本文選題：肝衰竭　切入點(diǎn)：血氨　出處：《鄭州大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：背景肝臟是人體的內(nèi)最重要的器官之一,具有解毒、合成分解代謝、分泌、生物轉(zhuǎn)化以及免疫防御等功能,其血流量占心搏出量的30-40%,正常的肝臟能夠及時(shí)把血液中多余的、有害的物質(zhì)解毒或者滅活,并合成人體必需的各種營養(yǎng)物質(zhì),被譽(yù)為人體內(nèi)的“加工廠”,在我們生命活動中起著舉足輕重的作用。血氨(blood ammonia,BA)是氨基酸代謝的主要產(chǎn)物,主要來源于腸道產(chǎn)氨、腎臟泌氨、肌肉產(chǎn)氨等。過多的氨對人體的毒性很大,肝臟是氨的主要代謝場所,能及時(shí)將氨轉(zhuǎn)變成無毒或毒性小的物質(zhì)排出體外,保持氨含量的平衡。肝衰竭(hepatic failure,HF)是由于各種原因造成肝細(xì)胞損壞引起肝臟功能障礙不能滿足身體需要而引起的一組臨床癥候群,是臨床常見的危急重癥之一,嚴(yán)重患者可引起肝性腦病、肝腎綜合征、出血等,死亡率高達(dá)70%-80%。其中血氨升高可能是引起肝衰竭一系列異常表現(xiàn)的重要原因之一。然而血氨升高對機(jī)體細(xì)胞的毒性作用不是均等的,在相同濃度的血氨作用下,293、HDF、VeroPQXB1/2細(xì)胞系較少受到生長抑制或者不受到生長抑制,而McCoy、MDCK細(xì)胞系較多細(xì)胞受到生長抑制,但HeLa、BHK細(xì)胞系生長存在明顯的生長抑制,說明血氨對細(xì)胞的影響具有明顯細(xì)胞特異性。肝衰竭時(shí)血氨升高可以損傷神經(jīng)細(xì)胞或神經(jīng)膠質(zhì)細(xì)胞導(dǎo)致肝性腦病的發(fā)生,高血氨對肝細(xì)胞的損傷和毒性作用尚不清楚。進(jìn)一步研究高血氨在肝衰竭時(shí)對肝細(xì)胞損傷的作用及其機(jī)制具有重要的意義。從2005到2011在我們醫(yī)院觀察到在給予肝功能衰竭的患者降血氨治療時(shí)可以顯著減輕肝損傷,降低患者的死亡率,改善患者的預(yù)后。我們建立慢性高氨誘發(fā)的大鼠模型,發(fā)現(xiàn)肝臟的酶學(xué)指標(biāo)均顯著增高。肝臟病理結(jié)果顯示高氨血癥可誘導(dǎo)肝細(xì)胞損傷,早期以細(xì)胞凋亡的表現(xiàn)為主,而炎癥細(xì)胞的浸潤、炎癥因子的表達(dá)和細(xì)胞壞死并不明顯,說明高血氨可能首先引起肝細(xì)胞凋亡進(jìn)而參與肝損傷的進(jìn)展從而在肝衰竭的發(fā)生和發(fā)展中發(fā)揮進(jìn)一步的肝細(xì)胞損傷作用。目的建立大鼠急性肝衰竭模型,并給予早期降血氨治療。研究早期降血氨后對肝細(xì)胞損傷和凋亡的影響。并通過觀察氯化氨對培養(yǎng)大鼠肝細(xì)胞增值和凋亡的影響以及血氨對肝細(xì)胞線粒體功能及細(xì)胞內(nèi)鈣超載和細(xì)胞凋亡的影響及其相互關(guān)系,探討高血氨對肝細(xì)胞損傷的機(jī)制,為肝衰竭的進(jìn)一步治療提供新的途徑和靶點(diǎn)。方法動物實(shí)驗(yàn)1.雌性SD大鼠48只,隨機(jī)分為3組：1,模型組,給予D-氨基半乳糖(450mg/kg)聯(lián)合內(nèi)毒素(100μg/kg)腹腔內(nèi)注射構(gòu)建急性肝衰竭模型,2,對照組以生理鹽水腹腔注射作為空白對照,3,OA干預(yù)組給藥同時(shí)加用門冬氨酸鳥氨酸(1.5g/kg,間隔6h)腹腔內(nèi)注射。2.分別于給藥后12h及24h麻醉處死動物,心臟采血并取肝臟組織。血液標(biāo)本干化學(xué)法檢測血氨,ELISA檢測血清ALT、AST.TNF-α、IL-6;肝臟組織經(jīng)固定脫水石蠟包埋切片,HE染色觀察病理變化,TUNEL檢測細(xì)胞凋亡率；冰凍組織提取DNA,電泳觀察DNA Ladder,RT-PCR檢測P53及SPP1基因的相對表達(dá)量。細(xì)胞學(xué)研究1,通過應(yīng)用氯化氨(NH4C1)對大鼠肝細(xì)胞進(jìn)行處理,構(gòu)建高血氨細(xì)胞模型。2,培養(yǎng)細(xì)胞隨機(jī)分為三組,分別應(yīng)用胞內(nèi)高效鈣選擇性螫合劑BAPTA-AM和胞外鈣選擇性螯合劑乙二醇二乙醚二胺四乙酸阻斷細(xì)胞內(nèi)或外的鈣離子,應(yīng)用MTT法檢測上述兩種干預(yù)方法和未干預(yù)組細(xì)胞的生長能力,檢測線粒體膜通道孔(mitochondrial permeability transition pore；MPTP)開放情況,應(yīng)用流式細(xì)胞術(shù)檢測細(xì)胞凋亡,并應(yīng)用Western blot法測定細(xì)胞色素C和PARP(poly ADP-ribose polymerase)DNA修復(fù)酶的表達(dá)情況。應(yīng)用熒光定量PCR法檢測鈣調(diào)蛋白(calmodulin,CALM)、鈣調(diào)素依賴的蛋白激酶(CAMKⅡ)的RNA表達(dá)情況。ELISA法檢測CAMKⅡ的濃度。結(jié)果動物實(shí)驗(yàn)1 血氨檢測結(jié)果顯示大鼠血氨值12h達(dá)高峰,24h后下降,12h及24h肝衰模型組及OA干預(yù)組血氨值均較空白對照組明顯升高,OA干預(yù)組與肝衰模型組相比明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2 肝臟酶學(xué)相關(guān)指標(biāo)(ALT,AST)隨著給藥時(shí)間延長明顯升高,除空白對照組外,24h組均高于12h組,12h OA干預(yù)組較肝衰模型組均值降低,但差異無統(tǒng)計(jì)學(xué)意義(P0.05)；24hOA干預(yù)組與肝衰竭模型組相比明顯下降,差異有統(tǒng)計(jì)學(xué)意義,(P0.05),ALT、AST均與血氨值呈正相關(guān)(P0.01)。3 肝組織病理改變隨著給藥時(shí)間延長逐漸加重,12h肝衰模型組與OA干預(yù)組未見明顯差別,24h OA干預(yù)組較肝衰模型組病變減輕,空白對照未見異常病變。除空白對照組外,其余各組均可見凋亡細(xì)胞特異的DNALadder。4 TUNEL結(jié)果顯示：隨著給藥時(shí)間延長,肝衰模型組及OA干預(yù)組肝細(xì)胞凋亡率增加(24h12h,P0.01)。肝衰模型組及OA干預(yù)組凋亡率較空白對照組明顯升高,OA干預(yù)組與肝衰模型組相比明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05),凋亡率與血氨值呈正相關(guān)(P0.01)。5 IL-6、TNF-α、P53表達(dá)水平均隨著給藥時(shí)間增加(24h12h),其中肝衰模型組及OA干預(yù)組與空白對照組相比均明顯升高,OA干預(yù)組與肝衰竭模型組相比明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05),且其均與血氨值呈正相關(guān)(P0.01)。6 SPP1在急性肝衰大鼠模型高表達(dá),隨著給藥時(shí)間延長,SPP1基因相對表達(dá)水平隨時(shí)間增加(24h12h),其中肝衰模型組及OA干預(yù)組相對表達(dá)量與空白對照組相比均明顯升高；肝衰竭模型組與OA干預(yù)組相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)；SPP1與血氨值呈正相關(guān)(P0.01)。細(xì)胞學(xué)研究：1 MTT結(jié)果示：NH4Cl作用于鼠肝細(xì)胞后,隨時(shí)間延長和濃度增大,抑制率逐漸增加。EGTA+NH4Cl組(以下簡稱EGTA組)與NH4Cl組無明顯差異(p0.05)；在高濃度時(shí),BAPTA-AM組(以下簡稱BAPTA-AM組)較NH4Cl組明顯降低,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。2 流式細(xì)胞術(shù)結(jié)果示：NH4Cl作用于鼠肝細(xì)胞后,隨時(shí)間延長和濃度增大,凋亡細(xì)胞所占比率逐漸增加。BAPTA-AM組凋亡率較NH4Cl組和EGTA組降低,有統(tǒng)計(jì)學(xué)意義(p0.05),NH4Cl組和EGTA組之間無差異(p0.05)。3 Western blot結(jié)果示：NH4Cl組和EGTA組在5.0mM以上氯化氨作用72h后出現(xiàn)PARP蛋白被切割,BAPTA-AM組無變化；NH4Cl組和EGTA組Cyt C表達(dá)量有時(shí)間、濃度依賴性,而BAPTA-AM組的無差異。4 線粒體通透轉(zhuǎn)運(yùn)孔道(PTP)結(jié)果示：NH4Cl處理后,熒光峰值明顯左移,峰值左移隨時(shí)間延長而增加,BAPTA-AM組較NH4Cl組和EGTA組降低,有統(tǒng)計(jì)學(xué)意義(p0.05),NH4Cl組和EGTA組之間無差異(p0.05)。5 Real-time PC R結(jié)果示：在不同時(shí)間和不同濃度NH4Cl處理?xiàng)l件下,CAM的mRNA表達(dá)無差異,而CamK^JmRNA隨NH4Cl濃度的增加而減少；相同濃度的NH4Cl的情況下,CamK的rnRNA隨時(shí)間的延長而減少。6 ELISA結(jié)果示：不同時(shí)間和不同濃度的NH4Cl處理細(xì)胞后CAMKⅡ無差異。結(jié)論1.早期全程降血氨可降低肝細(xì)胞凋亡率,減輕肝損傷,并減少TNF-α、IL-6SPP1及凋亡相關(guān)基因P53的表達(dá)；2. TNF-α、IL-6、SPP1和P53的表達(dá)水平和血氨水平以及凋亡率相關(guān)3.高血氨可以引起鈣超載,并通過激活線粒體途徑引起細(xì)胞凋亡,但并不激活Ca2+-CaM-CaMKⅡ信號通路；4. 胞內(nèi)鈣離子螯合劑可有效保護(hù)高血氨所致的肝細(xì)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R575
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本文編號：1588114