卵巢癌患者尿液及紫杉醇处理HeLa细胞的代谢组学分析

发布时间：2019-01-27 11:10

【摘要】：卵巢癌是女性生殖系统的常见恶性肿瘤之一,死亡率居妇科恶性肿瘤之首。其发病隐匿,缺乏有效的早期诊断方法,多数患者发病时已处于晚期。卵巢癌的临床分期可为临床治疗提供准确的依据,对卵巢癌病情的判断、治疗方案的制定和预后的估计至关重要。代谢组学是继基因组学、转录组学、蛋白质组学之后系统生物学的一门新兴学科,主要研究生物体系受到刺激或扰动后小分子代谢物(分子量小于1000)的变化。其在肿瘤代谢标志物的寻找和发病机制的阐明等方面具有独特的优势和广泛的应用价值。由于临床分期可对卵巢癌的病情做出明确的界定,因此本研究利用代谢组学技术探查尿液代谢物构成与卵巢癌临床分期的相关性,以期为寻找简便的用于筛查卵巢癌患者和判断卵巢癌病情的方法提供线索。实验方法 1.收集15例正常人和56例原发性上皮性卵巢癌患者的晨尿,用甲醇提取尿液中的代谢物并用0.45μm滤膜过滤后低温冰箱保存。 2.采用高效液相色谱-质谱(HPLC-MS)联用技术对提取的代谢物进行检测,并提取三维数据。 3.用主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA)对质谱数据进行多元统计分析。 4.对质谱数据的相应分析结果进行数据库检索和比对,确定尿液中主要的差异代谢物。通过单因素方差分析(one-way ANOVA)和最小显著差(LSD)t检验对代谢物进行分析。实验结果尿液样品经HPLC-MS检测后用PCA进行分析,结果显示健康人和不同临床分期卵巢癌患者的样品点在得分图中存在分离趋势。用PLS-DA作进一步分析,分类结果较PCA得到明显改善。通过对结合VIP值和相关系数得到的差异点进行非配对双尾t检验,及对有统计学意义的差异点进行数据库检索和比对,能够体现健康人与不同临床阶段卵巢癌患者之间代谢差别的尿中代谢物被筛选出来,它们是N-乙酰神经氨酸-9-磷酸、5’-甲硫腺苷、尿酸-3-核苷、假尿嘧啶核苷、L-缬氨酸、琥珀酸、L-脯氨酸及β-烟酰胺单核苷酸。这些化合物在正常人和处于不同临床阶段的卵巢癌患者尿液中含量存在明显差异(P0.05或0.01),且随着卵巢癌患者病情的发展呈现增高趋势。实验结论不同临床分期卵巢癌患者尿液代谢物构成存在差异,卵巢癌患者尿液代谢物构成与临床分期有相关性。宫颈癌是妇科肿瘤所致死亡的第二位常见原因,严重影响女性的健康。宫颈癌常用治疗方法有手术、放疗、化疗以及靶向治疗和免疫疗法等,其中化疗药物在宫颈癌治疗中占重要地位。紫杉醇(paclitaxel, PTX)是从紫衫树中提取的一种化合物,为最常用的抗肿瘤药物之一。PTX是宫颈癌化疗的首选药物之一,对宫颈癌有确切的疗效。 PTX可结合微管蛋白,增强微管蛋白的稳定性,阻碍细胞有丝分裂,进而发挥抗肿瘤作用。此外,PTX对免疫系统亦有调节作用,多项研究提示PTX可以影响癌细胞的代谢。代谢组学是研究生命体系受到刺激或者基因改变时,代谢产物动态变化规律的科学,旨在对有机体或生物流体内所有的代谢产物进行综合的定量和定性分析。本研究使用代谢组学技术,观察PTX对宫颈癌HeLa细胞代谢的影响,从细胞代谢水平探查PTX抗宫颈癌的机制。实验方法 1.用MTT试验分析PTX对HeLa细胞的细胞毒作用。根据PTX对HeLa细胞存活率的影响,确定代谢组学研究的合适的药物浓度及作用时间。 2.用PTX处理HeLa细胞,收集细胞并提取细胞内的代谢物。用快速高分辨液相色谱-四极杆飞行时间质谱(RRLC-Q-TOF/MS)联用技术对代谢物进行分析。 3.结合主成分分析(principal component analysis, PCA)(?)偏最小二乘法-判别分析(partial least squares discriminant analysis, PLS-DA)寻找差异代谢物。差异代谢物的统计分析采用非配对双尾t检验,P0.05时差异有统计学意义。 4.通过安捷伦METLIN个人化合物数据库和谱库(PCDL)及KEGG数据库对有统计学意义的代谢物进行检索比对,确定差异代谢物及相关代谢途径和关键酶。实验结果 1.PTX对HeLa细胞增殖的抑制作用呈时间和浓度效应关系。随着药物浓度的增加和处理时间的延长,PTX对HeLa细胞增殖的抑制作用逐渐增强。药物浓度为100nmol/L、处理时间为48h时,HeLa细胞的存活率为65%,选定该药物浓度及处理时间为细胞代谢组学研究处理条件。 2.PTX对HeLa细胞的代谢有广泛的影响。PTX可使HeLa细胞内壳多糖、N1-乙酰精胺、NADH、L-酪氨酸、由谷氨酰胺和两个半胱氨酸构成的三肽及由甘氨酸和赖氨酸构成的二肽的含量降低。同时,PTX亦可使HeLa细胞内鸟嘌呤核苷、鸟氨酸、尿素、乳酸、丝氨酸、脯氨酸以及由脯氨酸、赖氨酸、甘氨酸构成的三肽和由脯氨酸、赖氨酸、丙氨酸构成的三肽增多。实验结论 PTX对宫颈癌HeLa细胞的多种氨基酸代谢、核苷酸代谢、嘧啶代谢、丙酮酸盐代谢、聚胺代谢、氨基糖和核苷酸糖代谢有明显的影响,并对HeLa细胞内NADH及小肽的代谢有作用。
[Abstract]:Ovarian cancer is one of the most common malignant tumors of female reproductive system. The disease is hidden and the effective early diagnosis method is lacking, most of the patients have been in the late stage. The clinical stage of the ovarian cancer can provide an accurate basis for clinical treatment, and it is important to determine the condition of the ovarian cancer, the formulation of the treatment plan and the estimation of the prognosis. The study of metabolic group is a new subject of systematic biology following the study of genomics, transcriptome and proteomics, which mainly studies the change of the small molecular metabolite (molecular weight less than 1000) after stimulation or disturbance of the biological system. It has unique advantages and wide application price in the search of tumor metabolic markers and the elucidation of the pathogenesis. Value. Since the clinical stage can clearly define the condition of the ovarian cancer, the study uses the metabolic group to explore the correlation between the urine metabolite composition and the clinical stage of the ovarian cancer, with a view to providing a line for finding a simple method for screening ovarian cancer patients and for determining the condition of the ovarian cancer Soo. Methods 1. The morning urine of 15 normal persons and 56 patients with primary epithelial ovarian cancer were collected, and the metabolites in the urine were extracted with methanol and filtered with 0.45. m and the extracted metabolite is detected by a high-performance liquid chromatography-mass spectrometry (HPLC-MS) combination technology, 3. The mass spectra of the three-dimensional data were extracted by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). according to the corresponding analysis result of the mass spectrum data, Primary difference metabolites in urine. One-way ANOVA and minimum significant difference (LSD) were obtained by single-factor analysis of variance (one-way ANOVA). t test pair Metabolites were analyzed. The results of the experimental results were analyzed by HPLC-MS and analyzed by PCA. The results showed that healthy people and patients with different clinical stages of ovarian cancer The separation trend of the sample points in the score map is presented. The PLS-DA is used for further analysis. The result of classification is obviously improved than that of PCA. The non-paired double-tail t test is carried out on the difference points obtained by combining the VIP value and the correlation coefficient, and the database retrieval and comparison are carried out on the difference points with statistical significance, which can reflect the relationship between the healthy person and the patients with different clinical stage ovarian cancer. the metabolites in the urine of the metabolic difference are screened out, is N-glycinic acid-9-phosphoric acid, 5 '-methylthionein, uric acid-3-nuclear antigen, pseudo-allantoin, L-arginine, succinic acid, The levels of L-proline and L-proline and L-bichromine were significantly different in the urine of the normal and the patients with ovarian cancer at different clinical stages (P0.05 or 0.01), and with the eggs, Patients with nest cancer The results of the experiment show that there is a difference in the urine metabolites in the patients with different clinical stages of ovarian cancer. The composition of urine metabolites in cancer patients is related to the clinical stage. Cervical cancer is a gynecological tumor. The second common cause of death is the serious impact on the health of women. The common treatment methods of cervical cancer include surgery, radiotherapy, chemotherapy, and targeted therapy and immunotherapy. In the treatment of cervical cancer, the chemotherapy drugs play an important role in the treatment of cervical cancer. One of the most commonly used anti-tumor drugs. PTX is the cervix. One of the preferred drugs for cancer chemotherapy is the exact therapeutic effect on cervical cancer. PTX can bind to the tubulin and enhance the microtubule protein. In addition, PTX has a good effect on the immune system. A number of studies suggest that PTX can affect the metabolism of cancer cells. or the comprehensive quantitative and qualitative analysis of all the metabolites in the biological fluid. the shadow of metabolism A mechanism for detecting the anti-cervical cancer of PTX from the level of cellular metabolism. Method 1. The cytotoxic effect of PTX on HeLa cells was analyzed by MTT assay. The effect of cell viability was determined to determine the appropriate drug concentration and time of action for the metabolic group study. 2. The HeLa cells were treated with PTX and the cells were collected and the metabolites in the cells were extracted. Analysis of metabolites by time-mass spectrometry (RRLC-Q-TOF/ MS) Principal component analysis (PCA) (?) Partial least squares-discriminant analysis Differential analysis (PLS-DA) was used to find the difference metabolites. The statistical analysis of metabolites was based on the non-paired double-tail t test, and the difference was statistically significant at the time of P05. 4. By the Agilent METLIN Personal Compound Database and the Spectrum Library (PCDL) and the KEGG database, Statistics Determination of the difference metabolites and related metabolic pathways and critical metabolites by retrieval of the significant metabolites The inhibitory effect of PTX on the proliferation of HeLa cells was in the form of time and concentration. With the increase of drug concentration and the extension of treatment time, the inhibitory effect of PTX on the proliferation of HeLa cells was gradually enhanced. The drug concentration was 100nmol/ L and the treatment time was 48h. the survival rate of eLa cells was 65%, the concentration of the drug was selected, and The treatment time is the treatment condition of the cell metabolism group. and the content of the dipeptide composed of glycine and lysine is reduced, and the PTX can also make the bird terin the HeLa cell nucleon, ornithine, urea, lactic acid, serine and preserved. and The three-peptide consisting of proline, lysine and glycine and the tripeptide composed of proline, lysine and alanine are increased.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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