TSP-1及其受体CD47在大鼠肾缺血再灌注损伤大鼠模型中的作用及机制初步探讨
发布时间：2019-01-06 19:58
【摘要】：目的:肾缺血再灌注损伤（ischemia reperfusion injury, IRI）是急性肾损伤(acute kidney injury,AKI)重要的组织病理学机制。由IRI引起的AKI是一个重要的临床问题，尽管AKI的治疗方法不断改进，但是肾IRI仍与AKI的发病率和死亡率密切相关。肾IRI的发生机制很复杂，且并未完全阐明，与氧自由基、内皮功能紊乱、炎症损伤、细胞坏死和凋亡等多方面均有关。已证实氧化应激产生的活性氧(reactive oxygenspecies, ROS)在肾IRI中发挥了重要作用。在正常肾脏组织，基质蛋白血小板反应蛋白-1(thrombospondin-1, TSP-1)表达量很少。但在缺氧条件下，多种细胞可以合成TSP-1。有研究表明，TSP-1与AKI的发生和发展有关，同样也与慢性肾脏病的进展关系密切。因此，人们越来越多的关注TSP-1在急慢性肾脏病变中的地位。CD47又被称为整合素相关蛋白(integrin-associated protein, IAP)，是TSP-1的同源受体之一，它在肾脏广泛表达，但它在肾实质和间质细胞促进肾损伤的机制还不清楚。因此，本研究通过构建肾IRI大鼠模型，观察TSP-1及其受体CD47在IRI大鼠肾脏的分布情况及表达变化，以及血ROS的变化和肾组织氧化应激指标的变化，研究TSP-1、CD47在肾IRI时的表达及分布，以及与氧化应激的关系，探讨其在肾IRI发病机制中的作用。 方法:成年雄性SD大鼠42只，随机分为正常对照组（n=5）、假手术组(sham group, SG)（n=5）和模型组（n=32），模型组下设IRI后1h、6h、12h、24h4个时间点，每个时间点8只大鼠。手术方式:将大鼠麻醉后，逐层开腹暴露双肾，用玻璃分针分离双侧肾动脉，将已经消毒的压脉带片段包裹在肾动脉外层，将手术线系在压脉带片段外迅速打结阻断肾血流，观察肾脏颜色的变化。阻断肾血流45min后，剪断手术线，恢复肾脏供血，观察肾脏颜色变化。逐层关腹，复苏。观察IRI后各组大鼠尿量变化，采用全自动生化分析仪检测各组大鼠的肾功能，通过苏木精-伊红染色法(hematoxylin-eosin staining, HE)、糖原染色(periodicacid-schiff stain, PAS)了解肾脏病理损害，通过透射电镜观察足细胞有无损伤、系膜基质是否增生。采用流式细胞仪检测血ROS变化，采用酶联免疫吸附法测定肾组织中羟自由基和丙二醛的浓度，了解肾IRI损伤时的氧化应激状态。通过肾组织免疫组化，观察TSP-1和CD47蛋白在肾脏的表达及变化情况，通过免疫荧光双染了解TSP-1及其受体CD47在肾IRI时是否存在共表达。 结果:与正常对照组和假手术组相比，IRI各组大鼠尿量明显减少；肾IRI各组血肌酐（serum creatinine, Scr）和尿素氮（urea nitrogen, BUN）明显升高，差异具有统计学意义（P＜0.05）；随着缺血再灌注后时间的延长，，肾小管出现不同程度的扩张、变性与坏死，间质炎性细胞浸润、充血水肿等变化。各组间肾小管HE染色病理损伤评分差异显著，差异具有统计学意义（P＜0.05）；通过透射电镜观察发现肾IRI各组大鼠肾小球足细胞足突消失、系膜基质增生明显，足细胞计数差异具有统计学意义（P＜0.05），而肾IRI各组间差异无统计学意义（P＞0.05）；红细胞中ROS荧光强度阳性率逐渐升高，肾组织中的羟自由基和丙二醛的浓度呈逐渐升高趋势。在肾IRI早期，肾小管TSP-1、CD47表达即明显增加，至肾IRI24h时TSP-1、CD47在残存肾小管仍有较高表达。通过免疫荧光双染发现，在肾IRI肾小管TSP-1和CD47存在共表达。相关性分析发现ROS荧光强度阳性率与羟自由基和丙二醛的浓度呈正相关，与Scr、BUN、肾小管损伤评分、TSP-1积分光密度、CD47积分光密度呈正相关，与足细胞计数呈负相关。 结论:通过简易手术阻断双侧肾动脉血流，可以成功建立大鼠肾IRI模型。肾IRI后引起肾组织和全身的氧化应激反应，引起肾脏组织结构损害，足细胞损伤，肾功能异常。肾IRI时大鼠肾脏TSP-1和CD47表达明显升高，与肾组织和全身的氧化应激反应密切相关。TSP-1、CD47是肾小管损伤早期的一个预警指标，并伴随肾IRI整个过程持续表达，与ROS共同参与了肾IRI肾脏损伤过程。
[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.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5
本文编号：2403257
[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.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5
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本文编号：2403257
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