高血氨对肝细胞损伤和凋亡机制的研究

发布时间：2018-03-09 10:20

  本文选题：肝衰竭　切入点：血氨　出处：《郑州大学》2014年博士论文　论文类型：学位论文

【摘要】：背景肝脏是人体的内最重要的器官之一,具有解毒、合成分解代谢、分泌、生物转化以及免疫防御等功能,其血流量占心搏出量的30-40%,正常的肝脏能够及时把血液中多余的、有害的物质解毒或者灭活,并合成人体必需的各种营养物质,被誉为人体内的“加工厂”,在我们生命活动中起着举足轻重的作用。血氨(blood ammonia,BA)是氨基酸代谢的主要产物,主要来源于肠道产氨、肾脏泌氨、肌肉产氨等。过多的氨对人体的毒性很大,肝脏是氨的主要代谢场所,能及时将氨转变成无毒或毒性小的物质排出体外,保持氨含量的平衡。肝衰竭(hepatic failure,HF)是由于各种原因造成肝细胞损坏引起肝脏功能障碍不能满足身体需要而引起的一组临床症候群,是临床常见的危急重症之一,严重患者可引起肝性脑病、肝肾综合征、出血等,死亡率高达70%-80%。其中血氨升高可能是引起肝衰竭一系列异常表现的重要原因之一。然而血氨升高对机体细胞的毒性作用不是均等的,在相同浓度的血氨作用下,293、HDF、VeroPQXB1/2细胞系较少受到生长抑制或者不受到生长抑制,而McCoy、MDCK细胞系较多细胞受到生长抑制,但HeLa、BHK细胞系生长存在明显的生长抑制,说明血氨对细胞的影响具有明显细胞特异性。肝衰竭时血氨升高可以损伤神经细胞或神经胶质细胞导致肝性脑病的发生,高血氨对肝细胞的损伤和毒性作用尚不清楚。进一步研究高血氨在肝衰竭时对肝细胞损伤的作用及其机制具有重要的意义。从2005到2011在我们医院观察到在给予肝功能衰竭的患者降血氨治疗时可以显著减轻肝损伤,降低患者的死亡率,改善患者的预后。我们建立慢性高氨诱发的大鼠模型,发现肝脏的酶学指标均显著增高。肝脏病理结果显示高氨血症可诱导肝细胞损伤,早期以细胞凋亡的表现为主,而炎症细胞的浸润、炎症因子的表达和细胞坏死并不明显,说明高血氨可能首先引起肝细胞凋亡进而参与肝损伤的进展从而在肝衰竭的发生和发展中发挥进一步的肝细胞损伤作用。目的建立大鼠急性肝衰竭模型,并给予早期降血氨治疗。研究早期降血氨后对肝细胞损伤和凋亡的影响。并通过观察氯化氨对培养大鼠肝细胞增值和凋亡的影响以及血氨对肝细胞线粒体功能及细胞内钙超载和细胞凋亡的影响及其相互关系,探讨高血氨对肝细胞损伤的机制,为肝衰竭的进一步治疗提供新的途径和靶点。方法动物实验1.雌性SD大鼠48只,随机分为3组：1,模型组,给予D-氨基半乳糖(450mg/kg)联合内毒素(100μg/kg)腹腔内注射构建急性肝衰竭模型,2,对照组以生理盐水腹腔注射作为空白对照,3,OA干预组给药同时加用门冬氨酸鸟氨酸(1.5g/kg,间隔6h)腹腔内注射。2.分别于给药后12h及24h麻醉处死动物,心脏采血并取肝脏组织。血液标本干化学法检测血氨,ELISA检测血清ALT、AST.TNF-α、IL-6;肝脏组织经固定脱水石蜡包埋切片,HE染色观察病理变化,TUNEL检测细胞凋亡率；冰冻组织提取DNA,电泳观察DNA Ladder,RT-PCR检测P53及SPP1基因的相对表达量。细胞学研究1,通过应用氯化氨(NH4C1)对大鼠肝细胞进行处理,构建高血氨细胞模型。2,培养细胞随机分为三组,分别应用胞内高效钙选择性螫合剂BAPTA-AM和胞外钙选择性螯合剂乙二醇二乙醚二胺四乙酸阻断细胞内或外的钙离子,应用MTT法检测上述两种干预方法和未干预组细胞的生长能力,检测线粒体膜通道孔(mitochondrial permeability transition pore；MPTP)开放情况,应用流式细胞术检测细胞凋亡,并应用Western blot法测定细胞色素C和PARP(poly ADP-ribose polymerase)DNA修复酶的表达情况。应用荧光定量PCR法检测钙调蛋白(calmodulin,CALM)、钙调素依赖的蛋白激酶(CAMKⅡ)的RNA表达情况。ELISA法检测CAMKⅡ的浓度。结果动物实验1 血氨检测结果显示大鼠血氨值12h达高峰,24h后下降,12h及24h肝衰模型组及OA干预组血氨值均较空白对照组明显升高,OA干预组与肝衰模型组相比明显降低,差异有统计学意义(P0.05)。2 肝脏酶学相关指标(ALT,AST)随着给药时间延长明显升高,除空白对照组外,24h组均高于12h组,12h OA干预组较肝衰模型组均值降低,但差异无统计学意义(P0.05)；24hOA干预组与肝衰竭模型组相比明显下降,差异有统计学意义,(P0.05),ALT、AST均与血氨值呈正相关(P0.01)。3 肝组织病理改变随着给药时间延长逐渐加重,12h肝衰模型组与OA干预组未见明显差别,24h OA干预组较肝衰模型组病变减轻,空白对照未见异常病变。除空白对照组外,其余各组均可见凋亡细胞特异的DNALadder。4 TUNEL结果显示：随着给药时间延长,肝衰模型组及OA干预组肝细胞凋亡率增加(24h12h,P0.01)。肝衰模型组及OA干预组凋亡率较空白对照组明显升高,OA干预组与肝衰模型组相比明显降低,差异有统计学意义(P0.05),凋亡率与血氨值呈正相关(P0.01)。5 IL-6、TNF-α、P53表达水平均随着给药时间增加(24h12h),其中肝衰模型组及OA干预组与空白对照组相比均明显升高,OA干预组与肝衰竭模型组相比明显下降,差异有统计学意义(P0.05),且其均与血氨值呈正相关(P0.01)。6 SPP1在急性肝衰大鼠模型高表达,随着给药时间延长,SPP1基因相对表达水平随时间增加(24h12h),其中肝衰模型组及OA干预组相对表达量与空白对照组相比均明显升高；肝衰竭模型组与OA干预组相比差异无统计学意义(P0.05)；SPP1与血氨值呈正相关(P0.01)。细胞学研究：1 MTT结果示：NH4Cl作用于鼠肝细胞后,随时间延长和浓度增大,抑制率逐渐增加。EGTA+NH4Cl组(以下简称EGTA组)与NH4Cl组无明显差异(p0.05)；在高浓度时,BAPTA-AM组(以下简称BAPTA-AM组)较NH4Cl组明显降低,差异有统计学意义(p0.05)。2 流式细胞术结果示：NH4Cl作用于鼠肝细胞后,随时间延长和浓度增大,凋亡细胞所占比率逐渐增加。BAPTA-AM组凋亡率较NH4Cl组和EGTA组降低,有统计学意义(p0.05),NH4Cl组和EGTA组之间无差异(p0.05)。3 Western blot结果示：NH4Cl组和EGTA组在5.0mM以上氯化氨作用72h后出现PARP蛋白被切割,BAPTA-AM组无变化；NH4Cl组和EGTA组Cyt C表达量有时间、浓度依赖性,而BAPTA-AM组的无差异。4 线粒体通透转运孔道(PTP)结果示：NH4Cl处理后,荧光峰值明显左移,峰值左移随时间延长而增加,BAPTA-AM组较NH4Cl组和EGTA组降低,有统计学意义(p0.05),NH4Cl组和EGTA组之间无差异(p0.05)。5 Real-time PC R结果示：在不同时间和不同浓度NH4Cl处理条件下,CAM的mRNA表达无差异,而CamK^JmRNA随NH4Cl浓度的增加而减少；相同浓度的NH4Cl的情况下,CamK的rnRNA随时间的延长而减少。6 ELISA结果示：不同时间和不同浓度的NH4Cl处理细胞后CAMKⅡ无差异。结论1.早期全程降血氨可降低肝细胞凋亡率,减轻肝损伤,并减少TNF-α、IL-6SPP1及凋亡相关基因P53的表达；2. TNF-α、IL-6、SPP1和P53的表达水平和血氨水平以及凋亡率相关3.高血氨可以引起钙超载,并通过激活线粒体途径引起细胞凋亡,但并不激活Ca2+-CaM-CaMKⅡ信号通路；4. 胞内钙离子螯合剂可有效保护高血氨所致的肝细胞凋亡。
[Abstract]:The background of the liver is one of the most important organs of the body with detoxification, synthesis metabolism, secretion, biotransformation and immune defense functions, the blood flow for the cardiac output 30-40%, normal liver can timely take blood in excess or harmful substances, detoxification inactivated, nutrients and synthesis the necessary human, known as the body's "factory", plays an important role in the activities of our lives. The blood ammonia (blood ammonia BA) is the main product of amino acid metabolism, mainly from intestinal ammonia production, kidney urinary ammonia, ammonia and other toxic. Muscle producing excess ammonia on the human body the great, the liver is a major metabolic site of ammonia, ammonia will promptly turn into non-toxic or less toxic substances excreted, keep the ammonia content balance. Liver failure (hepatic failure HF) is due to various causes of liver cell damage caused by liver function Obstacles cannot meet the needs of the body caused by a group of clinical syndrome, is one of the common clinical emergency, patients can cause severe hepatic encephalopathy, hepatorenal syndrome, bleeding, the mortality rate as high as 70%-80%. which elevated blood ammonia may be a series of abnormal expression of one of the important reasons is the cause of liver failure. However, elevated blood ammonia toxicity the body cells are not equal, in the same concentration of blood ammonia, 293, HDF, VeroPQXB1/2 cell line is less affected by growth inhibition or by growth inhibition, and McCoy, a multicellular growth inhibition by MDCK, but the HeLa cell line, BHK cells have obvious growth inhibition effect of blood ammonia the cell has obvious cell specific liver failure. Elevated blood ammonia can damage nerve cells or glial cells leads to the occurrence of hepatic encephalopathy, damage and toxicity of high blood ammonia on liver cells The role is not clear. In liver failure of liver cell injury effect and its mechanism has important significance to the further study of high blood ammonia. From 2005 to 2011 in our hospital were observed in the treatment of blood ammonia for patients with liver failure can significantly reduce liver damage, reduce mortality, improve the prognosis of the patients. We establish a rat model of chronic high ammonia induced liver enzymes, found increased significantly. The liver pathologic results showed that liver cell injury induced by hyperammonemia, in the early stage of apoptosis and expression, inflammatory cell infiltration, inflammatory factor expression and cell necrosis is not obvious, the progress of high blood ammonia can first cause liver cell apoptosis and involved in liver injury and liver cell injury play a further role in the occurrence and development of liver failure. Objective to establish acute hepatic failure rat model, and to To early fall blood ammonia treatment. The damage of liver cell apoptosis and the effects of early blood ammonia and ammonium chloride. By observing the effect on cultured liver cell proliferation and apoptosis in rats and blood ammonia effect on mitochondrial function and intracellular calcium overload and apoptosis and their relationship, to explore the mechanism of high blood ammonia of liver cell injury, provide new ways and targets for further treatment of liver failure. Methods 1. animal experiment 48 female SD rats were randomly divided into 3 groups: 1 model group, given D- galactosamine (450mg/kg) combined with endotoxin (100 g/kg) model of acute liver failure, construction intraperitoneal injection of 2, the control group by intraperitoneal injection of normal saline as the control, 3, OA intervention group was administered with L-ornithine-L-aspartate (1.5g/kg interval, 6h) intraperitoneal injection of.2. after administration, 12h and 24h were sacrificed animal, heart blood and liver Dirty tissue. Blood samples of dry blood ammonia chemical detection method, detection of serum ALT, ELISA, AST.TNF- alpha, IL-6; liver tissues were fixed and paraffin embedded sections to observe the pathological changes of HE staining, TUNEL detection of cell apoptosis; frozen tissue DNA extraction and electrophoresis were used to observe the DNA Ladder, the relative expression of SPP1 gene and detection of P53 RT-PCR 1. Cytological study, through the application of ammonium chloride (NH4C1) treatment on rat liver cells, construct high blood ammonia cell model of.2 cells were randomly divided into three groups, respectively, using efficient intracellular calcium glycol selective chelating agent BAPTA-AM and extracellular calcium selective chelating two ether amine four acetic acid two calcium blockers ion inside or outside the cell, the growth ability of the MTT method was used to detect the two kinds of intervention and non intervention group cells, detection of MPTP (mitochondrial permeability transition pore; MPTP) opened, using flow cytometry Cell apoptosis detection technique, and determination of cytochrome C by Western blot and PARP (poly ADP-ribose polymerase) expression of DNA repair enzyme. The detection of calmodulin by fluorescence quantitative PCR (calmodulin, CALM), calmodulin dependent protein kinase (CAMK II) RNA expression of CAMK II.ELISA concentration detection method the results of animal experiments. 1 blood ammonia detection results showed that the rats blood ammonia value peaked at 12h, 24h and 24h decreased after 12h, liver failure model group and OA group were blood ammonia value is higher than the blank control group, OA intervention group and alf model group were significantly decreased, the difference was statistically significant (P0.05).2 related indicators of hepatic enzymes (ALT, AST) with the delivery time increases significantly, except for the blank control group, 24h group were higher than that of 12h group, 12h OA intervention group than in the model group decreased the mean hepatic failure, but the difference was not statistically significant (P0.05); 24hOA intervention group and the liver Failure model was significantly lower than that in group, the difference was statistically significant, (P0.05), ALT, AST and blood ammonia value (P0.01) of.3 was positively correlated with pathological changes in liver tissues with the delivery time aggravated liver failure, 12h model group and OA intervention group had no obvious difference, 24h OA intervention group than in liver disease model the blank control group, no abnormal lesions. Except control group, other groups were apoptotic cell specific DNALadder.4 TUNEL results showed that: with the prolongation of injection, liver failure model group and OA group of liver cell apoptosis rate increased (24h12h, P0.01). ALF model group and OA group apoptosis rate is blank control group increased significantly, OA intervention group and alf model group were significantly decreased, the difference was statistically significant (P0.05), the rate of apoptosis was positively correlated with blood ammonia (P0.01).5 IL-6, TNF- alpha, P53 expression levels were increased with the delivery time (24h12h), which ALF model group and OA group compared with the blank control group were significantly increased, OA intervention group and hepatic failure model was significantly lower than that in group, the difference was statistically significant (P0.05), and the blood ammonia value was positively correlated with.6 (P0.01) SPP1 in acute liver failure rat model with high expression, to time, the relative SPP1 gene expression level increased with time (24h12h), the liver failure model group and OA group relative expression compared with the blank control group were significantly increased; liver failure in model group and OA intervention group had no significant difference (P0.05); SPP1 and blood ammonia value was positively correlated (P0.01) cytological studies: 1. The results of MTT showed that the effect of NH4Cl on rat liver cells, with time and concentration increased, the inhibition rate increased gradually in.EGTA+NH4Cl group (EGTA group) with no significant difference between NH4Cl group (P0.05); at high concentrations, BAPTA-AM group (hereinafter referred to as BAPTA-AM Group) was lower than that in NH4Cl group, the difference was statistically significant (P0.05).2 flow cytometry results showed that: the effect of NH4Cl on rat liver cells, with time and concentration increasing, the percentage of apoptotic cells gradually increased the apoptosis rate of.BAPTA-AM group compared with NH4Cl group and EGTA group decreased, with statistical significance (P0.05), no difference between group NH4Cl and group EGTA (P0.05).3 Western blot results showed: NH4Cl group and EGTA group of ammonium chloride, the role of 72h in 5.0mM after more than PARP protein was cut, no change in BAPTA-AM group; NH4Cl group and EGTA group Cyt C expression with time, concentration dependent, and the BAPTA-AM group no difference in.4 of mitochondrial permeability transition pore (PTP) results showed: after NH4Cl treatment, the fluorescence peak was shifted to the left, the left peak increases with the time prolonging, BAPTA-AM group compared with NH4Cl group and EGTA group decreased, with statistical significance (P0.05), no difference between group NH4Cl and group EGTA (P0.05).5 Rea L-time PC R results showed: in different time and different concentrations of NH4Cl under the condition of CAM, the expression of mRNA and CamK^JmRNA had no difference, with the increase of NH4Cl concentration decreased; the same concentration of NH4Cl, CamK and rnRNA with time and reduce the.6 ELISA result shows that the different time and different concentration NH4Cl cells after CAMK II no difference. Conclusion 1. early stage lowering blood ammonia can decrease hepatocyte apoptosis, reduce liver damage, and reduce the expression of TNF- alpha, IL-6SPP1 and apoptosis related gene P53; 2. TNF- alpha, IL-6, SPP1 and the expression of P53 and blood ammonia level and apoptosis rate of 3. high blood ammonia can cause calcium overload, and through activation of the mitochondrial pathway induced apoptosis, but do not activate the Ca2+-CaM-CaMK II signal pathway; 4. intracellular calcium chelator to liver cell apoptosis induced by high blood ammonia of effective protection.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575
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本文编号：1588114