【作者】 陈志；

【导师】 刘群；

【作者基本信息】 吉林大学 ， 神经病学， 2010， 博士

【摘要】 近年来,尿毒症的发病人数逐渐增多。尿毒症,是各种原发或继发的慢性肾脏疾病不断恶化、进展的最终阶段,也称终末期肾病(ESRD),此时由于肾脏功能受损,大量代谢产物在体内蓄积、钠水潴留,引起水、电解质、酸碱平衡紊乱及全身各系统功能失调,其中心脑血管系统受累最重,也是影响尿毒症患者预后的主要因素。尿毒症对心血管系统的影响研究得较多,近年有关尿毒症对神经系统的影响逐渐引起学者们的重视。然而,有关尿毒症病人发生脑缺血再灌注损伤后的病理生理变化尚未见报道。本文以尿毒症大鼠为研究对象,从多个角度探讨尿毒症合并脑缺血再灌注损伤后脑组织的病理变化、抗氧化能力的变化、炎症因子的影响及神经细胞凋亡的情况,其中运用了RT-PCR技术、免疫组化技术等多种方法,最终发现尿毒症合并脑缺血再灌注损伤的大鼠除神经细胞凋亡与单纯脑缺血再灌注无显著不同外,其余几方面的改变均重于单纯脑缺血再灌注大鼠,提示尿毒症患者体内长期存在的大量毒素使机体免疫功能下降,机体长期处于过度氧化应激水平及微炎症状态之下,当机体合并其他系统疾病特别是脑血管病时,预后极差。通过这一研究,使我们再次认识到对尿毒症病人进行抗氧化治疗、纠正微炎症状态的必要性,这是和血液净化治疗同等重要的治疗手段,是改善尿毒症病人预后的必要手段。更多还原

【Abstract】 The incidence of chronic kidney disease (CKD) and cerebrovascular disease was increased with obesity, hypertension, diabetes and the growing elderly population in recent years. It has become the serious public health problem for humanity. Uremia is the final stage of deterioration of primary or secondary chronic kidney disease. The kidney function is damaged seriously, a large number of metabolites accumulate in the body, combined with sodium and water retention, resulting in acid-base balance disorders and dysfunction of various systems in uremic patients. Cardiovascular system is the most severely affected and is the important factor influencing uremia prognosis. Higher incidence rate of cerebrovascular disease was found in Uremic patients, its mortality rate even in dialysis patients also occupy a large proportion. At present, there are a lot of researches about the impact of uremia on the cardiovascular system, and the impact of uremia on the nervous system has been in favor of scholars gradually. However, the pathological changes and mechanisms after cerebral ischemia-reperfusion injury of uremic patients have not been reported. Experimental studies of the feature and mechanism of the cerebral ischemia reperfusion in uremic rats in the level of whole animal, cellular, molecular, genetic were made in this paper in order to find the appropriate treatment to reduce the incidence of uremic patients with cerebral ischemia and reduce morbidity and mortality and improve the prognosis.Methods:(1) Experimental animals and groups:90 clean grade healthy male Wistar rats were divided into uremic group (18), uremia with cerebral ischemia-reperfusion group (72) randomly, uremia models were made. The other 90 rats were divided into normal control group (18) and ischemia-reperfusion group (72) randomly. Rats of the uremia with cerebral ischemia-reperfusion group and cerebral ischemia and reperfusion group were made into middle cerebral artery occlusion models. Reperfusion after 2 hours cerebral ischemia. according to different time points after reperfusion (Oh，2h，12h,24h), each group was divided into four sub-groups(18 each sub-group). Rats of normal control group and uremic group were also divided into four sub-groups according to different time points after reperfusion.(2) Animal Model:1) Methods of uremic rat model:The classic 5/6 nephrectomy2) Methods of focal cerebral ischemia-reperfusion injury model:the classic filament induced by reversible middle cerebral artery occlusion method (MCAO).(3) Observe indicators and detecting methods:1) The research of pathological changes in brain tissueHistopathological examination:6 rats were taken from each sub-group, whole body perfusion fixed quickly, coronal cut to prepare wax block in the hippocampal area, paraffin block cut into 4um consecutive coronal sections, stained, brain tissue pathological changes observed by light microscope.2) Comparative study of nerve cell damageOther 6 rats were taken from each sub-group,2/3 of cortical tissue of ischemic side were taken to make 10% brain tissue homogenate.①Determination of brain energy metabolism:Determination of lactic acid in homogenate activity levels and changes in ATPase.②Determination of antioxidant enzymes and lipid peroxidation products:Determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity and malondialdehyde (MDA) content by colorimetry3) Changes of inflammatory cytokines of rats brain tissueThe expression of IL-1β, IL-6, TNF-amRNA detected by RT-PCR in 4) The research of rats nerve cell apoptosis ①Apoptosis analysed by TUNEL assay:The experimental animals were anesthetized, heart perfusion, fixation, heads were removed, brain slices were taken in the hippocampal area, apoptotic cells were detected by TUNEL in situ label of DNA fragments.②Immunohistochemical detection of Bcl-2. Bax expression:Immunohistochemical staining method using SABC. DAB coloration.(4) Statistical analysis:All experimental data were analyzed using SPSS 16.0, the data were presented as mean= standard deviation (χ±s) and t-test was used to compare the two groups in the same time point.(5) Results:1) The pathological changes in ratsNormal hippocampal structure, no congestion, edema were found in control group, uremia group at each time point. Pyramidal cells were normal, boundary clear, large round nucleus, nucleolus and nuclear membrane were clearly visible. Edema hippocampal tissues were found in ischemia-reperfusion group, the number of pyramidal cells reduced, disorganized, and no hierarchical structure, cell gap widened, eosinophilic cytoplasm, nucleus dissolution. Compared with ischemia-reperfusion group, hippocampus structure of rats were damaged obviously by cell body swelling, angular, nuclear condensation or nuclear dissolution, nucleoli disappeared, cells ill-defined in uremia with cerebral ischemia and reperfusion group. With time prolonged, these changes gradually worsened in both two groups of rats with ischemia-reperfusion.2) Comparative study of nerve cell damage①Determination of brain energy metabolism:Compared with the control group, LA content was higher and ATPase activity declined in uremia group at every time points, but no significant difference (P>0.01). LA content decreased gradually and ATPase activity increased gradually of the other two groups of rats in the 2-24h after reperfusion; Compared with cerebral ischemia group, LA content was higher and ATPase activity declined in uremia with cerebral ischemia reperfusion group at every time points (P＜0.01).②Determination of antioxidant enzymes and lipid peroxidation products of brain tissues:Compared with the control group. MDA content was higher and SOD and GSH-PX activity decreased in uremia group at every time points, but no significant difference (P> 0.01); MDA content increased gradually and SOD and GSH-PX activity decreased gradually of the other two groups of rats in the 2-24h after reperfusion; Compared with cerebral ischemia group. MDA content increased significantly and SOD and GSH-PX activity decreased significantly in uremia with cerebral ischemia reperfusion group at every time points (P＜0.01).3) Changes of inflammatory cytokines of rats nerve cells①TNF-αmRNA:the expression of TNF-amRNA increased in uremia group at every time points than the control group, but no significant difference (P> 0.01); the expression of TNF-αmRNA reached the peak at 2h time points and decreased gradually, but still higher than control group (P<0.01); the expression of TNF-amRNA increased in uremia with cerebral ischemia reperfusion group at every time points (P<0.01), compared with cerebral ischemia group.②IL-1βmRNA, IL-6 mRNA:the expression of IL-1βmRNA. IL-6 mRNA increased in uremia group at every time points than the control group, but no significant difference (P> 0.01); the expression of IL-1βmRNA, IL-6 mRNA increased gradually in both other groups with reperfusion time prolonged; Compared with cerebral ischemia group, the expression of IL-1βmRNA, IL-6 mRNA increased significantly in uremia with cerebral ischemia reperfusion group at every time points (P<0.01).4) The research of rats nerve cell apoptosis①Apoptosis analysed by TUNEL assay:Apoptotic cells were found occasionally in hippocampal area in uremic group at each time point. Apoptotic cells were found in hippocampal area in ischemia-reperfusion group at 2h time points, increased gradually with time prolonged, reached the peak at 24h time points; Compared with cerebral ischemia group, the Apoptotic cells increased in uremia with cerebral ischemia reperfusion group at every time points., but no significant difference between the two groups (P> 0.01).②Immunohistochemical detection of Bcl-2, Bax expression:The expression of Bcl-2 and Bax were rare in control group and the uremic group at each time point, no significant difference between the two groups (P> 0.01). The expression of Bcl-2 reached the peak at 12h time points in the other two groups, and then decreased gradually, but still expressed at 24h: the number of Bcl-2 positive cells in uremia with cerebral ischemia reperfusion group less than ischemia reperfusion group at every time points, but no significant difference (P> 0.01); the expression of Bax reached the peak at 24h in other 2 groups, the number of Bcl-2 positive cells in uremia with cerebral ischemia reperfusion group more than ischemia reperfusion group at every time points, but no significant difference (P> 0.01)Conclusions(1) Pathological changes of brain tissue in uremia with cerebral ischemia reperfusion group are more evident than it in cerebral ischemia-reperfusion group.(2) Disturbance of Energy Metabolism is more significant in uremic rats after cerebral ischemia-reperfusion, the oxidative stress injury is greater, the antioxidant capacity is weaker.(3) High expression of TNF-a in the early stage of cerebral ischemia confirmed that it is the initiating factor of the inflammatory response; IL-1β, IL-6 were expressed during reperfusion process revealed the close relationship of inflammation and cerebral ischemia-reperfusion injurys; inflammatory response has been strengthened in uremic rats with cerebral ischemia-reperfusion injury confirmed that the mini-inflammatory state of uremic rats has continuing effects on the nervous system.(4) The neuronal apoptosis of uremic rats with cerebral ischemia and reperfusion injury is not obvious than cerebral ischemia rats.(5) Uremia may be independent risk factors of ischemia-reperfusion injury.更多还原