本文選題：急性腎損傷 + Corin��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：研究目的與意義急性腎損傷(acute kidney injury,AKI)是一種嚴重的臨床綜合征,具有很高的發(fā)病率和死亡率。引發(fā)AKI的因素有很多,.比如腎低灌注、腎毒性藥物、外科手術(shù)以及敗血癥等等。而在許多臨床手術(shù)中腎缺血再灌注損傷是導(dǎo)致急性腎功能衰竭的主要風(fēng)險因素,比如腎臟移植、主動脈的心臟搭橋手術(shù)以及心肺的外科手術(shù)等。據(jù)有關(guān)文獻報道我國住院人群中20%患有急性腎損傷,而ICU病房中急性腎損傷的患病率高達40%-60%,但臨床上目前并沒有有效的治療方法,因此開發(fā)新型的預(yù)防和治療急性腎損傷的策略迫在眉睫。Corin是一種新近發(fā)現(xiàn)的Ⅱ型跨膜絲氨酸蛋白酶,其結(jié)構(gòu)是由一個短的氨基末端胞漿區(qū)、一個跨膜區(qū)和胞外區(qū)組成,胞外區(qū)含有兩個卷曲結(jié)構(gòu)域、八個低密度脂蛋白受體結(jié)構(gòu)域、一個清道夫受體結(jié)構(gòu)域和一個胰蛋白酶樣的蛋白酶結(jié)構(gòu)域,其主要表達于心臟,主要功能是通過將心鈉肽前體切割活化成有活性的ANP,進而調(diào)節(jié)血壓和心臟功能。但近期發(fā)現(xiàn)Corin也表達于腎臟的腎小管中,那么Corin在腎小管病變的急性腎損傷中是否起到作用,我們無從得知。有關(guān)實驗表明,基因變異和功能障礙導(dǎo)致Corin的活性降低,可能與一系列心血管疾病如心肌肥厚、高血壓的發(fā)病有關(guān),所以Corin在腎缺血再灌注損傷中的作用更值得我們?nèi)ヌ接懞脱芯�。研究方�? Corin在腎缺血再灌注損傷模型中的表達變化1.1腎缺血再灌注損傷小鼠模型的構(gòu)建及檢測Corin的表達情況選取9周齡的野生型(wild type,WT)C57BL/6小鼠,用戊巴比妥鈉麻醉小鼠后,沿中線切開腹腔,用動脈夾夾住雙側(cè)腎蒂45分鐘,45分鐘后去掉動脈夾,在雙腎上滴上37℃生理鹽水,觀察灌注情況,確保血液復(fù)流,然后開始計時,分別在24h、48h、72h時處死小鼠取材。分別用蛋白質(zhì)免疫印跡法(Western Blot,WB)、Real-time RT-PCR以及免疫組織化學(xué)法(immunohistochemistry,IHC)檢測Corin在腎缺血再灌注損傷模型小鼠腎臟組織中的表達變化情況。1.2 Corin在多種腎小管中的表達情況 采用組織免疫熒光(immunofluoresce,IF)的方法同時對Corin以及腎小管不同部位標記物蛋白進行熒光雙染共定位。1.3體外細胞模型構(gòu)建及檢測Corin表達情況體外培養(yǎng)人的腎小管上皮細胞(HK-2),采用低氧培養(yǎng)箱培育1.5小時后轉(zhuǎn)為正常培養(yǎng)的方法,體外模擬腎缺血再灌注模型。采用Western Blot和Real-time RT-PCR的方法檢測Corin在HK-2細胞中的變化情況。2 Corin在小鼠腎缺血再灌注損傷中的作用2.1野生型小鼠與Corin-/-小鼠腎臟功能學(xué)指標對比下腔靜脈取血,檢測血清中肌酐尿素氮的含量。2.2野生型小鼠與Corin-/-小鼠腎臟形態(tài)學(xué)損傷對比用蘇木素-伊紅染色的方法(hematoxylin-eosin staining,HE)對野生型小鼠和Corin-/-小鼠腎臟石蠟切片進行染色,然后進行形態(tài)學(xué)損傷評分。用TUNEL對野生型和Corin-/-小鼠腎臟組織石蠟切片就行熒光染色,檢測腎臟細胞凋亡情況。2.3檢測野生型小鼠和Corin-/-小鼠腎缺血再灌注模型中炎癥相關(guān)指標的情況用免疫組化的方法對野生型小鼠和Corin-/-小鼠腎臟組織石蠟切片染色,Ly6B標記中性粒細胞、CD68標記巨噬細胞。2.4體外培養(yǎng)細胞轉(zhuǎn)染Corin過表達質(zhì)粒對低氧低糖培養(yǎng)HK-2細胞凋亡及炎癥的影響情況用Real-time RT-PCR的方法檢測過表達Corin后HK-2細胞在低氧低糖條件下炎癥因子的變化情況。用流式細胞技術(shù)檢測過表達Corin后HK-2細胞在低氧低糖條件下凋亡的變化情況。3 Corin機制的初步探討用Western Blot的方法,檢測腎臟勻漿中β-catenin的表達變化。研究結(jié)果1 Corin在腎缺血再灌注模型中的表達變化1.1 Corin在腎缺血再灌注小鼠模型中的表達水平顯著降低Real-time RT-PCR和Western Blot檢測小鼠腎臟中Corin的表達,結(jié)果表明在蛋白和mRNA水平模型組中Corin的表達明顯比假手術(shù)中降低。免疫組化結(jié)果也印證了 Corin的表達在腎臟組織中是顯著降低的。1.2 Corin主要表達于腎臟的近曲小管和遠曲小管中免疫熒光結(jié)果表明,Corin主要表達于腎臟的近曲小管和遠曲小管中,髓質(zhì)的集合管中表達較少。1.3體外模擬條件下Corin的表達含量顯著降低用Real-time RT-PCR和Western Blot檢測低氧低糖處理的HK-2細胞中Corin的表達顯著降低。2 Corin在腎缺血再灌注損傷中起到保護作用2.1 Corin的缺失加重了腎缺血再灌注的損傷 Corin的缺失導(dǎo)致血清中的肌酐尿素氮顯著升高、腎臟的損傷更重、中性粒細胞和巨噬細胞的浸潤明顯增加、細胞凋亡數(shù)明顯增多。2.2過表達Corin后低氧低糖條件下的HK-2凋亡減少、炎癥反應(yīng)降低HK-2細胞過表達Corin后,在低氧低糖的條件下用細胞流式技術(shù)檢測細胞的凋亡數(shù)明顯減少,用Real-time PT-PCR檢測細胞炎癥反應(yīng)明顯降低。3 Corin機制的初步探討腎缺血再灌損傷后激活Wnt/β-catenin信號通路,使β-catenin的表達升高,Corin的缺失導(dǎo)致β-catenin的表達更高。研究結(jié)論與創(chuàng)新性本研究首次表征Corin在腎臟疾病中的變化,發(fā)現(xiàn)在腎缺血再灌注損傷中Corin的表達是顯著降低的,而且Corin的缺失導(dǎo)致腎缺血再灌注損傷加重。本研究為設(shè)計和開發(fā)Corin相關(guān)蛋白質(zhì)藥物并用于AKI的防治提供了實驗基礎(chǔ)和理論依據(jù)。
[Abstract]:The purpose and significance of acute kidney injury (AKI) is a severe clinical syndrome with high morbidity and mortality. There are many factors causing AKI, such as renal hypoperfusion, nephrotoxic drugs, surgery and sepsis, etc. and renal ischemia reperfusion injury is the cause of acute renal injury in a number of clinical operations. Major risk factors for failure, such as kidney transplantation, aortic bypass surgery, and cardiopulmonary surgery, are reported to be 20% of our hospitalized patients with acute renal injury, while the incidence of acute renal injury in the ICU ward is up to 40%-60%, but there is no effective treatment at present. The new strategy for the prevention and treatment of acute renal injury is an imminent.Corin, a newly discovered type II transmembrane serine protease, which consists of a short amino terminal cytoplasmic region, a transmembrane region and an extracellular domain, with two convoluted domains, eight low density lipoprotein receptor domains, and a scavenger. A domain of body structure and a trypsin like protease domain that is mainly expressed in the heart. The main function is to activate the active ANP by cutting the precursor of the heart natriuretic peptide to regulate blood pressure and cardiac function. But the recent discovery of Corin is also expressed in the renal tubules of the kidney, and whether Corin is in the acute renal injury of renal tubular lesions. We do not know that the effect of gene mutation and dysfunction leads to lower activity of Corin, which may be related to a series of cardiovascular diseases such as myocardial hypertrophy and hypertension, so the role of Corin in renal ischemia-reperfusion injury is more worthy of our study and study. Method 1 Corin in renal ischemia and reperfusion. The construction of 1.1 renal ischemia reperfusion injury mice model in the perfusion injury model and the construction of the mice model of renal ischemia reperfusion injury and the detection of the expression of Corin, the 9 weeks old wild type (wild type, WT) C57BL/6 mice were selected. After the pentobarbital sodium was anaesthetized in mice, the abdominal cavity was cut along the middle line, the double kidney pedicle was sandwiched with the artery clamp for 45 minutes, and the artery clips were removed after 45 minutes, and the double kidneys were removed after 45 minutes. At 37 degrees of physiological saline, the blood was observed to ensure the reflow of blood, and then the time was started, and the mice were killed at 24h, 48h and 72h, respectively. The protein immunoblotting (Western Blot, WB), Real-time RT-PCR and immunohistochemistry (immunohistochemistry, IHC) were used to detect the kidney of renal ischemia reperfusion injury model mice kidney respectively. Expression changes in dirty tissue.1.2 Corin expression in various renal tubules by using tissue immunofluorescence (Immunofluoresce, IF) method for the simultaneous localization of Corin and the protein of different parts of renal tubules by fluorescence double staining co localization.1.3 in vitro cell model and detection of Corin expression in human renal tubules in vitro Epithelial cells (HK-2), using hypoxia incubator for 1.5 hours to turn to normal culture and simulate renal ischemia reperfusion model in vitro. The changes of Corin in HK-2 cells were detected by Western Blot and Real-time RT-PCR. The effect of.2 Corin on renal ischemia-reperfusion injury in mice 2.1 wild type and Corin-/- mice The renal functional indexes compared with the inferior vena cava, the serum creatinine urea nitrogen content was detected in.2.2 wild type mice and Corin-/- mice. The renal paraffin section of wild type and Corin-/- mice was stained by hematoxylin-eosin staining (HE), and then the morphology of the kidney was stained in the wild type and Corin-/- mice. The injury score. Using TUNEL to stain the paraffin section of the kidney tissue of the wild and Corin-/- mice, the apoptosis of the renal cells was detected by.2.3, and the inflammatory related indexes in the renal ischemia reperfusion model of the wild type and Corin-/- mice were detected by the immunohistochemical method to the kidney tissue of the wild and Corin-/- mice. Slice staining, Ly6B labeled neutrophils, CD68 labeled macrophage.2.4 in vitro culture cells transfected with Corin overexpressed plasmid on the apoptosis and inflammation of HK-2 cells in low oxygen and low sugar culture, the changes of the inflammatory factors under the hypoxia and low sugar strip were detected by Real-time RT-PCR method. Flow cytometry was used. Changes in apoptosis of HK-2 cells after expression of Corin in low oxygen and low glucose conditions.3 Corin mechanism: a preliminary study of the changes in the expression of beta -catenin in renal homogenate by Western Blot. The results of the 1 Corin expression in the renal ischemia-reperfusion model were 1.1 Corin in the mouse model of renal ischemia reperfusion The expression level was significantly reduced by Real-time RT-PCR and Western Blot to detect the expression of Corin in the kidney of mice. The results showed that the expression of Corin in the protein and mRNA level group was significantly lower than that in the sham operation. The immunohistochemical results also confirmed that the.1.2 Corin of Corin expression in the renal tissue was significantly lower than that of the kidney. The results of immunofluorescence in the canaliculus and distal convoluted tubules showed that Corin was mainly expressed in the proximal tubules and distal tubules of the kidney. The expression of Corin in the medullary collection tube was less.1.3 in vitro, and the expression of Corin was significantly reduced by the Real-time RT-PCR and Western Blot in the HK-2 cells treated with low oxygen and low sugar treatment. The protective effect of low.2 Corin on renal ischemia reperfusion injury 2.1 Corin loss aggravates the impairment of renal ischemia reperfusion injury Corin loss in serum creatinine urea nitrogen significantly increased, renal damage is more serious, neutrophils and macrophage infiltration increased significantly, the number of cell apoptosis increased significantly after the.2.2 overexpression of Corin low The apoptosis of HK-2 under oxygen low sugar decreased, and the inflammatory response reduced the overexpression of Corin in HK-2 cells. The number of apoptotic cells was significantly reduced by flow cytometry in low oxygen and low glucose conditions. The mechanism of.3 Corin was obviously reduced by Real-time PT-PCR, and the primary discussion on the activation of Wnt/ beta -catenin after renal ischemia-reperfusion injury was preliminarily discussed. The signal pathway increased the expression of beta -catenin, and the deletion of Corin led to the higher expression of beta -catenin. The findings and innovative studies were the first to characterize the changes in Corin in renal diseases. It was found that the expression of Corin was significantly reduced during renal ischemia-reperfusion injury, and the absence of Corin resulted in the aggravation of renal ischemia reperfusion injury. The study provides experimental basis and theoretical basis for designing and developing Corin related protein drugs and for the prevention and treatment of AKI.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692

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9 李U，

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