PGRN在脓毒症诱导的急性肾损伤中的功能研究

发布时间：2018-05-06 22:05

本文选题：脓毒症 + 急性肾损伤　；参考：《山东大学》2016年硕士论文

【摘要】：脓毒症是一种由感染引起的,以全身性炎症反应为特征的复杂的临床综合征。脓毒症通常导致广泛的组织损伤和多器官功能紊乱。急性肾损伤(acute kidney injury, AKI)是脓毒症最常见的并发症之一,不仅增加了疾病的复杂性和护理费用,也是引起脓毒症相关死亡的高风险因素。脓毒症诱导的AKI的病理学机制十分复杂,涉及肾脏内血流动力学变化、内皮细胞失调、肾实质组织的炎性细胞浸润、肾小球血栓和坏死细胞与碎片引起的肾小管障碍等多种因素。Progranulin (PGRN)是一种新型的分泌生长因子,在快速增殖的上皮细胞和特定类型的神经细胞中高表达,多种其他组织和细胞类型中也存在PGRN表达,包括脂肪组织、造血细胞、软骨细胞及免疫细胞等。PGRN具有多种生理功能,不仅维持和调控正常组织的发生发展、增殖、再生及宿主防御,也参与多种类型的疾病过程,包括自身免疫性疾病、癌症和神经退行性疾病等。近年来,PGRN在炎症方面的作用受到广泛关注,另有研究报道,PGRN在肾缺血／再灌注小鼠模型中具有肾脏保护作用。脓毒症是急性肾损伤最主要的病因之一,而PGRN在脓毒症诱导的急性肾损伤中的作用尚不明确。本文利用两种常用的脓毒症相关急性肾损伤研究模型：LPS腹腔注射诱导内毒素血症和盲肠结扎穿孔术诱导多细菌感染建立脓毒症小鼠模型,初步探讨PGRN在脓毒症诱导的急性肾损伤中的功能。目的：明确PGRN在脓毒症相关的急性肾损伤中的作用,并探讨PGRN对脓毒症相关肾损伤中肾组织炎症应答与肾小管上皮细胞死亡的影响。方法：分别使用小鼠腹腔注射细菌脂多糖(lipopolysaccharides, LPS)和小鼠盲肠结扎再穿孔(cecal ligation and puncture, CLP)的方法,构建脓毒症模型。在建模6小时和24小时后,使用实时定量PCR检测肾组织中PGRN的mRNA水平,Western blot和免疫组化检测PGRN的蛋白表达,酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测血清与肾组织匀浆中PGRN的蛋白水平；在建模2小时前,野生型鼠外源性注射rPGRN和无菌PBS,其他野生型鼠和PGRN基因敲除鼠直接建模,为了检测脓毒症诱导的肾损伤的程度,血生化分析法检测血清中肌酐和尿素氮含量,HE染色法检测肾组织的损伤；免疫组化法检测肾组织中中性粒细胞和巨噬细胞浸润；实时定量PCR法检测肾组织炎性细胞因子的mRNA水平；TUNEL法检测肾组织细胞的凋亡；Western blot检测肾组织中凋亡蛋白的表达水平。结果：在两种脓毒症小鼠模型研究中,本研究发现脓毒症小鼠肾组织中PGRN的蛋白水平和mRNA水平均高于对照组小鼠；与野生型鼠相比,PGRN基因敲除的脓毒症小鼠的血清中肌酐和尿素氮水平更高；HE染色显示肾组织损伤更加严重；中性粒细胞和巨噬细胞浸润更加严重；促炎细胞因子IL-6、TNF-α、IL-1p的mRNA水平升高；肾组织细胞凋亡更显著,凋亡蛋白的表达水平升高；而与注射PBS组相比,外源性注射rPGRN组,小鼠肾功能障碍和肾组织损伤均明显减轻；注射rPGRN能够有效抑制肾组织中炎症应答及肾小管上皮细胞的凋亡性死亡。结论：PGRN在脓毒症小鼠肾组织及外周血中表达水平升高；PGRN敲除导致脓毒症相关急性肾损伤更加严重；而外源性注射重组PGRN后肾损伤明显改善。本研究采用野生型与PGRN基因敲除小鼠或外源注射rPGRN等方法,利用LPS注射和CLP脓毒症小鼠模型,明确了PGRN对脓毒症相关急性肾损伤的保护性作用,并从炎症应答和肾脏实质细胞死亡的角度分析了PGRN肾保护作用的机制,为临床脓毒症尤其是脓毒症诱导的急性肾损伤的防治,提供了新的理论依据。
[Abstract]:In recent years , PGRN is one of the most common complications of sepsis , including fatty tissue , hematopoietic cells , chondrocytes and immune cells . ELISA was used to detect the level of PGRN protein in serum and renal tissue homogenate ;
In order to detect the degree of renal injury induced by sepsis , blood biochemical analysis was used to detect serum creatinine and urea nitrogen content and HE staining was used to detect renal tissue injury .
Immunohistochemical method was used to detect the infiltration of neutrophils and macrophages in renal tissue .
The mRNA level of inflammatory cytokines in renal tissue was measured by real - time quantitative PCR .
TUNEL method was used to detect the apoptosis of renal tissue cells .
The expression level of apoptotic protein in renal tissue was detected by Western blot . Results : In the study of two types of septic mice , the protein level and mRNA level of PGRN in renal tissue of septic mice were higher than those in control group .
Compared with wild - type mice , the level of creatinine and urea nitrogen was higher in the serum of septic mice with PGRN knockout mice .
HE staining showed that renal tissue injury was more serious .
The infiltration of neutrophils and macrophages was more serious ;
The mRNA levels of pro - inflammatory cytokines IL - 6 , TNF - 伪 and IL - 1p were increased .
Apoptosis of renal tissue cells was more significant , and the level of apoptosis protein was increased .
Compared with PBS group , the rPGRN group , renal dysfunction and renal tissue injury in mice were significantly reduced .
Conclusion : PGRN can effectively inhibit the inflammatory response in renal tissue and death of renal tubular epithelial cells . Conclusion : The expression of PGRN in renal tissue and peripheral blood of septic mice is increased .
PGRN knockout resulted in more severe acute renal injury associated with sepsis ;
In this study , the protective effects of PGRN on acute renal injury associated with sepsis were identified by using wild - type and PGRN knockout mice or exogenous injected rPGRN , and the mechanism of PGRN kidney protection was analyzed from the angle of inflammatory response and renal parenchyma cell death , which provided a new theoretical basis for the prevention and treatment of acute renal injury induced by sepsis , especially sepsis .

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R459.7;R692.5

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