【摘要】：溴隱亭(Bromocriptine, BC)耐藥是垂體泌乳素腺瘤藥物治療無效或復(fù)發(fā)的關(guān)鍵因素,探索垂體泌乳素腺瘤對藥物BC產(chǎn)生耐藥性的原因并對耐藥垂體泌乳素腺瘤患者進(jìn)行準(zhǔn)確診斷有利于實施個體化治療,提高治療效果。本論文首先采用高效液相色譜(HPLC)與串聯(lián)質(zhì)譜(MS/MS)聯(lián)用技術(shù),建立了靈敏、快速的適用于臨床垂體泌乳素腺瘤患者的血漿和腫瘤組織中藥物BC的定量分析方法。采用該方法比較分析了耐藥型與敏感型垂體泌乳素腺瘤患者的血漿及腫瘤組織中藥物BC的分布差異,發(fā)現(xiàn)藥物BC在耐藥患者體內(nèi)也能夠被有效地吸收和轉(zhuǎn)運(yùn)至腫瘤組織,在腫瘤組織中維持較高的含量,耐藥型垂體泌乳素腺瘤患者經(jīng)BC治療無效的原因可能是腫瘤細(xì)胞自身對藥物產(chǎn)生的耐藥性。在上述研究基礎(chǔ)上,進(jìn)一步采用基于LC-MS/MS技術(shù)的代謝組學(xué)方法,分別以臨床垂體泌乳素腺瘤患者的血漿和大鼠垂體泌乳素腺瘤細(xì)胞為研究對象,開展了垂體泌乳素腺瘤藥物BC的耐藥性研究。篩選出一批與耐藥相關(guān)的潛在生物標(biāo)志物,并利用發(fā)現(xiàn)的生物標(biāo)志物進(jìn)行了代謝通路分析,在BC耐藥垂體泌乳素腺瘤患者和耐藥腫瘤細(xì)胞中發(fā)現(xiàn)了與耐藥相關(guān)的發(fā)生紊亂的代謝通路。本研究結(jié)果為臨床BC耐藥垂體泌乳素腺瘤的耐藥性機(jī)制提供了分子基礎(chǔ)及重要依據(jù)。本論文的研究內(nèi)容主要包括以下三個部分：1.臨床患者血漿及垂體泌乳素腺瘤組織中藥物溴隱亭的定量分析采用LC-MS/MS技術(shù),通過對色譜條件和MRM檢測模式下質(zhì)譜參數(shù)的系統(tǒng)優(yōu)化,建立了靈敏、快速、專屬性強(qiáng),適用于臨床人體血漿和腫瘤組織中藥物BC的定量分析方法。采用該方法對耐藥型和敏感型垂體泌乳素腺瘤患者的血漿及腫瘤組織中藥物BC進(jìn)行了定量分析,發(fā)現(xiàn)耐藥患者口服BC 2,4和6h后,體內(nèi)血漿藥物濃度明顯高于敏感患者；服藥3個月后的耐藥患者腫瘤組織中的BC濃度也明顯高于敏感患者,表明BC在耐藥患者體內(nèi)也能夠被有效地吸收,并能夠被有效地從血漿轉(zhuǎn)運(yùn)至腫瘤組織,在腫瘤組織中維持較高的藥物含量。本研究顯示腫瘤細(xì)胞自身對藥物BC產(chǎn)生的抵抗可能是導(dǎo)致耐藥型垂體泌乳素腺瘤患者經(jīng)藥物BC治療無效的原因。2.溴隱亭耐藥垂體泌乳素腺瘤的血漿代謝組學(xué)研究采用正、負(fù)離子檢測模式相結(jié)合的LC-MS/MS分析方法,對BC耐藥型和敏感型垂體泌乳素腺瘤患者的血漿樣本進(jìn)行了代謝組學(xué)分析。最終在耐藥患者和敏感患者的血漿中發(fā)現(xiàn)了64個具有顯著性差異的代謝物,即潛在耐藥標(biāo)志物。進(jìn)一步通過高分辨率MS譜及MS/MS譜分析,結(jié)合同位素豐度比、氮規(guī)則,以及網(wǎng)絡(luò)數(shù)據(jù)庫檢索和標(biāo)準(zhǔn)品對比等分析手段與步驟對這些潛在耐藥標(biāo)志物的結(jié)構(gòu)進(jìn)行了分析鑒定。目前,共鑒定出21個潛在耐藥標(biāo)志物的結(jié)構(gòu),包括氨基酸、鞘脂、脂肪酸類等代謝物,其中1-磷酸二氫鞘氨醇、C16鞘氨醇、1-脫氧C14二氫鞘氨醇等9個潛在耐藥標(biāo)志物具有較高的診斷準(zhǔn)確性(AUC0.9)。進(jìn)一步對這些潛在耐藥標(biāo)志物進(jìn)行了代謝通路分析,發(fā)現(xiàn)與BC敏感型垂體泌乳素腺瘤患者相比,耐藥患者體內(nèi)鞘脂類代謝,賴氨酸生物合成,氨酰tRNA生物合成,丙氨酸、天門冬氨酸和谷氨酸代謝,賴氨酸降解等多條代謝通路發(fā)生紊亂。3.溴隱亭耐藥垂體泌乳素腺瘤的細(xì)胞代謝組學(xué)研究以大鼠垂體泌乳素腺瘤耐藥型GH3細(xì)胞及敏感型MMQ細(xì)胞為研究對象,開展了藥物BC耐藥垂體泌乳素腺瘤的細(xì)胞代謝組學(xué)研究。首先,在細(xì)胞樣品前處理的過程中,采用液氮冷凍法對細(xì)胞進(jìn)行淬滅,并分別采用甲醇、甲醇、水對細(xì)胞樣品進(jìn)行3次提取。在此基礎(chǔ)上,對細(xì)胞數(shù)目和復(fù)溶溶劑進(jìn)行了考察,最終確定細(xì)胞樣品中的細(xì)胞數(shù)目應(yīng)≥5×106個,以80%甲醇水作為復(fù)溶溶劑,優(yōu)化了垂體泌乳素腺瘤細(xì)胞的代謝組學(xué)樣品前處理方法。其次,采用酶聯(lián)免疫吸附法(Enzyme-linked immunosorbent assay, ELISA)對藥物BC作用不同時間后MMQ和GH3細(xì)胞培養(yǎng)基中的泌乳素(Prolactin, PRL)進(jìn)行了檢測,考察了耐藥型GH3細(xì)胞經(jīng)BC作用后PRL水平的變化,并比較分析了藥物BC對兩種細(xì)胞PRL分泌的抑制效果,為潛在耐藥標(biāo)志物的篩選提供了依據(jù)。進(jìn)一步采用與血漿代謝組學(xué)相同的分析方法,對未經(jīng)藥物BC作用的MMQ和GH3細(xì)胞開展了代謝組學(xué)分析,通過多變量統(tǒng)計分析和相關(guān)性分析,發(fā)現(xiàn)了60個與耐藥相關(guān)的潛在生物標(biāo)志物,并鑒定出24個潛在耐藥標(biāo)志物的結(jié)構(gòu),其中包括膽堿、氨基酸、肉堿、核苷類等代謝物。代謝通路分析表明,與BC敏感型垂體泌乳素腺瘤細(xì)胞相比,耐藥腫瘤細(xì)胞中甘油磷脂代謝,纈氨酸、亮氨酸和異亮氨酸生物合成,磷酸肌醇代謝,谷胱甘肽代謝等多條代謝通路發(fā)生紊亂。其中,甘油磷脂代謝,泛酸酯和輔酶A生物合成,半胱氨酸和蛋氨酸代謝,甘氨酸、絲氨酸和蘇氨酸代謝,氨酰tRNA生物合成等5條代謝通路在耐藥垂體泌乳素腺瘤患者和耐藥腫瘤細(xì)胞中同時發(fā)生紊亂。
[Abstract]:Bromocriptine (BC) resistance is a key factor in the treatment of pituitary prolactinomas in the treatment of ineffective or recurrent, and the cause of the drug resistance of the Pituitary prolactinomas to the drug BC and the accurate diagnosis of the drug-resistant pituitary prolactinomas are beneficial to the individual treatment. And the treatment effect is improved. In this paper, high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/ MS) combined with tandem mass spectrometry (MS/ MS) were used to establish a sensitive and rapid method for quantitative analysis of drug BC in plasma and tumor tissues of patients with prolactinomas. By using the method, the distribution difference of the drug BC in the plasma and the tumor tissues of the drug-resistant and sensitive pituitary prolactinomas is analyzed, the drug BC can be effectively absorbed and transported to the tumor tissue in the drug-resistant patient, and the high content is maintained in the tumor tissue, The reason for the failure of the BC to treat the drug-resistant pituitary prolactinomas may be the drug resistance of the tumor cell itself to the drug. On the basis of the above-mentioned research, the drug resistance of the Pituitary prolactinoma drug BC was studied by using the method of LC-MS/ MS-based metabolomics, respectively, in the plasma of the patients with prolactinomas of the pituitary and the cells of the pituitary prolactinomas in rats. A number of potential biomarkers associated with drug resistance were screened and metabolic pathways were analyzed using the identified biomarkers, and metabolic pathways associated with drug resistance were identified in BC-resistant pituitary prolactinomas and drug-resistant tumor cells. The results of this study provide a molecular basis and an important basis for the drug-resistant mechanism of the clinical BC-resistant pituitary prolactinomas. The research content of this thesis mainly includes the following three parts:1. The quantitative analysis of the drug-bromocriptine in the plasma and prolactinoma tissues of the clinical patients adopts LC-MS/ MS technology, and the system has the advantages of sensitivity, rapidness and specificity through the system optimization of the mass spectrum parameters in the chromatographic conditions and the MRM detection mode, Which is suitable for the quantitative analysis method of the drug BC in the human plasma and the tumor tissue of the clinical human body. In the method, the drug BC in the plasma and tumor tissues of the drug-resistant and sensitive pituitary prolactinomas was quantitatively analyzed, and the plasma drug concentration in the body of the drug-resistant patients was significantly higher than that of the sensitive patients after 2,4 and 6 hours of oral administration of the drug-resistant patients. The BC concentration in the tumor tissue of the drug-resistant patient after 3 months of taking the medicine is also obviously higher than that of the sensitive patient, indicating that the BC can be effectively absorbed in the drug-resistant patient and can be effectively transferred from the plasma to the tumor tissue, and the high drug content is maintained in the tumor tissue. This study shows that the resistance of the tumor cells to the drug BC may be a cause of the ineffective treatment of the drug-resistant pituitary prolactinomas in patients with prolactinomas. The plasma metabolomics of the drug-resistant pituitary prolactinoma of the bromocriptine was studied by LC-MS/ MS method combined with positive and negative ion detection modes, and the plasma samples of the patients with the BC-resistant and sensitive pituitary prolactinomas were subjected to metabolomics analysis. In the end,64 metabolites with significant differences, i.e., potential drug-resistant markers, were found in plasma of drug-resistant patients and sensitive patients. The structure of these potential drug-resistant markers was analyzed by means of high resolution MS spectrum and MS/ MS spectrum analysis, isotopic abundance ratio, nitrogen rule, network database retrieval and standard product comparison. At present, the structure of 21 potential drug-resistant markers is identified, including the metabolites of amino acids, alicyclic and fatty acids, among which 9 potential drug-resistant markers such as 1-dihydro-aminol, C16-aminoalcohol,1-deoxy-C14-dihydro-aminoalcohol, and the like have high diagnostic accuracy (AUC0.9). The metabolic pathway analysis of these potential drug-resistant markers was further carried out, and it was found that in comparison with the BC-sensitive pituitary prolactinomas, the drug-resistant patient had lipid metabolism, lysine biosynthesis, aminoptRNA biosynthesis, alanine, aspartic acid and glutamic acid metabolism, A plurality of metabolic pathways, such as lysine degradation, are disturbed. The cell metabolomics of the drug-resistant type GH3 cells and sensitive MMQ cells of the rat pituitary prolactinomas were studied by the cell metabolomics of the drug-resistant pituitary prolactinomas in the bromocriptine, and the cell metabolomics of the drug-resistant pituitary prolactinomas was carried out. First, in the process of pretreatment of the cell sample, the cells were quenched by a liquid nitrogen freezing method, and the cell samples were extracted three times with methanol, methanol and water, respectively. On this basis, the number of cells and the reconstituted solvent were investigated, the number of cells in the cell sample was determined to be 5 to 106, and 80% of the methanol water was used as the reconstitution solvent, and the pre-treatment of the metabolomics samples of the pituitary prolactinoma cells was optimized. The prolactin (Prolactin, PRL) in MMQ and GH3 cells were detected by enzyme-linked immunosorbent assay (ELISA). The inhibitory effect of the drug BC on the PRL secretion of the two cells was compared and the basis for the selection of the potential drug-resistant marker was provided. By adopting the same analytical method as the plasma metabolomics, the metabolomics analysis of MMQ and GH3 cells without the effect of the drug BC was carried out, and 60 potential biomarkers related to drug resistance were found through multi-variable statistical analysis and correlation analysis. The structure of 24 potential drug-resistant markers was identified, including choline, amino acid, carnitine, and other metabolites. The analysis of the metabolic pathway showed that, compared with the BC-sensitive pituitary prolactinoma cells, the metabolism of the glycerol phospholipids, the amino acid, the leucine and the isoleucine biosynthesis, the metabolism of the phosphoinositide, the metabolism of the glutathione, and the like in the drug-resistant tumor cells were disordered. Among these,5 metabolic pathways, such as the metabolism of glycerol, pantothenate and coenzyme A, the metabolism of cysteine and methionine, the metabolism of glycine, serine and threonine, and the biosynthesis of ammonia and tRNA, are also disorders in drug-resistant pituitary prolactinomas and drug-resistant tumor cells.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R96

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本文編號：2501085