【摘要】：目的:腎缺血再灌注損傷（ischemia reperfusion injury, IRI）是急性腎損傷(acute kidney injury,AKI)重要的組織病理學(xué)機(jī)制。由IRI引起的AKI是一個重要的臨床問題，盡管AKI的治療方法不斷改進(jìn)，但是腎IRI仍與AKI的發(fā)病率和死亡率密切相關(guān)。腎IRI的發(fā)生機(jī)制很復(fù)雜，且并未完全闡明，與氧自由基、內(nèi)皮功能紊亂、炎癥損傷、細(xì)胞壞死和凋亡等多方面均有關(guān)。已證實(shí)氧化應(yīng)激產(chǎn)生的活性氧(reactive oxygenspecies, ROS)在腎IRI中發(fā)揮了重要作用。在正常腎臟組織，基質(zhì)蛋白血小板反應(yīng)蛋白-1(thrombospondin-1, TSP-1)表達(dá)量很少。但在缺氧條件下，多種細(xì)胞可以合成TSP-1。有研究表明，TSP-1與AKI的發(fā)生和發(fā)展有關(guān)，同樣也與慢性腎臟病的進(jìn)展關(guān)系密切。因此，人們越來越多的關(guān)注TSP-1在急慢性腎臟病變中的地位。CD47又被稱為整合素相關(guān)蛋白(integrin-associated protein, IAP)，是TSP-1的同源受體之一，它在腎臟廣泛表達(dá)，但它在腎實(shí)質(zhì)和間質(zhì)細(xì)胞促進(jìn)腎損傷的機(jī)制還不清楚。因此，本研究通過構(gòu)建腎IRI大鼠模型，觀察TSP-1及其受體CD47在IRI大鼠腎臟的分布情況及表達(dá)變化，以及血ROS的變化和腎組織氧化應(yīng)激指標(biāo)的變化，研究TSP-1、CD47在腎IRI時的表達(dá)及分布，以及與氧化應(yīng)激的關(guān)系，探討其在腎IRI發(fā)病機(jī)制中的作用。 方法:成年雄性SD大鼠42只，隨機(jī)分為正常對照組（n=5）、假手術(shù)組(sham group, SG)（n=5）和模型組（n=32），模型組下設(shè)IRI后1h、6h、12h、24h4個時間點(diǎn)，每個時間點(diǎn)8只大鼠。手術(shù)方式:將大鼠麻醉后，逐層開腹暴露雙腎，用玻璃分針分離雙側(cè)腎動脈，將已經(jīng)消毒的壓脈帶片段包裹在腎動脈外層，將手術(shù)線系在壓脈帶片段外迅速打結(jié)阻斷腎血流，觀察腎臟顏色的變化。阻斷腎血流45min后，剪斷手術(shù)線，恢復(fù)腎臟供血，觀察腎臟顏色變化。逐層關(guān)腹，復(fù)蘇。觀察IRI后各組大鼠尿量變化，采用全自動生化分析儀檢測各組大鼠的腎功能，通過蘇木精-伊紅染色法(hematoxylin-eosin staining, HE)、糖原染色(periodicacid-schiff stain, PAS)了解腎臟病理損害，通過透射電鏡觀察足細(xì)胞有無損傷、系膜基質(zhì)是否增生。采用流式細(xì)胞儀檢測血ROS變化，采用酶聯(lián)免疫吸附法測定腎組織中羥自由基和丙二醛的濃度，了解腎IRI損傷時的氧化應(yīng)激狀態(tài)。通過腎組織免疫組化，觀察TSP-1和CD47蛋白在腎臟的表達(dá)及變化情況，通過免疫熒光雙染了解TSP-1及其受體CD47在腎IRI時是否存在共表達(dá)。 結(jié)果:與正常對照組和假手術(shù)組相比，IRI各組大鼠尿量明顯減少；腎IRI各組血肌酐（serum creatinine, Scr）和尿素氮（urea nitrogen, BUN）明顯升高，差異具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；隨著缺血再灌注后時間的延長，，腎小管出現(xiàn)不同程度的擴(kuò)張、變性與壞死，間質(zhì)炎性細(xì)胞浸潤、充血水腫等變化。各組間腎小管HE染色病理損傷評分差異顯著，差異具有統(tǒng)計(jì)學(xué)意義（P＜0.05）；通過透射電鏡觀察發(fā)現(xiàn)腎IRI各組大鼠腎小球足細(xì)胞足突消失、系膜基質(zhì)增生明顯，足細(xì)胞計(jì)數(shù)差異具有統(tǒng)計(jì)學(xué)意義（P＜0.05），而腎IRI各組間差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）；紅細(xì)胞中ROS熒光強(qiáng)度陽性率逐漸升高，腎組織中的羥自由基和丙二醛的濃度呈逐漸升高趨勢。在腎IRI早期，腎小管TSP-1、CD47表達(dá)即明顯增加，至腎IRI24h時TSP-1、CD47在殘存腎小管仍有較高表達(dá)。通過免疫熒光雙染發(fā)現(xiàn)，在腎IRI腎小管TSP-1和CD47存在共表達(dá)。相關(guān)性分析發(fā)現(xiàn)ROS熒光強(qiáng)度陽性率與羥自由基和丙二醛的濃度呈正相關(guān)，與Scr、BUN、腎小管損傷評分、TSP-1積分光密度、CD47積分光密度呈正相關(guān)，與足細(xì)胞計(jì)數(shù)呈負(fù)相關(guān)。 結(jié)論:通過簡易手術(shù)阻斷雙側(cè)腎動脈血流，可以成功建立大鼠腎IRI模型。腎IRI后引起腎組織和全身的氧化應(yīng)激反應(yīng)，引起腎臟組織結(jié)構(gòu)損害，足細(xì)胞損傷，腎功能異常。腎IRI時大鼠腎臟TSP-1和CD47表達(dá)明顯升高，與腎組織和全身的氧化應(yīng)激反應(yīng)密切相關(guān)。TSP-1、CD47是腎小管損傷早期的一個預(yù)警指標(biāo)，并伴隨腎IRI整個過程持續(xù)表達(dá)，與ROS共同參與了腎IRI腎臟損傷過程。
[Abstract]:Objective: The renal ischemia-reperfusion injury (IRI) is an important pathological mechanism of acute kidney injury (AKI). AKI induced by IRI was an important clinical problem, although the treatment of AKI was continuously improved, but the renal IRI was still closely related to the incidence and mortality of AKI. The mechanism of renal IRI is complicated and is not fully set forth, and it is related to oxygen free radical, endothelial dysfunction, inflammatory injury, cell necrosis and apoptosis. Reactive oxygen species (ROS), which have been shown to be responsible for oxidative stress, play an important role in the renal IRI. In normal kidney, the expression of thrombospondin-1 (TSP-1) was very low. However, under the condition of hypoxia, a plurality of cells can synthesize the TSP-1. The research shows that TSP-1 is related to the development and development of AKI, and is also closely related to the development of chronic kidney disease. Therefore, more and more attention is paid to the position of TSP-1 in acute and chronic kidney diseases. CD47, which is also known as integrin-associated protein (IAP), is one of the homologous receptors of TSP-1, which is widely expressed in the kidney, but it is not clear in the mechanism of renal parenchymal and interstitial cells to promote renal injury. Therefore, the expression and distribution of TSP-1 and CD47 in the renal IRI and the relationship with oxidative stress were studied by constructing the renal IRI rat model, observing the distribution and expression of the TSP-1 and its receptor CD47 in the kidney of the IRI rat, and the change of the blood ROS and the change of the oxidative stress index of the renal tissue. To explore its role in the pathogenesis of renal IRI. Methods: 42 adult male SD rats were randomly divided into normal control group (n = 5), sham group (SG) (n = 5) and model group (n = 32). Rats. Methods of operation: After the rats were anesthetized, the double-side renal artery was exposed by laparotomy layer by layer, the double-side renal artery was separated by a glass separation needle, and the sterilized pressure pulse band was wrapped in the outer layer of the renal artery, and the operation line was quickly knotted off the renal blood flow outside the section of the pressure pulse band, and the change of the color of the kidney was observed. After the renal blood flow was blocked for 45min, the operation line was cut, the kidney was recovered for blood, and the color of the kidney was observed. To close the abdomen layer by layer, double The changes of urine volume in all groups after IRI were observed. The renal function of each group was detected by a full-automatic biochemical analyzer, and the renal pathological lesions were obtained by hematoxylin-eosin staining (HE) and glycogen staining (PAS). injury, whether the film matrix is increased or not The changes of the blood ROS were detected by flow cytometry. The concentration of hydroxyl radical and malondialdehyde (MDA) in the renal tissue was determined by enzyme-linked immunosorbent assay, and the oxidative stress in the renal IRI injury was found. State. The expression and change of TSP-1 and CD47 protein in the kidney were observed by immunohistochemistry. The presence of TSP-1 and its receptor CD47 in the renal IRI was determined by immunofluorescent double staining. Results: Compared with the normal control group and the sham-operation group, the urine volume of the rats in the IRI group was significantly decreased, and there was a significant difference between the blood myostatin (Scr) and the urea nitrogen (urea nitrogen, BUN) in the renal IRI group (P <0.05), and with the time of the ischemia-reperfusion, The extension of the renal tubular has different degree of expansion, degeneration and necrosis, interstitial inflammatory cell infiltration, hyperemia and edema. The results showed that there was a significant difference in the scores of renal tubular HE staining in each group, and the difference was statistically significant (P <0.05). There was no significant difference between the two groups (P <0.05). The positive rate of ROS fluorescence intensity in the red blood cell was gradually increased, and the concentration of hydroxyl radical and malondialdehyde in the renal tissue gradually increased. The expression of TSP-1 and CD47 in renal tubular TSP-1 was significantly increased in the early stage of renal IRI. High expression. The presence of TSP-1 and CD47 in the renal IRI tubules was found by immunofluorescent double staining. The correlation analysis showed that the positive rate of ROS fluorescence intensity was positively correlated with the concentration of hydroxyl radical and malondialdehyde (MDA), and correlated with the optical density of Scr, BUN, renal tubular injury, TSP-1 integral optical density and CD47 integral optical density. Conclusion: The blood flow of the double-side renal artery can be blocked by simple operation, and the kidney of the rat can be successfully established. IRI model. The renal tissue and the whole body oxidative stress reaction after the renal IRI cause damage to the structure of the kidney, damage of the foot cells, Renal function was abnormal. The expression of TSP-1 and CD47 in the kidney of rats with renal IRI increased significantly, and it was contrary to the oxidative stress of renal tissue and whole body. It should be closely related. The TSP-1 and CD47 are an early warning index in the early stage of renal tubular injury.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692.5

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