發(fā)布時間：2018-05-04 13:58

  本文選題：術(shù)后認(rèn)知功能障礙 + 海馬��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：背景與目的：術(shù)后認(rèn)知功能障礙(Postoperative cognitive dysfunction, POCD)是一種常見的術(shù)后并發(fā)癥。據(jù)流行病學(xué)資料,其發(fā)病率在接受心臟手術(shù)的病人中高達20-79%,在接受非心臟手術(shù)的病人中也達到了4.1-22.3%。POCD與高死亡率、住院時間延長、對醫(yī)療資源的大量消耗以及生活質(zhì)量下降等不良事件密切相關(guān)。POCD主要在老年病人中發(fā)病較多,一般認(rèn)為老齡是導(dǎo)致POCD發(fā)病最為重要的危險因素。盡管POCD的發(fā)病機制和病理生理變化尚未完全明確,但是有證據(jù)表明了該疾病與其他神經(jīng)退行性疾病的密切關(guān)系。鐵在腦內(nèi)的增齡性沉積是神經(jīng)退行性疾病的共同病理特征之一。過量的鐵可導(dǎo)致氧化應(yīng)激損傷和小膠質(zhì)細(xì)胞過度激活,從而對神經(jīng)細(xì)胞造成損傷。鐵螫合劑去鐵胺(Deferoxamine,DFO)已被用于多種腦內(nèi)鐵過載疾病和神經(jīng)退行性疾病的動物模型研究,并發(fā)現(xiàn)了其良好的應(yīng)用前景。盡管鐵沉積在神經(jīng)退行性疾病中發(fā)揮了重要作用,并且鐵螯合劑的應(yīng)用為預(yù)防和治療帶來了曙光,但是鐵沉積在POCD發(fā)生發(fā)展中的潛在機制還缺乏深入研究,DFO對POCD的作用效應(yīng)和作用機制也尚未闡明。因此,本研究擬通過動物實驗揭示海馬內(nèi)鐵沉積影響術(shù)后認(rèn)知功能的原因,探討DFO是否能改善術(shù)后認(rèn)知功能及其潛在的作用機制。方法：1.通過電感耦合等離子體質(zhì)譜(Inductively coupled plasma-mass spectrometry, ICP-MS)檢測組織內(nèi)總鐵含量。Western blot檢測ferritin表達量。以此評估成年和老年大鼠海馬鐵沉積的情況,并觀察剖腹探查術(shù)對海馬內(nèi)鐵沉積的影響。2.用Morris水迷宮(Morris water maze,MWM)評價海馬內(nèi)鐵沉積對大鼠術(shù)后認(rèn)知功能的影響。3.采用術(shù)前腹腔注射DFO的方法,觀察該藥物能否改善術(shù)后海馬鐵沉積以及對術(shù)后認(rèn)知功能是否具有保護作用。4.通過免疫組化染色(Immunohistochemical staining, IHC)和real-time PCR檢測海馬內(nèi)鐵轉(zhuǎn)運相關(guān)蛋白的表達、用試劑盒檢測氧化應(yīng)激和抗氧化應(yīng)激指標(biāo)、IHC檢測小膠質(zhì)細(xì)胞活化標(biāo)記物OX-42表達、Western blot檢測凋亡相關(guān)蛋白的表達和采用原位末端標(biāo)記法(Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, TUNEL)檢測凋亡細(xì)胞數(shù)量,以闡明術(shù)后海馬鐵沉積加重的潛在原因及DFO改善術(shù)后認(rèn)知功能的作用機制。結(jié)果：1.與成年大鼠相比,老年大鼠海馬組織總鐵含量和ferritin表達量顯著升高并在術(shù)后進一步加重。MWM測試發(fā)現(xiàn),老年大鼠術(shù)后潛伏期、游泳距離較成年大鼠明顯增加,而目標(biāo)象限停留時間百分比、中心區(qū)域停留時間百分比、平臺穿越次數(shù)較成年大鼠明顯減少。2.術(shù)前連續(xù)6天給予DFO可顯著減少成年及老年大鼠術(shù)后海馬組織總鐵含量及ferritin表達量,并且在老年大鼠中減少的程度明顯大于成年大鼠。MWM測試發(fā)現(xiàn),DFO可減少老年大鼠術(shù)后潛伏期和游泳距離,增加目標(biāo)象限停留時間百分比、中心區(qū)域停留時間百分比和平臺區(qū)域穿越次數(shù)；但是在成年大鼠中上述指標(biāo)無明顯改善。3.老年大鼠術(shù)后TfR mRNA及蛋白表達明顯減少,DMT1、hepcidin的mRNA及蛋白表達均顯著增加,Fpnl雖然mRNA表達增加但蛋白表達急劇下降。術(shù)前給予DFO后雖使TfR表達增加,但可明顯抑制DMT1和hepcidin的增加,防止Fpnl減少。4.DFO可抑制術(shù)后海馬ROS及MDA的產(chǎn)生,保護總SOD活性；顯著減少海馬OX-42的表達；抑制Caspase-3的表達及活化,抑制促凋亡蛋白Bax的表達,增加抗凋亡蛋白Bcl-2的表達并提高Bcl-2/Bax比值,還可顯著減少術(shù)后海馬TUNEL陽性細(xì)胞的數(shù)量。結(jié)論：1.大鼠海馬組織中存在增齡性鐵沉積,手術(shù)刺激可使鐵沉積加重并損害術(shù)后認(rèn)知功能。提示海馬內(nèi)鐵沉積可能與術(shù)后認(rèn)知功能下降相關(guān)。2.鐵螯合劑DFO可能有助于降低海馬內(nèi)鐵沉積,并改善老年大鼠術(shù)后認(rèn)知功能,但對成年大鼠作用不明顯。3.老年大鼠術(shù)后海馬鐵轉(zhuǎn)運蛋白表達改變使得細(xì)胞內(nèi)鐵外流減少,同時伴有細(xì)胞外的鐵內(nèi)流增加,可能是術(shù)后海馬鐵沉積增加的原因之一。4.DFO通過螫合作用和對鐵轉(zhuǎn)運相關(guān)蛋白的調(diào)控緩解了術(shù)后海馬內(nèi)鐵沉積；同時對海馬內(nèi)氧化應(yīng)激和小膠質(zhì)細(xì)胞激活的抑制,減少了細(xì)胞凋亡的發(fā)生,從而改善了術(shù)后認(rèn)知功能。5.DFO對海馬內(nèi)鐵沉積的緩解和對術(shù)后認(rèn)知功能的改善為預(yù)防和治療POCD提供了新的策略。
[Abstract]:Background and purpose: postoperative cognitive dysfunction (Postoperative cognitive dysfunction, POCD) is a common postoperative complication. According to epidemiological data, the incidence is as high as 20-79% in patients undergoing cardiac surgery. In patients receiving non cardiac surgery, the incidence of 4.1-22.3%.POCD and high mortality is also achieved, and the time of hospitalization is prolonged. The large consumption of medical resources and the decline of adverse events, such as the decline of the quality of life, are closely related to.POCD mainly in the elderly patients. It is generally believed that aging is the most important risk factor for the pathogenesis of POCD. Although the pathogenesis and pathophysiological changes of POCD are not completely clear, there is evidence that the disease and other nerves have been shown. The close relationship of degenerative disease. AGE deposition in the brain is one of the common pathological features of neurodegenerative diseases. Excessive iron can cause oxidative stress and microglia to excessively activate and cause damage to nerve cells. Iron sting mixture (Deferoxamine, DFO) has been used in a variety of brain iron overload diseases and The animal model of neurodegenerative disease has been studied and its good prospects are found. Although iron deposition plays an important role in neurodegenerative diseases, and the application of iron chelating mixture has brought dawn to prevention and treatment, the potential mechanism of iron deposition in the development of POCD is still lack of in-depth study and the effect of DFO on POCD The effects and mechanisms of action have not yet been elucidated. Therefore, this study is intended to reveal the effects of iron deposition in the hippocampus on cognitive function in the hippocampus and to explore whether DFO can improve postoperative cognitive function and its potential mechanisms. Methods: 1. through inductively coupled plasma mass spectrometry (Inductively coupled plasma-mass spectrometry, ICP) -MS) detection of total iron content in the tissue.Western blot detection of ferritin expression. In order to evaluate the iron deposition in hippocampus of adult and old rats, and the effect of exploratory laparotomy on the iron deposition in the hippocampus,.2. using Morris water maze (Morris water maze, MWM) to evaluate the effect of iron deposition on the cognitive function of rats in the hippocampus.3. before the operation. The method of intraperitoneal injection of DFO to observe whether the drug can improve the post operation hippocampal iron deposition and the protective effect of postoperative cognitive function on.4. through immunohistochemical staining (Immunohistochemical staining, IHC) and real-time PCR to detect the expression of iron transport related proteins in the hippocampus, and test the oxidative stress and antioxidant stress finger by the kit. IHC was used to detect the expression of OX-42 in microglia activation markers, Western blot was used to detect the expression of apoptosis related proteins and the number of apoptotic cells were detected by in situ terminal labeling (Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) in order to elucidate the potential cause of the aggravation of hippocampal iron deposition after operation and the improvement of the operation. Results: 1. compared with adult rats, the total iron content and ferritin expression in the hippocampus of the aged rats were significantly increased and the.MWM test was further aggravated after the operation. The incubation period of the aged rats was significantly increased in the incubation period than in the adult rats, while the percentage of the target quadrant residence time and the center area stayed in the aged rats. The total iron content and ferritin expression in hippocampus of adult and aged rats were significantly reduced by the time percentage of time compared with adult rats after.2. operation 6 days before operation, and the degree of decrease in old rats was significantly greater than that in adult rats. DFO could reduce the latency and the latency of postoperatively in old rats. The percentage of swimming distance, the percentage of stay in the target quadrant, the percentage of residence time in the central region and the number of regional crossing of the platform, but in the adult rats, the expression of TfR mRNA and protein in the.3. old rats decreased obviously, the mRNA and protein expression of DMT1, hepcidin increased significantly, and the expression of mRNA was increased in Fpnl. The expression of additive protein was sharply decreased. Although the expression of TfR increased before DFO, the increase of DMT1 and hepcidin was obviously inhibited, and Fpnl reduced.4.DFO could inhibit the production of ROS and MDA in the hippocampus, protect the total SOD activity, reduce the expression of OX-42 in hippocampus, inhibit the expression and activation of Caspase-3, and inhibit the expression of apoptotic protein Bax. Increase the expression of anti apoptotic protein Bcl-2 and increase the Bcl-2/Bax ratio, and significantly reduce the number of hippocampal TUNEL positive cells after operation. Conclusion: there is aging iron deposition in the hippocampus of 1. rats. The operation stimulation can aggravate the iron deposition and damage the postoperative cognitive function. It is suggested that the iron deposition in the hippocampus may be related to the decline of postoperative cognitive function. .2. iron chelating agent DFO may help to reduce the iron deposition in the hippocampus and improve the cognitive function of the aged rats, but the changes in the expression of iron transporter in the hippocampus of the adult rats are not obvious in the adult rats after operation, and the iron Exodus in the cells is reduced, and the increase of the iron internal flow outside the cells is also associated with the increase of the iron deposition in the hippocampus after the operation. One of the causes of.4.DFO can alleviate the iron deposition in the hippocampus after the chelation and the regulation of iron transport related proteins. Meanwhile, the inhibition of oxidative stress in the hippocampus and the inhibition of the activation of microglia reduces the occurrence of apoptosis, thus improving the remission of the postoperative cognitive function.5.DFO to the iron deposition in the hippocampus and the modification of postoperative cognitive function. It provides a new strategy for the prevention and treatment of POCD.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R614

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本文編號：1843225