基于UFLC-IT-TOF MS技術(shù)的乙型肝炎病毒慢性感染后肝病不同臨床階段的血清代謝組學(xué)研究
發(fā)布時間：2018-08-22 20:52
【摘要】：目的:利用超快速液相色譜-離子阱-飛行時間串聯(lián)質(zhì)譜分析(UFLC-IT-TOF MS)技術(shù),建立了分析檢測肝癌患者血清中廣譜小分子代謝物的代謝組學(xué)新方法。并將該方法應(yīng)用于測定乙肝病毒感染后肝病的不同階段患者及健康志愿者血清中的代謝產(chǎn)物,分析其間的差異和變化,探討其在機體內(nèi)的代謝特征,為揭示其發(fā)病機制及輔助診斷提供可參考的代謝組學(xué)信息。方法:1利用UFLC-IT-TOF MS技術(shù),建立檢測小分子代謝產(chǎn)物新方法隨機選取肝癌患者80例、健康者20例,留取晨起空腹外周靜脈血3ml,置于標(biāo)準(zhǔn)一次性采血玻璃管中,離心后取血清1.5ml存放至EP管密封,置于-80℃冰箱中冷凍保存?zhèn)溆�。根�?jù)標(biāo)準(zhǔn)物質(zhì)的質(zhì)譜相應(yīng)值確定各物質(zhì)檢測的離子模式,配制內(nèi)標(biāo)溶液備用。比較不同的溶劑系統(tǒng)種類、與血清的體積比和預(yù)存溫度,觀察沉淀蛋白的效果;比較不同色譜-質(zhì)譜條件下,有效信號、干擾噪音峰形及離子化效率,確定最佳方案,并通過在肝癌組混合血清樣本中加入標(biāo)準(zhǔn)溶液,經(jīng)多次平行測定驗證方法良好。將上述80例肝癌患者和20例健康者的混合血清樣本按該方法進(jìn)行UFLC-IT-TOF MS檢測。檢測結(jié)果應(yīng)用LCMS Solution(版本3.60)等軟件對上述三維數(shù)據(jù)進(jìn)行多維統(tǒng)計分析,以篩選出有統(tǒng)計學(xué)意義的變量。2具有潛在臨床應(yīng)用價值的代謝標(biāo)志物分析隨機選取處于肝病不同階段的HBV感染患者197例,同期選擇58例健康志愿者,入選受試者共255例。診斷標(biāo)準(zhǔn),樣本制備方法同上。應(yīng)用上述方法,檢測上述各組共255例血清單個樣本,得到的數(shù)值應(yīng)用IBM SPSS(版本24.0)軟件進(jìn)行分析。結(jié)果:1用于檢測血清中小分子代謝產(chǎn)物的檢測方法得到優(yōu)化通過對比成分、比例不同的溶劑,比較提取離子流色譜圖的峰強度、分離度和檢測物質(zhì)峰數(shù),通過對比溶劑與血清不同體積比的柱壓情況,結(jié)合環(huán)境保護的原則,通過對比不同預(yù)存溫度下的提取效率,確定最佳的有機蛋白沉淀方案。通過評價不同流動相的獲得信號、減少噪音、峰形的靈敏度和分離度繼離子化效率,確定最佳流動相的體系、組成、流速和柱溫。并利用標(biāo)準(zhǔn)溶液,平行在不同時間點、連續(xù)測定多個樣本,計算RSD值,進(jìn)行PCA分析,結(jié)果表明該方法精密度、穩(wěn)定性、可靠性良好。2在肝癌患者與健康人混合血清中篩選出有價值的潛在標(biāo)志物通過應(yīng)用多變量統(tǒng)計分析方法,分析肝癌患者與對照組的代謝物指紋圖譜,篩選出8種有臨床價值的肝癌潛在標(biāo)志代謝產(chǎn)物,確定為:甘氨膽酸、甘油磷酸酯、十四酰胺、乙酰苯丙氨酸、油酰胺、乙酰肉毒堿、溶血卵磷脂、甘氨鵝去氧膽酸。3應(yīng)用上述方法檢測HBV感染各組及健康對照組單個樣本確定有臨床價值的代謝標(biāo)志物通過上述方法,測定上述255例受試者的單個血清樣本中上述8種代謝產(chǎn)物的含量。發(fā)現(xiàn)其中4種物質(zhì)含量極低,各個樣本間差別較大,多數(shù)樣本測定數(shù)值為0,無法進(jìn)行統(tǒng)計學(xué)歸納。另外4種代謝產(chǎn)物:溶血卵磷脂、乙酰肉毒堿、油酰胺、甘氨膽酸,通過非參數(shù)檢驗,確定其在各組間的差別有統(tǒng)計學(xué)意義。對乙酰肉毒堿在肝硬化和肝癌患者中較為顯著的變化,應(yīng)用ROC曲線,計算AUC值,得到最佳界定點的靈敏的和特異性。結(jié)論:1通過優(yōu)化對肝癌患者與健康組混合血清樣本的預(yù)處理方式,建立了基于UFLC-IT-TOF MS技術(shù)的新方法,符合高通量的篩選代謝組學(xué)成分的研究方法要求,適合對待研究的生物樣本進(jìn)行大批量篩選代謝組學(xué)成分的研究。2溶血卵磷脂、乙酰肉毒堿、油酰胺有望成為肝癌的代謝標(biāo)志物。其中,溶血卵磷脂在肝癌患者血液中的含量較其他各組明顯降低,與另外2種代謝物相比較,AUC值最大,診斷效能好,為輔助臨床診斷及開展科學(xué)研究提供可參考的代謝組學(xué)信息。
[Abstract]:OBJECTIVE:To establish a new method for the determination of broad-spectrum small molecule metabolites in serum of patients with hepatocellular carcinoma by ultra-fast liquid chromatography-ion trap-time-of-flight mass spectrometry(UFLC-IT-TOF MS). METHODS: 1 Using UFLC-IT-TOF MS, a new method was established to detect small molecule metabolites in 80 patients with hepatocellular carcinoma (HCC) and 20 healthy volunteers. Peripheral venous blood was stored in a standard disposable glass tube for 3 ml. After centrifugation, 1.5 ml of serum was taken and sealed in an EP tube. The plasma was frozen in a refrigerator at - 80 C for reserve. Ion patterns of each substance were determined according to the corresponding mass spectrometry values of the standard substances, and internal standard solutions were prepared. The effect of precipitated protein was observed at pre-storage temperature, and the best scheme was determined by comparing the effective signal, noise peak shape and ionization efficiency under different conditions of GC-MS. The method was verified by adding standard solution into mixed serum samples of hepatocellular carcinoma group, and was verified by many parallel tests. Serum samples were tested by UFLC-IT-TOF MS. The results were analyzed by LCMS Solution (Version 3.60) and other software to screen statistically significant variables. 97 healthy volunteers and 255 healthy volunteers were selected at the same time. The diagnostic criteria and sample preparation methods were the same as above. Using the above methods, 255 serum samples from each group were detected. The values obtained were analyzed by IBM SPSS (version 24.0). Through comparing the components and the proportion of different solvents, the peak intensity, separation degree and the number of detection substances were compared. By comparing the column pressure of different volume ratio of solvent and serum, and combining with the principle of environmental protection, the best Precipitation Scheme of organic protein was determined by comparing the extraction efficiency under different storage temperature. The system, composition, flow rate and column temperature of the best mobile phase were determined by evaluating the signals obtained from different mobile phases, reducing noise, peak shape sensitivity and separation efficiency. Reliability is good. 2 Select valuable potential markers in the mixed serum of patients with liver cancer and healthy people. Analyse the metabolite fingerprint of patients with liver cancer and control group by using multivariate statistical analysis method. Screen out 8 potential metabolites of liver cancer markers with clinical value, which are: glycocholic acid, glycerophosphate, 14. Amide, acetylphenylalanine, oleamide, acetylcarnitine, lysophosphatidylcholine, glycine-geese deoxycholic acid. 3 The above methods were used to detect the metabolic markers of HBV infection in each group and in a single sample of healthy control group to determine the content of the above 8 metabolites in a single serum sample of the 255 subjects. The other four metabolites, hemolytic lecithin, acetylcarnitine, oleamide, glycocholic acid, were determined by non-parametric test, and the difference between the groups was statistically significant. Conclusion: 1. A new method based on UFLC-IT-TOF MS was established by optimizing the pretreatment method of mixed serum samples from patients with hepatocellular carcinoma and healthy subjects, which was in line with high throughput screening of metabolomic components. 2. Hemolytic lecithin, acetylcarnitine and oleamide are expected to be metabolic markers of hepatocellular carcinoma. Among them, the content of hemolytic lecithin in the blood of patients with hepatocellular carcinoma is significantly lower than that of the other two metabolites. It has the highest diagnostic value and good diagnostic efficacy, and can provide reference metabonomics information for clinical diagnosis and scientific research.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.62
本文編號：2198271
[Abstract]:OBJECTIVE:To establish a new method for the determination of broad-spectrum small molecule metabolites in serum of patients with hepatocellular carcinoma by ultra-fast liquid chromatography-ion trap-time-of-flight mass spectrometry(UFLC-IT-TOF MS). METHODS: 1 Using UFLC-IT-TOF MS, a new method was established to detect small molecule metabolites in 80 patients with hepatocellular carcinoma (HCC) and 20 healthy volunteers. Peripheral venous blood was stored in a standard disposable glass tube for 3 ml. After centrifugation, 1.5 ml of serum was taken and sealed in an EP tube. The plasma was frozen in a refrigerator at - 80 C for reserve. Ion patterns of each substance were determined according to the corresponding mass spectrometry values of the standard substances, and internal standard solutions were prepared. The effect of precipitated protein was observed at pre-storage temperature, and the best scheme was determined by comparing the effective signal, noise peak shape and ionization efficiency under different conditions of GC-MS. The method was verified by adding standard solution into mixed serum samples of hepatocellular carcinoma group, and was verified by many parallel tests. Serum samples were tested by UFLC-IT-TOF MS. The results were analyzed by LCMS Solution (Version 3.60) and other software to screen statistically significant variables. 97 healthy volunteers and 255 healthy volunteers were selected at the same time. The diagnostic criteria and sample preparation methods were the same as above. Using the above methods, 255 serum samples from each group were detected. The values obtained were analyzed by IBM SPSS (version 24.0). Through comparing the components and the proportion of different solvents, the peak intensity, separation degree and the number of detection substances were compared. By comparing the column pressure of different volume ratio of solvent and serum, and combining with the principle of environmental protection, the best Precipitation Scheme of organic protein was determined by comparing the extraction efficiency under different storage temperature. The system, composition, flow rate and column temperature of the best mobile phase were determined by evaluating the signals obtained from different mobile phases, reducing noise, peak shape sensitivity and separation efficiency. Reliability is good. 2 Select valuable potential markers in the mixed serum of patients with liver cancer and healthy people. Analyse the metabolite fingerprint of patients with liver cancer and control group by using multivariate statistical analysis method. Screen out 8 potential metabolites of liver cancer markers with clinical value, which are: glycocholic acid, glycerophosphate, 14. Amide, acetylphenylalanine, oleamide, acetylcarnitine, lysophosphatidylcholine, glycine-geese deoxycholic acid. 3 The above methods were used to detect the metabolic markers of HBV infection in each group and in a single sample of healthy control group to determine the content of the above 8 metabolites in a single serum sample of the 255 subjects. The other four metabolites, hemolytic lecithin, acetylcarnitine, oleamide, glycocholic acid, were determined by non-parametric test, and the difference between the groups was statistically significant. Conclusion: 1. A new method based on UFLC-IT-TOF MS was established by optimizing the pretreatment method of mixed serum samples from patients with hepatocellular carcinoma and healthy subjects, which was in line with high throughput screening of metabolomic components. 2. Hemolytic lecithin, acetylcarnitine and oleamide are expected to be metabolic markers of hepatocellular carcinoma. Among them, the content of hemolytic lecithin in the blood of patients with hepatocellular carcinoma is significantly lower than that of the other two metabolites. It has the highest diagnostic value and good diagnostic efficacy, and can provide reference metabonomics information for clinical diagnosis and scientific research.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.62
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