肝癌发生历程中溶血卵磷脂代谢异常的机制及意义
发布时间:2018-05-01 19:00
本文选题:肝病 + 溶血卵磷脂 ; 参考:《第二军医大学》2017年硕士论文
【摘要】:研究背景和目的肝脏是人体重要的新陈代谢器官,参与多种物质的生物转化。在各种代谢通路中,肝脏代谢多种生物大分子,生成不同的小分子物质。从正常-肝炎-肝硬化-肝癌的疾病发展过程中,肝脏代谢功能必然受到影响。肝脏功能的检测指标包括甲胎蛋白、ALT、AST、TBIL、GGT、AKP等,这些检测指标在肝病发展早期常常阴性,而出现明显阳性变化时可能已经是肝癌晚期。因此,寻找早期诊断的生物标志物及灵敏的检测方法显得非常迫切。脂质代谢是肝脏重要的生理功能之一。胆固醇在肝脏分解代谢产生胆汁酸,肝病时胆汁酸出现代谢异常。当胆固醇与胆汁酸及卵磷脂的比值升高时,胆固醇因饱合而结晶析出形成胆石,卵磷脂在磷脂酶A的作用下生成溶血卵磷脂。因此,胆汁酸、溶血卵磷脂、不饱和脂肪酸是肝脏的核心代谢物,定量检测这些代谢物的水平,有可能对肝病的发展阶段进行判断。但由于这些物质代谢快,常规检测方法不灵敏等,故临床还没有将这类检测作为反映肝脏疾病的发展阶段指标。本论文建立了一种用UPLC-MS/MS质谱定量测定这些代谢物在血液、尿液中的浓度变化的方法,该方法快速、方便、准确率高,有望为肝癌发展历程中疾病状态的评估提供有价值的参考依据。研究发现,溶血磷脂酰胆碱(LPC)在肝脏从正常到癌变的发展过程中存在异常变化,但是其原因机制及其与肝癌发生的关系目前还不清楚。LPC在磷脂酰胆碱酰基转移酶1(LPCAT1)的作用下生成磷脂酰胆碱(PC),PC在磷脂酶A2(PLA2)的作用下生成LPC和多不饱和脂肪酸,PC在卵磷脂胆固醇酰基转移酶(LCAT)的作用下生成LPC及胆固醇酯。LPC是这些代谢通路的重要中间环节,因而我们将LPCAT1、LCAT、PLA2三者与LPC的异常结合起来检测,希望找到LPC异常变化的原因。另外,在这些酶的活性调节过程中,还涉及到AKT、ERK、JNK等反应通路的调控。因此,我们同时对这些通路的活性进行研究,验证其与LPC的关系。用ELISA、免疫组化、Western blot、质谱分析等研究方法,探索LPC异常的原因,希望能够阐明其是否与肝癌的发生发展有关系。研究方法1.收集不同肝病患者的血液、尿液标本:收集2013-2016年间在东方肝胆外科医院临床住院的肝炎(50例)、肝硬化(50例)、肝癌(50例)患者的血液、尿液标本,离心后取上清,保存于-80度冰箱。同时收集健康体检的正常人(66例)的血液、尿液作对照。2.不同肝病患者的血清质谱方法学分析:在上海交通大学质谱分析检测中心检测正常人肝炎患者、肝硬化患者、肝癌患者的血和尿标本,探索储存方法及不同检测方法的稳定性和敏感性。3.elisa检测不同肝病患者的血清蛋白酶水平:收集正常人、肝炎患者、肝硬化患者、肝癌患者的血清,elisa检测lcat、lpcat1、spla2-Ⅱa、cpla2α四种酶含量的变化。4.免疫组化检测相关蛋白酶指标:挑选正常肝组织(取自肝良性血管瘤手术标本)、肝癌组织及其癌旁为肝硬化组织、肝癌组织及其癌旁为肝炎组织,石蜡包埋切片,免疫组化检测lpcat1、lcat、cpla2α、spla2-Ⅱa四种酶指标的表达。5.相关蛋白在细胞内的表达变化:选取正常肝细胞系l02及wrl-68、肝星状细胞系lx2、肝癌细胞系hep-3b、hepg2、huh-7、qgy-7701、smmc-7721及hcc-lm3,培养细胞并提取蛋白,westernblot检测细胞系lpcat1、lcat、cpla2α、spla2-Ⅱa四种蛋白酶表达变化,同时检测了四种蛋白酶上游的akt、erk、jnk等表达情况。6.细胞内外lpc质谱分析:用质谱方法定量检测培养的不同细胞系的细胞内及培养上清中lpc含量变化,将此结果与上述westernblot蛋白表达结果相对比。研究结果1.质谱方法学:运用uplc-ms/ms检测溶血卵磷脂、胆汁酸、不饱和脂肪酸在血液和尿液中的含量变化,方法快速灵敏,容易操作。通过比较血浆及血清中代谢物含量差异,结果血浆更接近于实际全血状态,因此,我们的研究认为血浆作为生物样本用于上述血液中代谢物的检测分析是比较好的选择。2.lpcat1、lcat、pla2免疫组化与elisa检测结果的对比:临床样本检测发现,血清中(胞外)lcat与lpc的含量变化趋势在肝病历程中最相近;免疫组织中(胞内)lpcat1与lpc的含量变化趋势在肝病历程中最相近。cpla2及spla2-Ⅱa与胞内外lpc的变化趋势差异性均比较大。3.细胞系质谱分析及westernblot结果分析:细胞学实验分析lcat、lpcat1、cpla2α、spla2四种酶的表达,并与质谱结果做对比,结果发现,细胞培养液中(胞外)lcat与lpc的含量变化在肝病历程中相关性最高、细胞内lpcat1与lpc的含量变化在肝病历程中相关性最高。而cpla2α及其磷酸化状态的含量变化趋势与lpc变化趋势似乎相反。4.lpc相关其它上游通路的蛋白指标:总akt、p-akt、总erk、p-erk、jnk及其磷酸化蛋白表达趋势与细胞内lpc的表达趋势差异较大,p-jnk、p-p38有一定相关性。研究结论本课题以肝脏的核心代谢物质胆汁酸、溶血卵磷脂、不饱和脂肪酸为切入点,研究了如何利用uplc-ms/ms定量检测肝病及肝癌患者血尿代谢物的变化,研究证实该方法快速、便捷、灵敏性高,且将血浆作为样本优于血清检测。从代谢物中最核心的lpc出发,探究了其在肝病患者发展进程中变化的原因。为了探索正常、肝炎、肝硬化、肝癌历程中lpc异常变化的原因,我们从cpla2α、p-cpla2α、spla2-Ⅱ、lcat四种酶的变化出发,结果发现肝病进程中lcat主导胞外(血清及培养液)lpc的含量变化,而lpcat1主导胞内(组织)lpc的含量变化。cpla2α、p-cpla2α、spla2-Ⅱa对肝病进程中细胞内外lpc的异常变化影响较小。这种在细胞内外由不同酶主导lpc变化的机制原因需要进一步探索。另外,p38、akt、p-akt、erk、p-erk、jnk与lpc的异常变化关系不大,p-p38及p-jnk与其有一定的相关性,也需要进一步的探索。
[Abstract]:The liver is an important metabolic organ of the human body and participates in the biological transformation of a variety of substances. In various metabolic pathways, the liver metabolizes a variety of biological macromolecules to produce different small molecular substances. The liver metabolic function is bound to be affected from the development of normal hepatitis cirrhosis liver cancer. Liver function The detection indexes include alpha fetoprotein, ALT, AST, TBIL, GGT, AKP and so on. These detection indexes are often negative in the early stage of liver disease, and the obvious positive changes may already be advanced in the liver cancer. Therefore, it is very urgent to find biomarkers and sensitive detection methods for early diagnosis. Lipid metabolism is one of the important physiological functions of the liver. Cholesterin produces bile acids in the liver and abnormal metabolism of bile acids during liver disease. When the ratio of cholesterol to bile acids and lecithin increases, the cholesterol is crystallized to form cholelithiasis, and lecithin produces hemolytic lecithin under the action of phospholipase A. Therefore, bile acids, hemolytic lecithin, unsaturated fatty acids are liver. It is possible to determine the level of these metabolites, which can be used to determine the level of these metabolites, and it is possible to judge the development stage of liver disease. However, because of the rapid metabolism and insensitivity of conventional detection methods, this kind of detection has not been used as an indicator of the development stage of liver disease. A quantitative measurement of UPLC-MS/MS mass spectrometry has been established in this paper. The method of determining the change in the concentration of these metabolites in the blood and urine is fast, convenient and accurate. It is expected to provide valuable reference for the assessment of the disease state in the development of liver cancer. The study found that the LPC has abnormal changes in the development of the liver from normal to cancerous, but the cause of this is the reason. The mechanism and its relationship with the occurrence of liver cancer is not yet clear that.LPC produces phosphatidylcholine (PC) under the action of phosphatidylcholinyltransferase 1 (LPCAT1). PC produces LPC and polyunsaturated fatty acids under the action of phospholipase A2 (PLA2). PC produces LPC and cholesteryl ester.LPC under the action of phosphatidylcholine acyl transferase (LCAT). The important intermediate link of metabolic pathway, so we combine the abnormal combination of LPCAT1, LCAT, PLA2 three and LPC, and hope to find the cause of the abnormal changes of LPC. In addition, in the process of regulating the activity of these enzymes, it also involves the regulation of AKT, ERK, JNK and other reaction pathways. The relationship with LPC. Using ELISA, immunohistochemical, Western blot, mass spectrometry, and other research methods to explore the causes of abnormal LPC, and hope to clarify whether it is related to the development of liver cancer. Method 1. collect blood from patients with different liver diseases, urine specimens: collect the hepatitis in the hospital in the Eastern Department of hepatobiliary surgery (50). (50 Cases of liver cirrhosis (50 cases), liver cancer (50 cases) blood, urine specimen, centrifugation and supernatant, preserved in the -80 degree refrigerator. Meanwhile, the blood of normal persons (66 cases) of healthy physical examination was collected and the serum mass spectrometry analysis of the patients with different.2. liver diseases was analyzed in the urine, and the normal human hepatitis patients were detected at the mass spectrometry analysis center of Shanghai Jiao Tong University. Blood and urine samples from patients with liver cirrhosis and liver cancer, explore the stability and sensitivity of storage methods and different methods of detection.3.elisa detection of serum protease levels in patients with different liver diseases: Serum of normal people, hepatitis, liver cirrhosis, liver cancer, ELISA, LCAT, lpcat1, spla2- II A, and cPLA2 a content of four enzymes .4. immunohistochemical detection related protease indexes: select normal liver tissue (taken from the surgical specimens of liver benign hemangioma), liver cancer tissue and its paracancerous liver cirrhosis tissue, liver cancer tissue and its paracancerous liver tissue, paraffin embedded section, immunohistochemical detection of lpcat1, LCAT, cPLA2 a, spla2- II a four enzyme indicators expressed.5. related protein in fine Changes in intracellular expression: the normal hepatocyte line L02 and WRL-68, hepatic stellate cell line lx2, hep-3b, HepG2, Huh-7, qgy-7701, SMMC-7721 and hcc-lm3 in the hepatoma cells were cultured and extracted, and Westernblot was used to detect the cell line lpcat1, LCAT, alpha, and four proteases were detected, and the upstream of the four proteases was detected. ERK, JNK and so on.6. cell LPC mass spectrometry analysis: quantitative detection of the changes of LPC content in the cell and culture supernatant of different cell lines by mass spectrometry. The results were compared with the results of the above Westernblot protein expression. The results of the 1. mass spectrometry: using uplc-ms/ms to detect the hemolytic lecithin, bile acid, and unsaturated. The changes in the content of fatty acids in the blood and urine are rapid, sensitive and easy to operate. By comparing the difference in the metabolite content in the plasma and serum, the plasma is closer to the actual whole blood state. Therefore, our study suggests that plasma as a biological sample for the detection and analysis of metabolites in the above blood is a better choice of.2.lpcat 1, LCAT, PLA2 immunohistochemistry and ELISA test results: clinical sample detection found that the change trend of serum (extracellular) LCAT and LPC was the closest in the course of liver disease; the change trend of the content of lpcat1 and LPC in the immune tissues (intracellular) was the difference between the most close.Cpla2 and spla2- II A and the LPC in the cell. The mass spectrometric analysis of large.3. cell lines and the analysis of Westernblot results: the expression of four enzymes in LCAT, lpcat1, cPLA2, and sPLA2 was analyzed by cytological experiments and compared with the mass spectrometry results. The results showed that the content of LCAT and LPC in the cell culture medium was the highest in the liver disease process, and the content of lpcat1 and LPC in the cells was changed in the liver record. The relationship between cPLA2 alpha and its phosphorylation status is the same as that of LPC, which seems to be contrary to the change trend of.4.lpc. The expression trend of total Akt, p-Akt, total ERK, p-ERK, JNK and its phosphorylated protein is different from the expression trend of LPC in the cell, and there is a certain correlation between p-JNK and p-p38. This topic uses the core metabolism substance bile acid, hemolytic lecithin and unsaturated fatty acid as the breakthrough point, and studies how to use uplc-ms/ms to quantify the changes in the metabolites of liver and liver cancer patients' hematuria. The research confirms that the method is fast, convenient and highly sensitive, and the plasma is better than the serum test. In order to explore the causes of abnormal changes of LPC in the course of normal, hepatitis, liver cirrhosis and liver cancer, we start from the changes in the four enzymes of cPLA2 alpha, p-cpla2 a, spla2- II, LCAT, and the results of the changes of LPC content of the LCAT dominant extracellular (serum and culture fluid) during the liver disease process. And the change of.Cpla2 alpha, p-cpla2 alpha and spla2- II A in the lpcat1 dominant intracellular (tissue) LPC has little influence on the abnormal changes of the intracellular and extracellular LPC in the process of liver disease. -jnk is related to it and needs further exploration.
【学位授予单位】:第二军医大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R735.7
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