本文選題：內(nèi)質(zhì)網(wǎng)應(yīng)激 + 凋亡��； 參考：《鄭州大學(xué)》2015年博士論文

【摘要】：肝臟移植是治療終末期肝臟疾病最有效的治療途徑。制約肝臟移植發(fā)展的最主要因素是肝臟供體的短缺。腦死亡供體是肝臟移植的主要供體來源。腦死亡是指包括腦干功能在內(nèi)的全腦功能不可逆和永久的喪失。腦死亡是一個(gè)復(fù)雜的病理生理過程,是一個(gè)直接影響器官形態(tài)和功能的動(dòng)態(tài)過程。研究表明腦死亡來源的供體較活體來源的供體移植術(shù)后并發(fā)癥多、生存率低。因此,腦死亡被認(rèn)為是造成供體器官損傷的一個(gè)獨(dú)立危險(xiǎn)因素。細(xì)胞凋亡的增加是導(dǎo)致腦死亡供體肝臟損傷的主要因素。引起細(xì)胞凋亡的途徑有三條,內(nèi)質(zhì)網(wǎng)應(yīng)激途徑引起凋亡是線粒體途徑和外源性途徑之外的第三條凋亡途徑。通常情況下,內(nèi)質(zhì)網(wǎng)通過未折疊蛋白反應(yīng)保護(hù)由外界刺激對(duì)細(xì)胞的損傷,但是當(dāng)應(yīng)激時(shí)間延長(zhǎng)或者應(yīng)激強(qiáng)度增大,不能通過未折疊蛋白反應(yīng)保護(hù)機(jī)制代償,最終會(huì)通過激活內(nèi)質(zhì)網(wǎng)應(yīng)激凋亡通路引起細(xì)胞凋亡。內(nèi)質(zhì)網(wǎng)類似激酶(PKR-like endoplasmic reticulum kinase,PERK)是一種絲氨酸蘇氨酸激酶,在內(nèi)質(zhì)網(wǎng)應(yīng)激啟動(dòng)后,通過磷酸化真核生物的翻譯起始因子2α(eukaryotic translation initiation factor 2α,e IF2α),介導(dǎo)細(xì)胞的凋亡。Salubrinal可通過選擇性誘導(dǎo)e IF2α磷酸化和抑制其去磷酸化來對(duì)抗內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress,ERs)誘導(dǎo)的細(xì)胞凋亡,并且其不保護(hù)與ERs無(wú)關(guān)的細(xì)胞凋亡刺激。研究表明Salubrinal能減輕細(xì)胞的凋亡。因此,研究應(yīng)用內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)凋亡抑制劑對(duì)腦死亡肝臟損傷的影響,有利于闡明腦死亡對(duì)肝臟損傷的機(jī)制,為腦死亡下器官功能的保護(hù)奠定理論基礎(chǔ)和提供藥物靶點(diǎn)。目的:探討內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑Salubrinal對(duì)大鼠腦死亡狀態(tài)下肝損傷的影響及分子機(jī)制方法:本研究分為三個(gè)部分:第一部分:大鼠腦死亡模型制作方法的改進(jìn):在傳統(tǒng)漸進(jìn)式誘導(dǎo)腦死亡模型的基礎(chǔ)上進(jìn)行改良,采用緩慢間歇顱內(nèi)加壓法,誘導(dǎo)腦死亡的發(fā)生。通過以下標(biāo)準(zhǔn)進(jìn)行腦死亡的判定:深昏迷、腦干反射消失、自主呼吸停止及腦電圖出現(xiàn)平直。在誘導(dǎo)腦死亡發(fā)生及維持腦死亡狀態(tài)時(shí)實(shí)時(shí)監(jiān)測(cè)顱內(nèi)壓、腦電圖、心電圖、平均動(dòng)脈壓、體溫等的變化。第二部分:內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)腦死亡狀態(tài)下肝損傷的機(jī)制研究:在改良的大鼠腦死亡模型的基礎(chǔ)上,將大鼠隨機(jī)分為假手術(shù)組及腦死亡組。通過免疫組織化學(xué)及蛋白質(zhì)免疫印跡檢測(cè)假手術(shù)組及腦死亡組大鼠肝臟中內(nèi)質(zhì)網(wǎng)應(yīng)激分子標(biāo)志物(Grp78和Xbp-1)及凋亡相關(guān)蛋白(Chop和Caspase-12)的變化。另外,利用原位細(xì)胞凋亡法檢測(cè)肝細(xì)胞的凋亡及透射電鏡觀察肝細(xì)胞的微觀變化。通過熒光定量PCR檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激凋亡相關(guān)基因的m RNA表達(dá)變化。第三部分:Salubrinal對(duì)大鼠腦死亡狀態(tài)下肝損傷的作用:將SD大鼠隨機(jī)分為3組:腦死亡組;腦死亡加DMSO組;腦死亡加Salubrinal組。腦死亡加DMSO組在誘導(dǎo)腦死亡前1小時(shí)腹腔注射DMSO;腦死亡加Salubrinal組在誘導(dǎo)腦死亡前1小時(shí)腹腔注射Salubrinal。q PCR檢測(cè)大鼠肝臟中Chop及Caspase-12 m RNA的表達(dá);Western blot檢測(cè)大鼠肝臟中PERK、p-e IF2α、e IF2α、Chop及Caspase-12蛋白的表達(dá);免疫組化檢測(cè)Chop及Caspase-12在大鼠肝組織中分布和表達(dá);全自動(dòng)生化分析儀檢測(cè)ALT及AST的表達(dá);TUNEL法檢測(cè)肝細(xì)胞的凋亡。結(jié)果:第一:建立了穩(wěn)定的大鼠腦死亡模型:經(jīng)有效的呼吸循環(huán)支持均能成功誘導(dǎo)腦死亡的發(fā)生并且均能維持腦死亡狀態(tài)6小時(shí)以上,平均動(dòng)脈壓在80mm Hg以上;第二:內(nèi)質(zhì)網(wǎng)應(yīng)激可能介導(dǎo)大鼠腦死亡狀態(tài)下的肝細(xì)胞損傷:腦死亡可以導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激的標(biāo)志物Grp78和Xbp-1表達(dá)增加,以及內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)的凋亡蛋白Chop和Caspase-12表達(dá)增加。另外,通過透射電鏡發(fā)現(xiàn)肝細(xì)胞出現(xiàn)細(xì)胞凋亡的特征以及通過原位細(xì)胞凋亡法檢測(cè)發(fā)現(xiàn)肝細(xì)胞凋亡增加;第三:Salubrinal減輕大鼠腦死亡狀態(tài)下的肝損傷:腦死亡組及DMSO加腦死亡組在Chop及Caspase-12的m RNA及蛋白表達(dá)水平以及PERK、e IF2α及p-e IF2α的蛋白表達(dá)沒有統(tǒng)計(jì)學(xué)差異。Salubrinal干預(yù)后相對(duì)腦死亡組p-e IF2α表達(dá)升高,e IF2α表達(dá)沒有明顯改變而P-PERK在腦死亡2h及6h表達(dá)降低。q PCR結(jié)果顯示:Salubrinal干預(yù)后Chop的m RNA表達(dá)在腦死亡4h明顯降低,Caspase-12的m RNA表達(dá)也明顯降低。我們也通過Western blot及免疫組織化學(xué)檢測(cè)發(fā)現(xiàn):Salubrinal干預(yù)后Chop及Caspase-12的蛋白表達(dá)也明顯下降。此外,Salubrinal干預(yù)后,肝功得到改善,肝細(xì)胞的凋亡減輕。結(jié)論:經(jīng)改良的緩慢間歇顱內(nèi)加壓法建立的大鼠腦死亡模型,較傳統(tǒng)的腦死亡模型相比具有維持腦死亡狀態(tài)時(shí)間長(zhǎng)、簡(jiǎn)單易復(fù)制、易于標(biāo)準(zhǔn)化、與臨床腦死亡相似的特點(diǎn)。首次發(fā)現(xiàn)大鼠腦死亡狀態(tài)下啟動(dòng)內(nèi)質(zhì)網(wǎng)應(yīng)激,并且內(nèi)質(zhì)網(wǎng)應(yīng)激的凋亡途徑介導(dǎo)腦死亡下的肝損傷。首次發(fā)現(xiàn)Salubrinal能明顯減輕腦死亡大鼠肝細(xì)胞的凋亡,可能通過PERK/e IF2α通路來實(shí)現(xiàn)其保護(hù)作用。
[Abstract]:Liver transplantation is the most effective treatment for end-stage liver disease. The main factor restricting the development of liver transplantation is the shortage of liver donor. Brain death donor is the main source of liver transplantation. Brain death refers to the irreversible and permanent loss of whole brain function including brain stem function. Brain death is a complex disease. Physical and physiological processes are a dynamic process that directly affects organ morphology and function. The study shows that brain death donors have more complications and lower survival rates than donor donor transplantation. Therefore, brain death is considered as an independent risk factor for donor organ damage. The increase of cell apoptosis is the leading brain death donor. The main factors of liver injury. There are three ways to induce apoptosis. The endoplasmic reticulum stress pathway causes apoptosis as the third apoptotic pathway outside the mitochondrial pathway and exogenous pathway. In general, the endoplasmic reticulum protects the cells by external stimulation by the reaction of unfolded proteins, but when the stress time is prolonged or Ying Jiqiang is prolonged. PKR-like endoplasmic reticulum kinase (PERK) is a serine threonine kinase (serine reticulum kinase, PERK). After the endoplasmic reticulum should be stimulated, the translational initiation of eukaryotic phosphorylated eukaryotes is initiated. Factor 2 alpha (eukaryotic translation initiation factor 2 alpha, e IF2 a), mediated cell apoptosis.Salubrinal can be induced by selectively inducing e IF2 alpha phosphorylation and inhibiting its dephosphorylation to counter endoplasmic reticulum stress induced apoptosis, and it does not protect cell apoptosis that is irrelevant. It shows that Salubrinal can reduce cell apoptosis. Therefore, the study of the effect of endoplasmic reticulum stress induced apoptosis inhibitor on brain death in the liver injury is beneficial to elucidate the mechanism of brain death to liver injury, lay a theoretical foundation for the protection of organ function under brain death and provide drug targets. Objective: to explore the endoplasmic reticulum stress inhibitor Salubri The effect and molecular mechanism of nal on brain death in rats: This study is divided into three parts: the first part: the improvement of the method of making brain death model in rats: Based on the traditional progressive induced brain death model, a slow intermittent intracranial pressure method is used to induce the occurrence of brain death. Determination of brain death: deep coma, brain stem reflex disappearance, autonomic breathing stop and electroencephalogram straight. The changes of intracranial pressure, electroencephalogram, electrocardiogram, mean arterial pressure and body temperature are monitored in real time when inducing brain death and maintenance of brain death. The second part: the mechanism of endoplasmic reticulum stress mediated liver injury in brain death: On the basis of the modified rat brain death model, rats were randomly divided into sham operation group and brain death group. The changes of endoplasmic reticulum stress molecular markers (Grp78 and Xbp-1) and apoptosis phase Guan Danbai (Chop and Caspase-12) in sham operation group and brain death group were detected by immunohistochemistry and protein immunoblotting. In situ apoptosis assay was used to detect the apoptosis of hepatocytes and the microscopic changes of liver cells by transmission electron microscopy. The changes in M RNA expression of endoplasmic reticulum stress apoptosis related genes were detected by fluorescence quantitative PCR. The third part: the effect of Salubrinal on liver injury in the brain death of rats: the SD rats were randomly divided into 3 groups: brain death group; brain death plus DMSO Group: brain death plus Salubrinal. Brain death and group DMSO were intraperitoneally injected with DMSO 1 hours before inducing brain death; brain death and Salubrinal group were intraperitoneally injected with Salubrinal.q PCR to detect the expression of Chop and Caspase-12 m RNA in rats' liver 1 hours before the induction of brain death; Western blot was used to detect the liver in rat liver. The expression of -12 protein; immunohistochemical detection of Chop and Caspase-12 in rat liver tissue distribution and expression; full automatic biochemical analyzer to detect the expression of ALT and AST; TUNEL method to detect the apoptosis of liver cells. Results: First: a stable rat brain death model was established: the brain death could be successfully induced by effective respiratory cycle support. All of them can maintain brain death for more than 6 hours and average arterial pressure above 80mm Hg; second: endoplasmic reticulum stress may mediate hepatocyte injury in rat brain death: brain death can lead to the increase of Grp78 and Xbp-1 expression of endoplasmic reticulum stress, and the increase of apoptosis protein Chop and Caspase-12 expression related to endoplasmic reticulum stress. In addition, the apoptosis of hepatocytes was found by transmission electron microscopy and apoptosis was detected by in situ cell apoptosis. Third: Salubrinal alleviated liver injury in the brain death of rats: the m RNA and protein expression levels of the brain death group and the DMSO brain death group in Chop and Caspase-12, and PERK, e IF2 alpha and P-E. There was no statistical difference in the expression of IF2 alpha protein. The expression of P-E IF2 alpha in the relative brain death group increased after.Salubrinal intervention, and the expression of E IF2 alpha was not significantly changed, while the P-PERK in 2H and 6h expression in brain died and.Q PCR showed that the expression of E was significantly lower in brain death. We also found that the protein expression of Chop and Caspase-12 was also significantly decreased after the Salubrinal intervention by Western blot and immunohistochemistry. Furthermore, the liver function was improved and the apoptosis of hepatocytes was alleviated by the prognosis of Salubrinal. Conclusion: the rat brain death model established by the modified slow intermittent intracranial pressure method was compared with the traditional brain death model. It has a long time of maintaining brain death, simple replicating, easy to standardize and similar to clinical brain death. It is the first time that endoplasmic reticulum stress is initiated in rat brain death, and the apoptosis pathway of endoplasmic reticulum stress mediates the liver damage under brain death. The first occurrence of Salubrinal can significantly reduce the liver cells of brain death rats. Apoptosis may be protected by PERK/e IF2 alpha pathway.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R657.3

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1 曹勝利;Salubrinal減輕大鼠腦死亡狀態(tài)下肝損傷的分子機(jī)制研究[D];鄭州大學(xué);2015年

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