本文選題：腦死亡 + 基因表達(dá)　； 參考：《鄭州大學(xué)》2016年博士論文

【摘要】：目前,肝臟移植的供肝主要來自腦死亡患者的捐獻(xiàn)。然而,實(shí)驗(yàn)和臨床研究發(fā)現(xiàn):與活體供肝相比,腦死亡供肝移植術(shù)后的缺血再灌注損傷重、急性排斥反應(yīng)發(fā)生率高,出現(xiàn)初始肝功能不良和原發(fā)性移植物無功能的風(fēng)險(xiǎn)大。因此,國內(nèi)外學(xué)者建立了大量動物腦死亡模型來研究腦死亡狀態(tài)對潛在供體器官的影響以及可能的干預(yù)措施。腦死亡是指“包括腦干在內(nèi)的全腦功能不可逆性的喪失”,可以引起機(jī)體復(fù)雜的病理生理變化,主要表現(xiàn)為血流動力紊亂、激素水平改變、炎癥和免疫系統(tǒng)被激活、補(bǔ)體和凝血系統(tǒng)被激活。這些不利因素導(dǎo)致線粒體和內(nèi)質(zhì)網(wǎng)應(yīng)激細(xì)胞凋亡通路被激活,使細(xì)胞凋亡增加,嚴(yán)重影響了腦死亡供肝的質(zhì)量。但是,腦死亡狀態(tài)下肝損傷的具體分子機(jī)制仍不清楚。因此,研究腦死亡狀態(tài)下肝臟組織中基因的變化,可以為減輕腦死亡狀態(tài)下肝損傷提供潛在的干預(yù)靶基因。血紅素加氧酶1(heme oxygenase 1,HO-1)又稱為熱休克蛋白32(heat shock protein,HSP32),是降解血紅素的限速酶,具有抗炎和抗凋亡的作用。研究發(fā)現(xiàn),腦死亡狀態(tài)下腎臟中HO-1和HSP70高表達(dá)。鈷原卟啉(cobalt protoporphyrin,CoPP)預(yù)處理腦死亡供體誘導(dǎo)HO-1高表達(dá),減輕移植腎中炎性細(xì)胞浸潤,并可以將腦死亡供腎移植后3個(gè)月的移植物存活率從20%顯著提高至50%。腦死亡狀態(tài)是否促進(jìn)肝臟中熱休克蛋白的表達(dá),ho-1誘導(dǎo)劑-copp是否能減輕腦死亡狀態(tài)下肝臟損傷,其分子作用機(jī)制是什么至今還不清楚。本課題擬從以下三個(gè)部分進(jìn)行研究,建立大鼠腦死亡模型,分析腦死亡狀態(tài)下大鼠肝組織中基因表達(dá)情況,探討copp對腦死亡狀態(tài)下大鼠肝損傷的分子作用機(jī)制,以期為提高腦死亡供肝質(zhì)量提供新的思路和方法,從而為臨床肝移植提供更多可用的腦死亡供體。第一部分:緩慢間斷顱內(nèi)加壓法建立大鼠腦死亡模型目的:為研究腦死亡狀態(tài)對肝臟質(zhì)量的影響,建立一種穩(wěn)定、可靠的大鼠腦死亡模型。方法:采用sd大鼠,顱骨鉆孔在硬膜外腔放置3ffogarty球囊導(dǎo)管,通過間斷緩慢加壓法誘導(dǎo)大鼠腦死亡。以自主呼吸停止、腦電波靜息、腦干反射消失和深昏迷作為腦死亡判定標(biāo)準(zhǔn)。尾靜脈補(bǔ)羥乙基淀粉溶液維持腦死亡6h期間平均動脈壓大于80mmhg。腦死亡組20只大鼠;假手術(shù)對照組(n=10)不行球囊加壓,其余操作同腦死亡組。監(jiān)測大鼠的動脈血壓、心率、呼氣末co2、補(bǔ)液量和尿量,檢測血清alt和ast水平,觀察肝組織病理變化。結(jié)果:大鼠腦死亡模型的成功率為90%,加壓誘導(dǎo)前準(zhǔn)備時(shí)間為70±10min,腦死亡加壓誘導(dǎo)時(shí)間為34±6min,加壓球囊所需的液體量為136±24μl。腦死亡組大鼠的羥乙基淀粉輸入量為36.46±17.85ml,尿量為32.77±18.44ml,血清中alt和ast分別為70.17±15.87u/l、201±23.31u/l,均明顯高于假手術(shù)對照(均p0.05)。光學(xué)顯微鏡下觀察可見,假手術(shù)對照組大鼠的肝臟組織形態(tài)正常;腦死亡組的大鼠肝臟呈現(xiàn)明顯的淤血和水腫,肝索增寬,肝竇變窄,存在散在的點(diǎn)狀壞死。第二部分:腦死亡狀態(tài)下大鼠肝組織中基因表達(dá)變化目的:分析腦死亡狀態(tài)下大鼠肝組織中基因表達(dá)變化,為揭示腦死亡狀態(tài)下肝損傷的分子機(jī)制提供實(shí)驗(yàn)依據(jù)。方法:分別在大鼠腦死亡后0h、1h、2h、4h、6h留取標(biāo)本,假手術(shù)對照組大鼠也在相對應(yīng)的時(shí)間點(diǎn)留取標(biāo)本,每個(gè)時(shí)間點(diǎn)6只大鼠。用agilent大鼠全基因組表達(dá)譜芯片分析腦死亡6h及假手術(shù)對照的大鼠肝臟組織中41012個(gè)基因的表達(dá)情況。qpcr檢測大鼠腦死亡后0h、1h、2h、4h、6h肝臟中ho-1、hsp27、hsp70、mcl-1、bcl-2、bnip3、caspase-3、hif-1α、hif-2α、hif-3α、arnt和glut-1的表達(dá)情況。westernblotting和免疫組化檢測大鼠腦死亡后肝臟中ho-1蛋白表達(dá)情況。結(jié)果:與假手術(shù)對照組比,腦死亡6h大鼠肝臟組織中有149個(gè)基因表達(dá)上調(diào)超過10倍,有136個(gè)基因表達(dá)下調(diào)超過10倍。腦死亡后0h、1h、2h、4h、6h的大鼠肝臟中ho-1的mrna和蛋白表達(dá)水平明顯上調(diào),ho-1蛋白表達(dá)在胞漿,在肝小葉中央靜脈周圍的肝臟細(xì)胞高表達(dá)。bnip3和caspase-3的mrna表達(dá)水平在腦死亡后0h、2h、4h、6h大鼠肝臟中明顯上調(diào)。第三部分:copp減輕大鼠腦死亡狀態(tài)下肝損傷的分子作用機(jī)制目的:探討ho-1誘導(dǎo)劑-copp對大鼠腦死亡狀態(tài)下肝損傷的作用及其分子機(jī)制。方法:將大鼠隨機(jī)分成4組,每組6只。腦死亡(b)組:大鼠腦死亡維持6h;假手術(shù)(s)組:不加壓球囊誘導(dǎo)腦死亡,其余操作同腦死亡組;copp預(yù)處理腦死亡(cb)組:術(shù)前24h腹腔注射copp5mg/kg,其余操作同腦死亡組;copp預(yù)處理假手術(shù)(cs)組:術(shù)前24h腹腔注射copp5mg/kg,其余操作同假手術(shù)組。全自動標(biāo)準(zhǔn)生化分析儀檢測血清中alt和ast的水平。westernblotting檢測肝臟中bcl-2、bax、mcl-1、chop、caspase-12、cytosoliccytochromec和cleaved-caspase-3蛋白的表達(dá)。tunel檢測大鼠肝臟中細(xì)胞凋亡情況。結(jié)果:copp預(yù)處理顯著誘導(dǎo)大鼠肝臟中ho-1蛋白高表達(dá)。與腦死亡組比,copp預(yù)處理腦死亡組大鼠的血清alt和ast水平明顯降低,肝臟中bcl-2和mcl-1蛋白表達(dá)明顯上調(diào),而bax、chop、caspase-12、cytosoliccytochromec和cleaved-caspase-3蛋白表達(dá)明顯下調(diào),肝臟中凋亡細(xì)胞明顯減少。結(jié)論:1.建立了穩(wěn)定、可靠、重復(fù)性好的大鼠腦死亡模型,為進(jìn)一步研究腦死亡狀態(tài)對供體器官的影響提供了基礎(chǔ)。2.首次全面分析了腦死亡狀態(tài)下大鼠肝臟組織中基因的表達(dá),為減輕腦死亡狀態(tài)下肝臟損傷提供了新的干預(yù)靶基因;腦死亡后肝臟組織中HO-1表達(dá)上調(diào),可能與腦死亡相關(guān)應(yīng)激啟動的保護(hù)性機(jī)制有關(guān)。3.HO-1誘導(dǎo)劑-CoPP可以顯著上調(diào)HO-1蛋白表達(dá),并通過調(diào)控線粒體和內(nèi)質(zhì)網(wǎng)凋亡途徑中關(guān)鍵蛋白,減輕腦死亡大鼠肝臟中細(xì)胞凋亡。HO-1有望成為提高腦死亡供肝質(zhì)量的潛在干預(yù)靶點(diǎn)。
[Abstract]:At present, donor liver transplantation is mainly from the donation of brain death patients. However, experimental and clinical studies have found that compared with living donor liver donor liver transplantation, cerebral death has a high incidence of ischemia-reperfusion injury, high incidence of acute rejection, high risk of initial liver dysfunction and primary graft without function. A large number of animal brain death models have been established to study the effects of brain death on the potential donor organs and possible interventions. Brain death refers to the loss of the irreversible function of the whole brain, including the brain stem, which can cause complex pathophysiological changes in the body, mainly characterized by disturbance of blood flow, changes in hormone levels, and inflammation. The disease and immune system are activated and the complement and coagulation system are activated. These adverse factors lead to the activation of the apoptosis pathway of mitochondria and endoplasmic reticulum stress cells, increase the apoptosis of cells and seriously affect the quality of brain death donor liver. However, the specific sub mechanism of liver injury in brain death is still unclear. Therefore, the study of brain death is in the state of brain death. The changes in the gene in the liver can provide potential intervention targets for reducing the liver injury in the brain. Heme oxygenase 1 (heme oxygenase 1, HO-1), also known as heat shock protein 32 (heat shock protein, HSP32), is a speed limiting enzyme that degrades heme, and has the effect of anti-inflammatory and anti apoptosis. HO-1 and HSP70 were highly expressed in the viscera. Cobalt protoporphyrin (CoPP) pretreated the brain death donor to induce the high expression of HO-1, alleviated the infiltration of inflammatory cells in the transplanted kidney, and could increase the graft survival rate from 20% to the death state of the brain 3 months after the transplantation of the brain to the death state of 50%. to promote the expression of heat shock protein in the liver. It is not clear what the molecular mechanism of the HO-1 inducer -copp can reduce the liver injury in the brain death state. This topic is to establish the rat brain death model from the following three parts, analyze the gene expression in the rat liver tissue under the brain death, and discuss the copp damage to the rat liver in the brain death state. Molecular mechanism of action, in order to provide new ideas and methods for improving the quality of brain death donor liver, provides more available brain death donors for clinical liver transplantation. Part 1: the aim of establishing rat brain death model with slow intermittent intracranial pressure method is to study the effect of brain death on the liver quality and establish a stable and reliable large scale. Method: the rat brain death model was used in SD rats. The 3ffogarty balloon catheter was placed in the epidural cavity in the skull hole, and the brain death was induced by intermittent slow compression. The brain death was determined by the stop breathing, the brain wave resting, the brain stem reflex disappearing and deep coma as the criteria for brain death. The tail vein supplementation hydroxyethyl starch solution maintained the brain death period of 6h. The mean arterial pressure was greater than that of 20 rats in the 80mmhg. brain death group; the sham operation control group (n=10) had no balloon pressure, the rest of the operation was with the brain death group. The blood pressure, heart rate, CO2, rehydration and urine volume of the rats were monitored, the levels of serum ALT and AST were detected, and the changes of liver tissue were observed. The results were as follows: the success rate of the rat brain death model was 90% The preparation time was 70 + 10min before induction, and the pressure induction time of brain death was 34 + 6min. The amount of liquid needed in the pressure balloon was 136 + 24 Mu L. brain death group, the hydroxyethyl starch input was 36.46 + 17.85ml, the urine volume was 32.77 + 18.44ml, and the serum ALT and ast were 70.17 + 15.87u/l and 201 + 23.31u/l respectively. All were significantly higher than those of the sham operation control (p0.0, all p0.0). 5). It was observed under the optical microscope that the liver tissue of the rats in the sham operation control group was normal, and the liver of the rats in the brain death group showed obvious congestion and edema, the widened hepatic cord, the narrowing of the hepatic sinus and the scattered necrosis. The second part: the change of gene expression in the rat liver tissue under the brain death: the analysis of the large brain death state. The changes in gene expression in rat liver tissue provide experimental basis for revealing the molecular mechanism of liver injury in the brain dead state. Methods: 0h, 1H, 2h, 4h, 6h were left after the death of the rat brain, and the rats in the sham operation control group were also left at the corresponding time points, and each time point was 6 rats. The whole genome expression chip of Agilent rats was used. Analysis of the expression of 41012 genes in the liver tissues of the brain dead 6h and the sham operation control rats.Qpcr detection of 0h, 1H, 2h, 4h, 6h liver HO-1, HSP27, HSP70, Mcl-1, Bcl-2, HSP70, Mcl-1,.Qpcr and immunohistochemical detection of the liver after the death of the rat brain after brain death The expression of medium HO-1 protein. Results: compared with the sham control group, the expression of 149 genes in the liver tissue of 6h rats was up to 10 times higher than that of the sham control group, and the expression of 136 genes was down 10 times. The mRNA and protein expression of HO-1 in 0h, 1H, 2h, 4h, 6h of the rat after brain death was up-regulated, HO-1 protein expressed in the cytoplasm and in the hepatic lobule. The expression level of high expression of.Bnip3 and Caspase-3 in the liver cells around the central vein was obviously up-regulated in the liver of 0h, 2h, 4h, 6h rats after brain death. The third part: copp alleviated the molecular mechanism of the liver injury in the brain death of rats: the effect of HO-1 inducer -copp on the liver injury in the rat brain and its molecular mechanism Method: the rats were randomly divided into 4 groups, 6 rats in each group. Brain death (b) group: rat brain death maintained 6h; sham operation (s) group: non pressurized balloon induced brain death, the rest of the same brain death group; copp pretreated brain death (CB) group: preoperative 24h intraperitoneal injection of copp5mg/kg, the rest of the same brain death group; copp preconditioning pseudopero operation (CS) group: preoperative 24h abdominal abdominal cavity Injection of copp5mg/kg, the rest of the operation with the sham operation group. Test the level of ALT and AST in serum by automatic standard biochemical analyzer to detect the expression of Bcl-2, Bax, Mcl-1, chop, caspase-12, cytosoliccytochromec and cleaved-caspase-3 protein in the liver. The high expression of HO-1 protein in the rat liver was induced. Compared with the brain death group, the level of serum ALT and AST in the rats with copp pretreated brain death group decreased significantly, the expression of Bcl-2 and Mcl-1 in the liver was obviously up, while the expression of Bax, chop, caspase-12, cytosoliccytochromec and cleaved-caspase-3 egg white was obviously down, and the apoptotic cells in the liver decreased significantly. Conclusion: 1. a stable, reliable and reproducible rat brain death model was established, which provided a basic.2. for the first time to analyze the expression of gene in the rat liver tissue in the brain dead state for the first time, and to provide a new target gene for reducing the liver injury in the brain death state. The up-regulated expression of HO-1 in the post liver tissue may be related to the protective mechanism of brain death related stress..3.HO-1 inducer -CoPP can significantly up-regulate the expression of HO-1 protein, and by regulating the key proteins in the apoptosis pathway of mitochondria and endoplasmic reticulum, the decrease of apoptosis.HO-1 in the liver of brain death rats is expected to improve the liver quality of brain death. Potential targets for quantitative intervention.
【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】：2016
【分類號】：R741;R657.3

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