【摘要】：卵巢癌是女性生殖系統(tǒng)的常見惡性腫瘤之一,死亡率居?jì)D科惡性腫瘤之首。其發(fā)病隱匿,缺乏有效的早期診斷方法,多數(shù)患者發(fā)病時(shí)已處于晚期。卵巢癌的臨床分期可為臨床治療提供準(zhǔn)確的依據(jù),對卵巢癌病情的判斷、治療方案的制定和預(yù)后的估計(jì)至關(guān)重要。 代謝組學(xué)是繼基因組學(xué)、轉(zhuǎn)錄組學(xué)、蛋白質(zhì)組學(xué)之后系統(tǒng)生物學(xué)的一門新興學(xué)科,主要研究生物體系受到刺激或擾動后小分子代謝物(分子量小于1000)的變化。其在腫瘤代謝標(biāo)志物的尋找和發(fā)病機(jī)制的闡明等方面具有獨(dú)特的優(yōu)勢和廣泛的應(yīng)用價(jià)值。由于臨床分期可對卵巢癌的病情做出明確的界定,因此本研究利用代謝組學(xué)技術(shù)探查尿液代謝物構(gòu)成與卵巢癌臨床分期的相關(guān)性,以期為尋找簡便的用于篩查卵巢癌患者和判斷卵巢癌病情的方法提供線索。 實(shí)驗(yàn)方法 1.收集15例正常人和56例原發(fā)性上皮性卵巢癌患者的晨尿,用甲醇提取尿液中的代謝物并用0.45μm濾膜過濾后低溫冰箱保存。 2.采用高效液相色譜-質(zhì)譜(HPLC-MS)聯(lián)用技術(shù)對提取的代謝物進(jìn)行檢測,并提取三維數(shù)據(jù)。 3.用主成分分析(PCA)和偏最小二乘法-判別分析(PLS-DA)對質(zhì)譜數(shù)據(jù)進(jìn)行多元統(tǒng)計(jì)分析。 4.對質(zhì)譜數(shù)據(jù)的相應(yīng)分析結(jié)果進(jìn)行數(shù)據(jù)庫檢索和比對,確定尿液中主要的差異代謝物。通過單因素方差分析(one-way ANOVA)和最小顯著差(LSD)t檢驗(yàn)對代謝物進(jìn)行分析。 實(shí)驗(yàn)結(jié)果 尿液樣品經(jīng)HPLC-MS檢測后用PCA進(jìn)行分析,結(jié)果顯示健康人和不同臨床分期卵巢癌患者的樣品點(diǎn)在得分圖中存在分離趨勢。用PLS-DA作進(jìn)一步分析,分類結(jié)果較PCA得到明顯改善。通過對結(jié)合VIP值和相關(guān)系數(shù)得到的差異點(diǎn)進(jìn)行非配對雙尾t檢驗(yàn),及對有統(tǒng)計(jì)學(xué)意義的差異點(diǎn)進(jìn)行數(shù)據(jù)庫檢索和比對,能夠體現(xiàn)健康人與不同臨床階段卵巢癌患者之間代謝差別的尿中代謝物被篩選出來,它們是N-乙酰神經(jīng)氨酸-9-磷酸、5’-甲硫腺苷、尿酸-3-核苷、假尿嘧啶核苷、L-纈氨酸、琥珀酸、L-脯氨酸及β-煙酰胺單核苷酸。這些化合物在正常人和處于不同臨床階段的卵巢癌患者尿液中含量存在明顯差異(P0.05或0.01),且隨著卵巢癌患者病情的發(fā)展呈現(xiàn)增高趨勢。 實(shí)驗(yàn)結(jié)論 不同臨床分期卵巢癌患者尿液代謝物構(gòu)成存在差異,卵巢癌患者尿液代謝物構(gòu)成與臨床分期有相關(guān)性。 宮頸癌是婦科腫瘤所致死亡的第二位常見原因,嚴(yán)重影響女性的健康。宮頸癌常用治療方法有手術(shù)、放療、化療以及靶向治療和免疫療法等,其中化療藥物在宮頸癌治療中占重要地位。紫杉醇(paclitaxel, PTX)是從紫衫樹中提取的一種化合物,為最常用的抗腫瘤藥物之一。PTX是宮頸癌化療的首選藥物之一,對宮頸癌有確切的療效。 PTX可結(jié)合微管蛋白,增強(qiáng)微管蛋白的穩(wěn)定性,阻礙細(xì)胞有絲分裂,進(jìn)而發(fā)揮抗腫瘤作用。此外,PTX對免疫系統(tǒng)亦有調(diào)節(jié)作用,多項(xiàng)研究提示PTX可以影響癌細(xì)胞的代謝。 代謝組學(xué)是研究生命體系受到刺激或者基因改變時(shí),代謝產(chǎn)物動態(tài)變化規(guī)律的科學(xué),旨在對有機(jī)體或生物流體內(nèi)所有的代謝產(chǎn)物進(jìn)行綜合的定量和定性分析。本研究使用代謝組學(xué)技術(shù),觀察PTX對宮頸癌HeLa細(xì)胞代謝的影響,從細(xì)胞代謝水平探查PTX抗宮頸癌的機(jī)制。 實(shí)驗(yàn)方法 1.用MTT試驗(yàn)分析PTX對HeLa細(xì)胞的細(xì)胞毒作用。根據(jù)PTX對HeLa細(xì)胞存活率的影響,確定代謝組學(xué)研究的合適的藥物濃度及作用時(shí)間。 2.用PTX處理HeLa細(xì)胞,收集細(xì)胞并提取細(xì)胞內(nèi)的代謝物。用快速高分辨液相色譜-四極桿飛行時(shí)間質(zhì)譜(RRLC-Q-TOF/MS)聯(lián)用技術(shù)對代謝物進(jìn)行分析。 3.結(jié)合主成分分析(principal component analysis, PCA)(?)偏最小二乘法-判別分析(partial least squares discriminant analysis, PLS-DA)尋找差異代謝物。差異代謝物的統(tǒng)計(jì)分析采用非配對雙尾t檢驗(yàn),P0.05時(shí)差異有統(tǒng)計(jì)學(xué)意義。 4.通過安捷倫METLIN個(gè)人化合物數(shù)據(jù)庫和譜庫(PCDL)及KEGG數(shù)據(jù)庫對有統(tǒng)計(jì)學(xué)意義的代謝物進(jìn)行檢索比對,確定差異代謝物及相關(guān)代謝途徑和關(guān)鍵酶。 實(shí)驗(yàn)結(jié)果 1.PTX對HeLa細(xì)胞增殖的抑制作用呈時(shí)間和濃度效應(yīng)關(guān)系。隨著藥物濃度的增加和處理時(shí)間的延長,PTX對HeLa細(xì)胞增殖的抑制作用逐漸增強(qiáng)。藥物濃度為100nmol/L、處理時(shí)間為48h時(shí),HeLa細(xì)胞的存活率為65%,選定該藥物濃度及處理時(shí)間為細(xì)胞代謝組學(xué)研究處理?xiàng)l件。 2.PTX對HeLa細(xì)胞的代謝有廣泛的影響。PTX可使HeLa細(xì)胞內(nèi)殼多糖、N1-乙酰精胺、NADH、L-酪氨酸、由谷氨酰胺和兩個(gè)半胱氨酸構(gòu)成的三肽及由甘氨酸和賴氨酸構(gòu)成的二肽的含量降低。同時(shí),PTX亦可使HeLa細(xì)胞內(nèi)鳥嘌呤核苷、鳥氨酸、尿素、乳酸、絲氨酸、脯氨酸以及由脯氨酸、賴氨酸、甘氨酸構(gòu)成的三肽和由脯氨酸、賴氨酸、丙氨酸構(gòu)成的三肽增多。 實(shí)驗(yàn)結(jié)論 PTX對宮頸癌HeLa細(xì)胞的多種氨基酸代謝、核苷酸代謝、嘧啶代謝、丙酮酸鹽代謝、聚胺代謝、氨基糖和核苷酸糖代謝有明顯的影響,并對HeLa細(xì)胞內(nèi)NADH及小肽的代謝有作用。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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