基于代谢组学方法的垂体泌乳素腺瘤药物溴隐亭的耐药性研究
发布时间：2019-06-17 15:31
【摘要】：溴隐亭(Bromocriptine, BC)耐药是垂体泌乳素腺瘤药物治疗无效或复发的关键因素,探索垂体泌乳素腺瘤对药物BC产生耐药性的原因并对耐药垂体泌乳素腺瘤患者进行准确诊断有利于实施个体化治疗,提高治疗效果。本论文首先采用高效液相色谱(HPLC)与串联质谱(MS/MS)联用技术,建立了灵敏、快速的适用于临床垂体泌乳素腺瘤患者的血浆和肿瘤组织中药物BC的定量分析方法。采用该方法比较分析了耐药型与敏感型垂体泌乳素腺瘤患者的血浆及肿瘤组织中药物BC的分布差异,发现药物BC在耐药患者体内也能够被有效地吸收和转运至肿瘤组织,在肿瘤组织中维持较高的含量,耐药型垂体泌乳素腺瘤患者经BC治疗无效的原因可能是肿瘤细胞自身对药物产生的耐药性。在上述研究基础上,进一步采用基于LC-MS/MS技术的代谢组学方法,分别以临床垂体泌乳素腺瘤患者的血浆和大鼠垂体泌乳素腺瘤细胞为研究对象,开展了垂体泌乳素腺瘤药物BC的耐药性研究。筛选出一批与耐药相关的潜在生物标志物,并利用发现的生物标志物进行了代谢通路分析,在BC耐药垂体泌乳素腺瘤患者和耐药肿瘤细胞中发现了与耐药相关的发生紊乱的代谢通路。本研究结果为临床BC耐药垂体泌乳素腺瘤的耐药性机制提供了分子基础及重要依据。本论文的研究内容主要包括以下三个部分：1.临床患者血浆及垂体泌乳素腺瘤组织中药物溴隐亭的定量分析采用LC-MS/MS技术,通过对色谱条件和MRM检测模式下质谱参数的系统优化,建立了灵敏、快速、专属性强,适用于临床人体血浆和肿瘤组织中药物BC的定量分析方法。采用该方法对耐药型和敏感型垂体泌乳素腺瘤患者的血浆及肿瘤组织中药物BC进行了定量分析,发现耐药患者口服BC 2,4和6h后,体内血浆药物浓度明显高于敏感患者；服药3个月后的耐药患者肿瘤组织中的BC浓度也明显高于敏感患者,表明BC在耐药患者体内也能够被有效地吸收,并能够被有效地从血浆转运至肿瘤组织,在肿瘤组织中维持较高的药物含量。本研究显示肿瘤细胞自身对药物BC产生的抵抗可能是导致耐药型垂体泌乳素腺瘤患者经药物BC治疗无效的原因。2.溴隐亭耐药垂体泌乳素腺瘤的血浆代谢组学研究采用正、负离子检测模式相结合的LC-MS/MS分析方法,对BC耐药型和敏感型垂体泌乳素腺瘤患者的血浆样本进行了代谢组学分析。最终在耐药患者和敏感患者的血浆中发现了64个具有显著性差异的代谢物,即潜在耐药标志物。进一步通过高分辨率MS谱及MS/MS谱分析,结合同位素丰度比、氮规则,以及网络数据库检索和标准品对比等分析手段与步骤对这些潜在耐药标志物的结构进行了分析鉴定。目前,共鉴定出21个潜在耐药标志物的结构,包括氨基酸、鞘脂、脂肪酸类等代谢物,其中1-磷酸二氢鞘氨醇、C16鞘氨醇、1-脱氧C14二氢鞘氨醇等9个潜在耐药标志物具有较高的诊断准确性(AUC0.9)。进一步对这些潜在耐药标志物进行了代谢通路分析,发现与BC敏感型垂体泌乳素腺瘤患者相比,耐药患者体内鞘脂类代谢,赖氨酸生物合成,氨酰tRNA生物合成,丙氨酸、天门冬氨酸和谷氨酸代谢,赖氨酸降解等多条代谢通路发生紊乱。3.溴隐亭耐药垂体泌乳素腺瘤的细胞代谢组学研究以大鼠垂体泌乳素腺瘤耐药型GH3细胞及敏感型MMQ细胞为研究对象,开展了药物BC耐药垂体泌乳素腺瘤的细胞代谢组学研究。首先,在细胞样品前处理的过程中,采用液氮冷冻法对细胞进行淬灭,并分别采用甲醇、甲醇、水对细胞样品进行3次提取。在此基础上,对细胞数目和复溶溶剂进行了考察,最终确定细胞样品中的细胞数目应≥5×106个,以80%甲醇水作为复溶溶剂,优化了垂体泌乳素腺瘤细胞的代谢组学样品前处理方法。其次,采用酶联免疫吸附法(Enzyme-linked immunosorbent assay, ELISA)对药物BC作用不同时间后MMQ和GH3细胞培养基中的泌乳素(Prolactin, PRL)进行了检测,考察了耐药型GH3细胞经BC作用后PRL水平的变化,并比较分析了药物BC对两种细胞PRL分泌的抑制效果,为潜在耐药标志物的筛选提供了依据。进一步采用与血浆代谢组学相同的分析方法,对未经药物BC作用的MMQ和GH3细胞开展了代谢组学分析,通过多变量统计分析和相关性分析,发现了60个与耐药相关的潜在生物标志物,并鉴定出24个潜在耐药标志物的结构,其中包括胆碱、氨基酸、肉碱、核苷类等代谢物。代谢通路分析表明,与BC敏感型垂体泌乳素腺瘤细胞相比,耐药肿瘤细胞中甘油磷脂代谢,缬氨酸、亮氨酸和异亮氨酸生物合成,磷酸肌醇代谢,谷胱甘肽代谢等多条代谢通路发生紊乱。其中,甘油磷脂代谢,泛酸酯和辅酶A生物合成,半胱氨酸和蛋氨酸代谢,甘氨酸、丝氨酸和苏氨酸代谢,氨酰tRNA生物合成等5条代谢通路在耐药垂体泌乳素腺瘤患者和耐药肿瘤细胞中同时发生紊乱。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96
本文编号：2501085
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96
【参考文献】
相关期刊论文 前2条
1 吴哲褒,于春江;侵袭性泌乳素腺瘤的治疗进展[J];国外医学.神经病学神经外科学分册;2004年06期
2 贾伟;蒋健;刘平;赵立平;陈闽军;周明眉;杨丽萍;王米渠;邱明丰;张永煜;;代谢组学在中医药复杂理论体系研究中的应用[J];中国中药杂志;2006年08期
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