【作者】 陈艳；

【导师】 张秀英；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2014， 硕士

【摘要】 肾缺血再灌注损伤(renal ischemia reperfusion injury, RIRI)是临床上常见的病理过程，也是急性肾损伤(acute kidney injury,AKI)最常见的一种病因之一。肾脏缺血再灌注损伤会引起急性肾小管坏死及晚期肾组织的纤维化。缺血后处理（postconditioning, POC）是近年来发现的一种新的方法，即在缺血后持续再灌注前，通过给予多次短暂的再灌注/缺血处理来减轻再灌注造成的损伤。由于其可以在缺血发生后应用，可行性高，因此具有很好临床应用前景本研究通过制备大鼠肾脏缺血再灌注损伤模型来研究POC对肾组织慢性纤维化的保护作用并阐明其保护机制，为其临床应用奠定理论基础。首先，本实验成功制备大鼠肾脏缺血再灌注损伤模型，统计分析各组动物生存情况及检测肾脏的功能。生存曲线的结果显示Sham组和POC组大鼠没有发生死亡，其生存率为100%，而I/R组大鼠在第一天和第二天发生死亡，其生存率为75%。血清中肌酐的检测结果表明，在缺血再灌注早期，POC可以大幅度地降低血清中Cr的含量，使其恢复正常水平，但是在再灌注一个月，POC组及I/R组中Cr的浓度无明显差异。其次，本实验对再灌注后三个月的动物肾脏组织结构及纤维化程度进行了观察及检测。结果显示，Sham组中肾组织形态正常，未发生纤维化。I/R组中肾组织结构发生病变，其特征为：部分肾小球硬化、纤维化，相应肾小管萎缩、消失间质纤维化。而POC组中肾组织的纤维化病变较轻，其程度明显低于I/R组。最后，通过检测肾脏组织Nitrotyrosine和8-OHdG的表达，推测POC的保护机制可能是通过减轻了细胞的氧化应激及线粒体DNA氧化损伤，从而对肾脏起到了保护作用。结果发现，Sham组Nitrotyrosine的表达量较低，8-OHdG不表达。I/R组Nitrotyrosine的表达量大幅度地增加，8-OHdG为强阳性表达且表达主要位于胞浆中。而POC组Nitrotyrosine和8-OHdG的表达明显低于I/R组。综上所述，本研究通过制备大鼠肾脏缺血再灌注损伤模型来研究POC对肾组织慢性纤维化的作用机制。结果表明POC降低了肾脏缺血再灌注损伤大鼠的死亡率，减轻了肾组织的纤维化程度，其机制在于减轻了细胞的氧化应激及线粒体DNA氧化损伤，从而对大鼠肾脏起到了保护作用。更多还原

【Abstract】 Renal ischemia-reperfusion injury (RIRI) is a clinically common pathologicalprocess and it is also one of the most common causes of acute kidney injury (AKI),which may occur in several clinical situations, including kidney transplantation,partial nephrectomy, renal artery angioplasty, sepsis, hydronephrosis, elective urologicoperations, cardiopulmonary bypass, aortic bypass surgery, liver transplantation etc.RIRI is mainly caused by a large amount of reactive oxygen species (ROS) andinfammatory response induced by reperfusion injury, which lead to a combination ofapoptosis and necrosis. RIRI can result in renal fibrosis, the fibrosis is almost thecommon ways and pathological basis of various types of chronic kidney diseaseprogress to the end-stage renal failure.Postconditioning (POC) is a new protective method in recent years, which isdefined as a series of brief reperfusion repeatedly to alleviate the damage at the earlyphase of reperfusion. It has been reported that POC can protect the brain, heart, liver,kidney, etc. we establish the model of renal ischemia-reperfusion injury in rat andclarify the protective effect and mechanisms of POC, which attenuates the renalfibrosis after ischemia reperfusion injury by the application of function,histomorphology and molecular biology.Methods: Adult male Sprague-Dawley rats were randomly divided into threegroups: sham group, ischemic reperfusion (I/R) group, postconditioning (POC) group.Sham group was the control group, left kidneys were exposed for45min and withoutany treatment. I/R group, rats were subjected to right nephrectomy and left kidneyswere subjected to45min of ischemia followed by reperfusion. POC group, rats weresubjected to right nephrectomy, left kidneys were subjected to three cycles of30s ofreperfusion followed by30s of ischemia immediately after45min of ischemia and7min short-time full reperfusion, then followed by reperfusion. Each group of animals were reared three months under clean conditions, then observing the animals in eachgroup to survive, drawing the animal survival curve. Collecting the serum in the twodays and one month and detecting the renal function. At three months, HE staining ofthe kidneys tissue structure, Masson staining of the renal tissue fibrosis, and theexpression of Nitrotyrosine and8-OHdG were detected by the immunohistochemistryand Western Blot.Results: Survival curves displayed that Sham rats and POC rats survived, thesurvival rate was100%, while death occurred in rats of the I/R group on the first dayand the second days, the survival rate was75%. From the results of animal serumcreatinine, at two days, serum levels of Cr in Sham rats maintained at the normallevels, serum levels of Cr in I/R rats were signifcantly higher (P＜0.01), while weresignifcantly lower in POC rats compared with I/R rats (P＜0.05). One month afterreperfusion, serum levels of Cr in these three groups were no significant difference.Three months after reperfusion, HE staining of paraffn sections demonstrated nosignifcant morphological changes in renal glomerular or tubular cells in the Shamgroup. The I/R group showed signifcant morphological changes and characterized inparts of glomerular sclerosis and fibrosis, the corresponding tubular atrophy anddisappearance, interstitial fibrosis. POC attenuated these severe renal damages.Masson staining showed that Sham group renal tissue no fibrosis, the I/R groupdemonstrated the apparent glomerular fibrosis, tubular fibrosis and interstitial fibrosis,POC attenuated the renal fibrosis. Immunohistochemistry staining of nitrotyrosineshowed that the Sham group with low expression and the I/R group was highlyexpressed, while the expression of POC group was significantly lower than the I/Rgroup. Western blot analyses of nitrotyrosine expression were same as above.Immunohistochemistry staining of8-OHdG demonstrated no expressed in the Shamgroup, strongly positive expressed in the I/R group, and expression localizedprimarily in the cytoplasm, while the expression of POC group was significantlylower than the I/R group.Conclusions: Postconditioning reduces rats mortality induced by the renalischemia-reperfusion injury, reducing the renal fibrosis, the mechanism is that alleviating cellular oxidative stress and mitochondria DNA oxidative damage, andthus playing a protective role in rat kidneys.更多还原