The final combination is: 7 days after transplantation: alanine + urea + tyrosine; 14 days after transplantation: alanine + urea + sorbitol + uric acid + palmitic acid monoglyceride + aspartic acid and glycerol is fifteen + acid + acid arachidonic acid; 28 days after transplantation: alanine serine + + sorbitol + oleic acid + glycine; 29 to 60 days after transplantation: lactic acid + oxalic acid + + threonine Propylene glycol. 7 days after transplantation the time point selected combination, the ROC curve of AUC reached 0.935, the sensitivity and specificity were respectively 90.5% and 81.8%, is the strongest predictive power of a GVHD in all combinations. Conclusion (1) metabolism model based on the GC-MS method can be used for blood a GVHD predicted stem cell transplantation. (2) hematopoietic stem cell transplantation selected after each time point can be selected to predict and distinguish the specific ability a GVHD metabolic markers.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R457.7

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本文編號(hào)：1746457