本文選題：Caspase-12 + NF-kB　； 參考：《廣西醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：在心臟外科的研究中,心肌保護一直是研究的熱點,心肌缺血再灌注損傷(MIRI)是在術(shù)中導(dǎo)致心肌細胞損傷的最主要因素,也是主要的研究方向。MIRI是一個多因素的病理生理過程,目前已知的通路主要有鈣離子超載、線粒體途徑、微血管創(chuàng)傷、炎癥反應(yīng)以及脂類物質(zhì)超氧化等機制,在此研究之上,近年來發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)可以影響細胞凋亡及組織的炎癥反應(yīng)并且在MIRI中發(fā)揮重要作用。在ERS中內(nèi)質(zhì)網(wǎng)通過激活未折疊蛋白反應(yīng)(UPR)以保護由ERS所引起的細胞損傷,恢復(fù)細胞功能。葡萄糖調(diào)節(jié)蛋白78(GRP78)可以促進內(nèi)質(zhì)網(wǎng)內(nèi)蛋白折疊、恢復(fù)內(nèi)質(zhì)網(wǎng)的功能,起到保護細胞的作用,同時也是內(nèi)質(zhì)網(wǎng)應(yīng)激啟動的信號,與介導(dǎo)凋亡途徑的跨膜蛋白結(jié)合,使其處于失活狀態(tài)。Caspase-12位于內(nèi)質(zhì)網(wǎng)胞漿面,該物質(zhì)是內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)凋亡的一種特異性介質(zhì),僅僅存在于內(nèi)質(zhì)網(wǎng)應(yīng)激中,并介通過激活Caspase-3介導(dǎo)凋亡。核因子-κB(NF-κB)是一個多向性核轉(zhuǎn)錄調(diào)節(jié)因子,在多種急性應(yīng)激反應(yīng)中表達,目前被認為是內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的炎癥反應(yīng)的關(guān)鍵因子,關(guān)于其的損傷心肌作用和心肌保護作用均有報道,上述三種蛋白及調(diào)控因子具體作用機制復(fù)雜且先前的研究多在細胞層面進行,關(guān)于動物實驗亦或人體中的實驗?zāi)壳吧写M一步研究。對于減少術(shù)中心肌損傷,體外循環(huán)下心臟不停跳手術(shù)有獨到的優(yōu)勢,其術(shù)式在90年代末開始開展應(yīng)用,并逐漸發(fā)展,已有了長足的進步,關(guān)于該術(shù)式的研究也充分證明了其有良好的預(yù)防MIRI作用,可以有效的進行腦、肺、腎臟等多臟器的保護。但關(guān)于其分子層面的研究目前尚少。本實驗建立體外循環(huán)的模型,探索心臟停跳及不停跳手術(shù)的情況下,ERS的反應(yīng)強度及細胞凋亡程度,探索ERS以及其中的三種調(diào)節(jié)因子在不同手術(shù)方式下的表達情況,并對比手術(shù)的優(yōu)缺點。方法：動物由廣西醫(yī)科大學(xué)動物實驗中心提供,為成年新西蘭大白兔共36只,將實驗動物隨機分為停跳組(體外循環(huán)心臟停跳組,n=12),不停跳組(體外循環(huán)心臟不停跳組,n=12)以及對照組(假手術(shù)組)。建立兔體外循環(huán)模型。假手術(shù)組僅作開關(guān)胸手術(shù),不做其他處理。不停跳組體外通過頸總動脈作為動脈灌注,頸總靜脈作為靜脈引流,進而建立循環(huán),維持體外循環(huán)約120min,整個實驗過程中不進行停跳液的灌注、不阻斷升主動脈及上下腔靜脈、冠狀動脈維持血流灌注。停跳組建立體外循環(huán)后,并行循環(huán)約15min,將實驗動物溫度降低至28℃～30℃時將主動脈阻斷,并從主動脈根部灌注St. Thomas Ⅱ停搏液(15ml/kg,約4℃),總共灌注3次St. Thomas Ⅱ停搏液(后兩次分別于30min和60min分別進行,灌注量為第一次的一半)。灌注完畢后緩慢復(fù)溫,于實驗動物處于34℃時基本恢復(fù)全流量,最后使心臟復(fù)跳并行體外循環(huán)。實驗動物生命體征平穩(wěn)后停止體外循環(huán)操作。實驗成功的標志為實驗動物在停止體外循環(huán)1個小時后仍存活,處死實驗動物,進行心肌標本的采集。根據(jù)免疫組化試劑盒的步驟分別對三組實驗動物心肌進行GRP78、Caspase-12以及NF-κB的表達檢測,并對三組動物的心肌行TUNEL法檢測凋亡率。結(jié)果：(1)GRP78的表達量：停跳組(72.24%±7.89%),不停跳組(16.53%±2.59%),假手術(shù)組(1.68%±1.19%),組間差別有明顯統(tǒng)計學(xué)意義(P0.01)。(2) NF-κB的表達量：停跳組(73.70%±6.70%),不停跳組(13.81%±1.79%),假手術(shù)組(2.42%±1.42%),組間差別有明顯統(tǒng)計學(xué)意義(P0.01)(3)Caspase-12的表達量：停跳組(73.74%±7.15%),不停跳組(14.49%±2.29%),假手術(shù)組(2.46%±1.56%),組間差別有明顯統(tǒng)計學(xué)意義(P0.01)(4)心肌細胞凋亡指數(shù)(AI)：心臟停跳組凋亡率為(10.11±0.53),心臟不停跳組(4.27±0.61),對照組為(1.33±0.35)(P0.01)。組間差別有統(tǒng)計學(xué)意義(P0.01)(5)相關(guān)性分析：心臟停跳組以及心臟不停跳組心肌細胞凋亡率與該組caspase-12表達量相關(guān)系數(shù)分別為為0.961(P0.01)以及0.912(P0.01),呈明顯的正相關(guān)；與該組GRP78表達量相關(guān)系數(shù)分別為0.827(P0.01)以及0.831(P0.01),呈正相關(guān),與該組NF-κB表達量相關(guān)系數(shù)分別為0.317(P0.05)以及0.281(P0.05),無明顯相關(guān)性；結(jié)論：(1)GRP78在心臟停跳及不停跳組中都明顯升高,說明內(nèi)質(zhì)網(wǎng)應(yīng)激參與了體外循環(huán)中細胞的損傷。(2)Caspase-12在兩個實驗組中明顯升高說明了體外循環(huán)中心肌細胞損傷明顯有心肌凋亡參與(3) NF-κB在兩組中表達明顯上調(diào)說明NF-κB通路參與了體外循環(huán)下的心肌內(nèi)質(zhì)網(wǎng)應(yīng)激及相關(guān)炎癥反應(yīng),其與心肌凋亡率相關(guān)性不明顯可以佐證其具有抗凋亡和促雙重作用。(4)心臟不停跳手術(shù)可以減少內(nèi)質(zhì)網(wǎng)應(yīng)激,減少心肌損傷,保護心功能。
[Abstract]:Objective: in the study of cardiac surgery, myocardial protection has always been a hot spot of research. Myocardial ischemia reperfusion injury (MIRI) is the most important factor in the injury of cardiac myocytes in the operation, and the main research direction.MIRI is a multi factor pathophysiological process. The main known pathways are calcium overload, mitochondrial pathway, and microsatellite pathway at present. In recent years, endoplasmic reticulum stress (ERS) can affect apoptosis and tissue inflammation and play an important role in MIRI. In ERS, endoplasmic reticulum (endoplasmic reticulum) can protect cell damage caused by ERS by activating unfolded protein reaction (UPR). The function of glucose regulating protein 78 (GRP78) can promote the internal protein folding in the endoplasmic reticulum, restore the function of endoplasmic reticulum, play a role in protecting the cells, and also signal the initiation of endoplasmic reticulum stress, combined with the transmembrane protein which mediates the apoptosis pathway, and make it inactive.Caspase-12 in the endoplasmic reticulum, and the substance is within. The mesoplasmic reticulum stress mediated apoptosis is a specific medium that only exists in endoplasmic reticulum stress and mediates apoptosis through activation of Caspase-3. Nuclear factor kappa B (NF- kappa B) is a multidirectional nuclear regulatory factor, expressed in a variety of acute stress responses, and is currently considered to be a key factor in the inflammatory response mediated by endoplasmic reticulum stress. There are reports of myocardial injury and myocardial protection. The specific mechanisms of these three proteins and regulatory factors are complex and previous studies are mostly at the cellular level. Animal experiments and human experiments are still to be further studied. For the reduction of cardiac muscle injury, cardiac arrest under cardiopulmonary bypass is performed. The unique advantage, its operation began to carry out the application in the late 90s, and gradually developed, has made great progress. The study of this type of operation has also fully proved that it has good preventive effect on MIRI and can effectively protect many organs such as brain, lung, kidney and so on. However, there are few studies on its molecular level. This experiment is established in vitro. To explore the reaction intensity and the degree of apoptosis of ERS, explore the expression of ERS and the three regulatory factors under different surgical methods, and compare the advantages and disadvantages of the operation. Methods: the animals were provided by the animal experiment center of Guangxi Medical University for adult New Zealand. A total of 36 white rabbits were randomly divided into stop jump group (cardiopulmonary bypass cardiac arrest group, n=12), non stop jump group (extracorporeal circulation heart beating group, n=12) and control group (sham operation group). The rabbit extracorporeal circulation model was established. The sham operation group was only operated on the switch chest operation and did not do other treatment. As a venous drainage, the total neck vein was used as a venous drainage, and the circulation was established to maintain an external circulation of about 120min. During the whole experiment, the blood perfusion was not carried out, the ascending aorta and the superior and inferior vena cava were not blocked. The coronary artery was maintained by the coronary artery. After the suspension of the external cycle, the parallel circulation was about 15min, and the temperature of the experimental animals was reduced to 28 to 30. The aorta was blocked, and the St. Thomas II cardioplegia (15ml/kg, about 4 C) was perfused from the root of the aorta, and 3 St. Thomas II cardioplegia was perfused (two times respectively in 30min and 60min, respectively, half of the first time respectively). After the perfusion was completed, the temperature was slowly rewarming, and the whole flow was basically recovered at 34 C, and the heart was finally made to the heart. The sign of the experiment was that the experimental animals were still alive after 1 hours of cessation of cardiopulmonary bypass. The experimental animals were killed and the myocardial specimens were collected. The GRP78 of the three groups of experimental animals was carried out according to the steps of the immunohistochemical kits, Caspa The expression of se-12 and NF- kappa B was detected, and TUNEL method was used to detect the apoptosis rate of three groups of animals. Results: (1) the expression of GRP78: (72.24% + 7.89%), non stop group (16.53% + 2.59%), sham operation group (1.68% + 1.19%), and there was significant difference between groups (P0.01). (2) the expression of NF- kappa B (73.70% + 6.70%), ceaselessly Jump group (13.81% + 1.79%), sham operation group (2.42% + 1.42%), there was significant difference between groups (P0.01) (3) Caspase-12 expression: stop jump group (73.74% + 7.15%), non stop jump group (14.49% + 2.29%), sham operation group (2.46% + 1.56%), there was significant difference between groups (P0.01) (4) myocardial apoptosis index (AI): cardiac arrest group apoptosis The rate was (10.11 + 0.53), the heart beating group was (4.27 + 0.61) and the control group was (1.33 + 0.35) (P0.01). The difference between the groups was statistically significant (P0.01) (5) correlation analysis: the myocardial apoptosis rate of cardiac arrest group and heart beating group was 0.961 (P0.01) and 0.912 (P0.01), respectively, and was obviously positive. Correlation: the correlation coefficient of GRP78 expression was 0.827 (P0.01) and 0.831 (P0.01), respectively, and the correlation coefficient of NF- kappa B expression was 0.317 (P0.05) and 0.281 (P0.05), respectively. Conclusion: (1) GRP78 was significantly increased in the cardiac arrest and non stop jump groups, indicating that endoplasmic reticulum stress was involved in the in vitro development. (2) the significant increase of Caspase-12 in the two experimental groups indicated that myocyte injury in cardiopulmonary bypass was significantly associated with myocardial apoptosis (3) the expression of NF- kappa B in the two groups indicated that NF- kappa B pathway was involved in cardiac endoplasmic reticulum stress and related inflammatory responses under cardiopulmonary bypass, which was not related to the rate of myocardial apoptosis. (4) beating heart surgery can reduce endoplasmic reticulum stress, reduce myocardial injury and protect heart function.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R654.2

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