本文選題：肝病 + 溶血卵磷脂　； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景和目的肝臟是人體重要的新陳代謝器官,參與多種物質(zhì)的生物轉(zhuǎn)化。在各種代謝通路中,肝臟代謝多種生物大分子,生成不同的小分子物質(zhì)。從正常-肝炎-肝硬化-肝癌的疾病發(fā)展過程中,肝臟代謝功能必然受到影響。肝臟功能的檢測指標(biāo)包括甲胎蛋白、ALT、AST、TBIL、GGT、AKP等,這些檢測指標(biāo)在肝病發(fā)展早期常常陰性,而出現(xiàn)明顯陽性變化時可能已經(jīng)是肝癌晚期。因此,尋找早期診斷的生物標(biāo)志物及靈敏的檢測方法顯得非常迫切。脂質(zhì)代謝是肝臟重要的生理功能之一。膽固醇在肝臟分解代謝產(chǎn)生膽汁酸,肝病時膽汁酸出現(xiàn)代謝異常。當(dāng)膽固醇與膽汁酸及卵磷脂的比值升高時,膽固醇因飽合而結(jié)晶析出形成膽石,卵磷脂在磷脂酶A的作用下生成溶血卵磷脂。因此,膽汁酸、溶血卵磷脂、不飽和脂肪酸是肝臟的核心代謝物,定量檢測這些代謝物的水平,有可能對肝病的發(fā)展階段進(jìn)行判斷。但由于這些物質(zhì)代謝快,常規(guī)檢測方法不靈敏等,故臨床還沒有將這類檢測作為反映肝臟疾病的發(fā)展階段指標(biāo)。本論文建立了一種用UPLC-MS/MS質(zhì)譜定量測定這些代謝物在血液、尿液中的濃度變化的方法,該方法快速、方便、準(zhǔn)確率高,有望為肝癌發(fā)展歷程中疾病狀態(tài)的評估提供有價(jià)值的參考依據(jù)。研究發(fā)現(xiàn),溶血磷脂酰膽堿(LPC)在肝臟從正常到癌變的發(fā)展過程中存在異常變化,但是其原因機(jī)制及其與肝癌發(fā)生的關(guān)系目前還不清楚。LPC在磷脂酰膽堿酰基轉(zhuǎn)移酶1(LPCAT1)的作用下生成磷脂酰膽堿(PC),PC在磷脂酶A2(PLA2)的作用下生成LPC和多不飽和脂肪酸,PC在卵磷脂膽固醇�；D(zhuǎn)移酶(LCAT)的作用下生成LPC及膽固醇酯。LPC是這些代謝通路的重要中間環(huán)節(jié),因而我們將LPCAT1、LCAT、PLA2三者與LPC的異常結(jié)合起來檢測,希望找到LPC異常變化的原因。另外,在這些酶的活性調(diào)節(jié)過程中,還涉及到AKT、ERK、JNK等反應(yīng)通路的調(diào)控。因此,我們同時對這些通路的活性進(jìn)行研究,驗(yàn)證其與LPC的關(guān)系。用ELISA、免疫組化、Western blot、質(zhì)譜分析等研究方法,探索LPC異常的原因,希望能夠闡明其是否與肝癌的發(fā)生發(fā)展有關(guān)系。研究方法1.收集不同肝病患者的血液、尿液標(biāo)本:收集2013-2016年間在東方肝膽外科醫(yī)院臨床住院的肝炎(50例)、肝硬化(50例)、肝癌(50例)患者的血液、尿液標(biāo)本,離心后取上清,保存于-80度冰箱。同時收集健康體檢的正常人(66例)的血液、尿液作對照。2.不同肝病患者的血清質(zhì)譜方法學(xué)分析:在上海交通大學(xué)質(zhì)譜分析檢測中心檢測正常人肝炎患者、肝硬化患者、肝癌患者的血和尿標(biāo)本,探索儲存方法及不同檢測方法的穩(wěn)定性和敏感性。3.elisa檢測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檢測lcat、lpcat1、spla2-Ⅱa、cpla2α四種酶含量的變化。4.免疫組化檢測相關(guān)蛋白酶指標(biāo):挑選正常肝組織(取自肝良性血管瘤手術(shù)標(biāo)本)、肝癌組織及其癌旁為肝硬化組織、肝癌組織及其癌旁為肝炎組織,石蠟包埋切片,免疫組化檢測lpcat1、lcat、cpla2α、spla2-Ⅱa四種酶指標(biāo)的表達(dá)。5.相關(guān)蛋白在細(xì)胞內(nèi)的表達(dá)變化:選取正常肝細(xì)胞系l02及wrl-68、肝星狀細(xì)胞系lx2、肝癌細(xì)胞系hep-3b、hepg2、huh-7、qgy-7701、smmc-7721及hcc-lm3,培養(yǎng)細(xì)胞并提取蛋白,westernblot檢測細(xì)胞系lpcat1、lcat、cpla2α、spla2-Ⅱa四種蛋白酶表達(dá)變化,同時檢測了四種蛋白酶上游的akt、erk、jnk等表達(dá)情況。6.細(xì)胞內(nèi)外lpc質(zhì)譜分析:用質(zhì)譜方法定量檢測培養(yǎng)的不同細(xì)胞系的細(xì)胞內(nèi)及培養(yǎng)上清中l(wèi)pc含量變化,將此結(jié)果與上述westernblot蛋白表達(dá)結(jié)果相對比。研究結(jié)果1.質(zhì)譜方法學(xué):運(yùn)用uplc-ms/ms檢測溶血卵磷脂、膽汁酸、不飽和脂肪酸在血液和尿液中的含量變化,方法快速靈敏,容易操作。通過比較血漿及血清中代謝物含量差異,結(jié)果血漿更接近于實(shí)際全血狀態(tài),因此,我們的研究認(rèn)為血漿作為生物樣本用于上述血液中代謝物的檢測分析是比較好的選擇。2.lpcat1、lcat、pla2免疫組化與elisa檢測結(jié)果的對比:臨床樣本檢測發(fā)現(xiàn),血清中(胞外)lcat與lpc的含量變化趨勢在肝病歷程中最相近;免疫組織中(胞內(nèi))lpcat1與lpc的含量變化趨勢在肝病歷程中最相近。cpla2及spla2-Ⅱa與胞內(nèi)外lpc的變化趨勢差異性均比較大。3.細(xì)胞系質(zhì)譜分析及westernblot結(jié)果分析:細(xì)胞學(xué)實(shí)驗(yàn)分析lcat、lpcat1、cpla2α、spla2四種酶的表達(dá),并與質(zhì)譜結(jié)果做對比,結(jié)果發(fā)現(xiàn),細(xì)胞培養(yǎng)液中(胞外)lcat與lpc的含量變化在肝病歷程中相關(guān)性最高、細(xì)胞內(nèi)lpcat1與lpc的含量變化在肝病歷程中相關(guān)性最高。而cpla2α及其磷酸化狀態(tài)的含量變化趨勢與lpc變化趨勢似乎相反。4.lpc相關(guān)其它上游通路的蛋白指標(biāo):總akt、p-akt、總erk、p-erk、jnk及其磷酸化蛋白表達(dá)趨勢與細(xì)胞內(nèi)lpc的表達(dá)趨勢差異較大,p-jnk、p-p38有一定相關(guān)性。研究結(jié)論本課題以肝臟的核心代謝物質(zhì)膽汁酸、溶血卵磷脂、不飽和脂肪酸為切入點(diǎn),研究了如何利用uplc-ms/ms定量檢測肝病及肝癌患者血尿代謝物的變化,研究證實(shí)該方法快速、便捷、靈敏性高,且將血漿作為樣本優(yōu)于血清檢測。從代謝物中最核心的lpc出發(fā),探究了其在肝病患者發(fā)展進(jìn)程中變化的原因。為了探索正常、肝炎、肝硬化、肝癌歷程中l(wèi)pc異常變化的原因,我們從cpla2α、p-cpla2α、spla2-Ⅱ、lcat四種酶的變化出發(fā),結(jié)果發(fā)現(xiàn)肝病進(jìn)程中l(wèi)cat主導(dǎo)胞外(血清及培養(yǎng)液)lpc的含量變化,而lpcat1主導(dǎo)胞內(nèi)(組織)lpc的含量變化。cpla2α、p-cpla2α、spla2-Ⅱa對肝病進(jìn)程中細(xì)胞內(nèi)外lpc的異常變化影響較小。這種在細(xì)胞內(nèi)外由不同酶主導(dǎo)lpc變化的機(jī)制原因需要進(jìn)一步探索。另外,p38、akt、p-akt、erk、p-erk、jnk與lpc的異常變化關(guān)系不大,p-p38及p-jnk與其有一定的相關(guān)性,也需要進(jìn)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 楊麗娜;溫靜;孫毅;梁佳佳;鄭衛(wèi)華;張麗麗;周于杰;熊志立;;四逆散抗肝損傷作用的大鼠血清UPLC-MS/MS代謝組學(xué)研究[J];藥學(xué)學(xué)報(bào);2014年03期

2 齊菲,顏光濤;胞漿型磷脂酶A_2與細(xì)胞增殖的關(guān)系[J];中華老年多器官疾病雜志;2005年01期

，

本文編號：1830657