Corin在急性肾损伤中的作用

发布时间：2018-05-21 02:23

本文选题：急性肾损伤 + Corin　；参考：《山东大学》2017年硕士论文

【摘要】：研究目的与意义急性肾损伤(acute kidney injury,AKI)是一种严重的临床综合征,具有很高的发病率和死亡率。引发AKI的因素有很多,.比如肾低灌注、肾毒性药物、外科手术以及败血症等等。而在许多临床手术中肾缺血再灌注损伤是导致急性肾功能衰竭的主要风险因素,比如肾脏移植、主动脉的心脏搭桥手术以及心肺的外科手术等。据有关文献报道我国住院人群中20%患有急性肾损伤,而ICU病房中急性肾损伤的患病率高达40%-60%,但临床上目前并没有有效的治疗方法,因此开发新型的预防和治疗急性肾损伤的策略迫在眉睫。Corin是一种新近发现的Ⅱ型跨膜丝氨酸蛋白酶,其结构是由一个短的氨基末端胞浆区、一个跨膜区和胞外区组成,胞外区含有两个卷曲结构域、八个低密度脂蛋白受体结构域、一个清道夫受体结构域和一个胰蛋白酶样的蛋白酶结构域,其主要表达于心脏,主要功能是通过将心钠肽前体切割活化成有活性的ANP,进而调节血压和心脏功能。但近期发现Corin也表达于肾脏的肾小管中,那么Corin在肾小管病变的急性肾损伤中是否起到作用,我们无从得知。有关实验表明,基因变异和功能障碍导致Corin的活性降低,可能与一系列心血管疾病如心肌肥厚、高血压的发病有关,所以Corin在肾缺血再灌注损伤中的作用更值得我们去探讨和研究。研究方法1 Corin在肾缺血再灌注损伤模型中的表达变化1.1肾缺血再灌注损伤小鼠模型的构建及检测Corin的表达情况选取9周龄的野生型(wild type,WT)C57BL/6小鼠,用戊巴比妥钠麻醉小鼠后,沿中线切开腹腔,用动脉夹夹住双侧肾蒂45分钟,45分钟后去掉动脉夹,在双肾上滴上37℃生理盐水,观察灌注情况,确保血液复流,然后开始计时,分别在24h、48h、72h时处死小鼠取材。分别用蛋白质免疫印迹法(Western Blot,WB)、Real-time RT-PCR以及免疫组织化学法(immunohistochemistry,IHC)检测Corin在肾缺血再灌注损伤模型小鼠肾脏组织中的表达变化情况。1.2 Corin在多种肾小管中的表达情况采用组织免疫荧光(immunofluoresce,IF)的方法同时对Corin以及肾小管不同部位标记物蛋白进行荧光双染共定位。1.3体外细胞模型构建及检测Corin表达情况体外培养人的肾小管上皮细胞(HK-2),采用低氧培养箱培育1.5小时后转为正常培养的方法,体外模拟肾缺血再灌注模型。采用Western Blot和Real-time RT-PCR的方法检测Corin在HK-2细胞中的变化情况。2 Corin在小鼠肾缺血再灌注损伤中的作用2.1野生型小鼠与Corin-/-小鼠肾脏功能学指标对比下腔静脉取血,检测血清中肌酐尿素氮的含量。2.2野生型小鼠与Corin-/-小鼠肾脏形态学损伤对比用苏木素-伊红染色的方法(hematoxylin-eosin staining,HE)对野生型小鼠和Corin-/-小鼠肾脏石蜡切片进行染色,然后进行形态学损伤评分。用TUNEL对野生型和Corin-/-小鼠肾脏组织石蜡切片就行荧光染色,检测肾脏细胞凋亡情况。2.3检测野生型小鼠和Corin-/-小鼠肾缺血再灌注模型中炎症相关指标的情况用免疫组化的方法对野生型小鼠和Corin-/-小鼠肾脏组织石蜡切片染色,Ly6B标记中性粒细胞、CD68标记巨噬细胞。2.4体外培养细胞转染Corin过表达质粒对低氧低糖培养HK-2细胞凋亡及炎症的影响情况用Real-time RT-PCR的方法检测过表达Corin后HK-2细胞在低氧低糖条件下炎症因子的变化情况。用流式细胞技术检测过表达Corin后HK-2细胞在低氧低糖条件下凋亡的变化情况。3 Corin机制的初步探讨用Western Blot的方法,检测肾脏匀浆中β-catenin的表达变化。研究结果1 Corin在肾缺血再灌注模型中的表达变化1.1 Corin在肾缺血再灌注小鼠模型中的表达水平显著降低Real-time RT-PCR和Western Blot检测小鼠肾脏中Corin的表达,结果表明在蛋白和mRNA水平模型组中Corin的表达明显比假手术中降低。免疫组化结果也印证了 Corin的表达在肾脏组织中是显著降低的。1.2 Corin主要表达于肾脏的近曲小管和远曲小管中免疫荧光结果表明,Corin主要表达于肾脏的近曲小管和远曲小管中,髓质的集合管中表达较少。1.3体外模拟条件下Corin的表达含量显著降低用Real-time RT-PCR和Western Blot检测低氧低糖处理的HK-2细胞中Corin的表达显著降低。2 Corin在肾缺血再灌注损伤中起到保护作用2.1 Corin的缺失加重了肾缺血再灌注的损伤 Corin的缺失导致血清中的肌酐尿素氮显著升高、肾脏的损伤更重、中性粒细胞和巨噬细胞的浸润明显增加、细胞凋亡数明显增多。2.2过表达Corin后低氧低糖条件下的HK-2凋亡减少、炎症反应降低HK-2细胞过表达Corin后,在低氧低糖的条件下用细胞流式技术检测细胞的凋亡数明显减少,用Real-time PT-PCR检测细胞炎症反应明显降低。3 Corin机制的初步探讨肾缺血再灌损伤后激活Wnt/β-catenin信号通路,使β-catenin的表达升高,Corin的缺失导致β-catenin的表达更高。研究结论与创新性本研究首次表征Corin在肾脏疾病中的变化,发现在肾缺血再灌注损伤中Corin的表达是显著降低的,而且Corin的缺失导致肾缺血再灌注损伤加重。本研究为设计和开发Corin相关蛋白质药物并用于AKI的防治提供了实验基础和理论依据。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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