本文關(guān)鍵詞： 糖尿病腎病 顆粒蛋白前體 足細(xì)胞 自噬　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:糖尿病腎病(diabetic nephropathy,DN)是導(dǎo)致終末期腎病(end stage renal disease,ESRD)的最重要的誘因之一,嚴(yán)重威脅人類的生命健康。糖尿病腎病的發(fā)病機(jī)制尤為復(fù)雜,主要包括糖脂代謝紊亂、血流動(dòng)力學(xué)、氧化應(yīng)激、炎癥反應(yīng)以及遺傳因素等。足細(xì)胞(podocyte)是維持腎小球內(nèi)環(huán)境穩(wěn)態(tài)的重要組成部分,足細(xì)胞與腎小球內(nèi)皮細(xì)胞和基底膜共同組成了腎小球?yàn)V過(guò)屏障,足細(xì)胞損傷是糖尿病腎病的早期事件,可導(dǎo)致濾過(guò)屏障遭到破壞,產(chǎn)生蛋白尿,最終加重腎臟損傷。但是,在糖尿病腎病中造成足細(xì)胞損傷的機(jī)制尚不完全明確。顆粒蛋白前體(progranulin,PGRN)是一種自分泌型生長(zhǎng)因子,廣泛表達(dá)分布于體內(nèi)各種組織與器官,例如神經(jīng)細(xì)胞、軟骨細(xì)胞、免疫細(xì)胞以及上皮細(xì)胞等。PGRN具有諸多生物學(xué)功能,廣泛參與到多種病理生理過(guò)程中,如腫瘤的發(fā)生發(fā)展、自身免疫類疾病、組織損傷修復(fù)、傷口愈合等。雖然課題組前期的研究證明PGRN可以通過(guò)負(fù)調(diào)控NOD2介導(dǎo)的信號(hào)通路在急性腎損傷中起到保護(hù)作用,但在糖尿病腎病中是否發(fā)揮作用尚不清楚。研究表明,自噬在糖尿病腎病的病理進(jìn)程中發(fā)揮著重要的作用,正常生理?xiàng)l件下足細(xì)胞保持著較高的自噬水平,糖尿病腎病發(fā)生時(shí)足細(xì)胞自噬水平降低導(dǎo)致足細(xì)胞受損進(jìn)而加重腎損傷。報(bào)道顯示,AMPK是自噬的激活劑,AMPK可以通過(guò)直接磷酸化自噬啟動(dòng)蛋白ULK1從而促進(jìn)自噬,進(jìn)而在糖尿病腎病中起到保護(hù)作用。因此本實(shí)驗(yàn)主要為了探究PGRN在糖尿病腎病中的作用,以及是否與自噬,AMPK信號(hào)通路有關(guān)。研究方法與結(jié)果1.PGRN在糖尿病腎病中的表達(dá)變化選取8-12周齡雄性野生型(wild type,WT)C57BL/6J小鼠,單腎摘除后恢復(fù)一周,腹腔注射鏈脲佐霉素(streptozotocin,STZ)100mg/kg連續(xù)三天誘導(dǎo)糖尿病腎病。當(dāng)小鼠模型構(gòu)建成功后,使用蛋白免疫印跡法(Western blot,WB)、Real-time RT-PCR、免疫熒光(immunofluorescence,IF)和免疫組化(immunohistochemistry,IHC),檢測(cè)PGRN在腎皮質(zhì)中表達(dá)變化及分布情況,結(jié)果顯示PGRN在糖尿病腎病模型組腎皮質(zhì)中與對(duì)照組相比表達(dá)明顯下降。同時(shí)體外高糖分別刺激人腎小球足細(xì)胞(humanpodocyte,HPC)、內(nèi)皮細(xì)胞(glomerular endothelial cell,GEnC)和大鼠系膜細(xì)胞(rat messangial cell,RMC)結(jié)果顯示,腎小球足細(xì)胞和內(nèi)皮細(xì)胞中的PGRN均表達(dá)下降。2.PGRN在糖尿病腎病損傷中的作用適齡野生型雄性C57BL/6小鼠和Grn基因敲除鼠(B6(Cg)-Grntml lAidi/J)隨機(jī)分組,分別構(gòu)建糖尿病腎病模型和對(duì)照組。通過(guò)PAS對(duì)野生型和Grn基因敲除組腎臟石蠟切片染色,透射電鏡觀察小鼠足細(xì)胞損傷。結(jié)果顯示,在糖尿病腎病組PGRN的缺失導(dǎo)致了腎皮質(zhì)中腎小球系膜基質(zhì)增生加重。透射電鏡結(jié)果顯示PGRN的缺失加重了腎小球足細(xì)胞損傷,導(dǎo)致足細(xì)胞足突的融合消失。IF標(biāo)記足細(xì)胞特異性蛋白Nephrin、Podocin結(jié)果顯示PGRN的缺失加重了足細(xì)胞功能蛋白的損傷。同時(shí)Real-time RT-PCR檢測(cè)野生型與Grn基因敲除鼠腎皮質(zhì)炎性因子與趨化因子的變化,結(jié)果顯示,PGRN的缺失導(dǎo)致腎皮質(zhì)中炎性因子與趨化因子較野生型糖尿病腎病組更高。體外流式細(xì)胞術(shù)檢測(cè)足細(xì)胞凋亡水平,結(jié)果顯示加入重組PGRN可以明顯降低高糖引起的細(xì)胞凋亡,WB檢測(cè)足細(xì)胞功能蛋白Nephrin,結(jié)果顯示加入重組PGRN可恢復(fù)高糖誘導(dǎo)的Nephrin蛋白水平的降低。3.PGRN在糖尿病腎病損傷中的作用機(jī)制WB檢測(cè)野生型與Grn基因敲除鼠腎皮質(zhì)中LC3B等自噬相關(guān)分子,結(jié)果顯示Grn基因敲除鼠糖尿病腎病組較野生型糖尿病腎病組的自噬水平更低。體外結(jié)果顯示足細(xì)胞高糖條件下自噬水平顯著降低,加入重組PGRN后自噬水平恢復(fù),Tandem實(shí)驗(yàn)觀察自噬流變化表明加入重組PGRN可以恢復(fù)足細(xì)胞高糖條件下自噬流水平。同時(shí)WB檢測(cè)自噬啟動(dòng)蛋白ULK1磷酸化水平,結(jié)果表明高糖條件下ULK1磷酸化水平降低,加入重組PGRN可以恢復(fù)磷酸化水平。進(jìn)一步WB檢測(cè)糖尿病腎病腎皮質(zhì)AMPK信號(hào)通路的水平,結(jié)果顯示糖尿病腎病組中Grn基因敲除較野生型AMPK信號(hào)通路進(jìn)一步降低。結(jié)論與創(chuàng)新性:1.糖尿病腎病小鼠模型中PGRN顯著降低,PGRN的缺失加重糖尿病腎病足細(xì)胞損傷。2.PGRN可以通過(guò)激活A(yù)MPK信號(hào)通路促進(jìn)ULK1磷酸化水平進(jìn)而啟動(dòng)足細(xì)胞自噬。本課題對(duì)指導(dǎo)設(shè)計(jì)PGRN作為靶點(diǎn)治療糖尿病腎病提供重要的依據(jù)。
[Abstract]:Background: diabetic nephropathy (diabetic, nephropathy, DN) is the leading cause of end-stage renal disease (end stage renal disease, ESRD) is the most important factor, a serious threat to human life and health. The pathogenesis of diabetic nephropathy is very complex, mainly including glucose and lipid metabolism disorders, hemodynamics, oxidative stress, inflammatory reaction and genetic factors podocyte. (podocyte) is an important part of maintaining homeostasis within the glomeruli, glomerular podocytes and endothelial cells and basement membrane composed of glomerular filtration barrier, podocyte injury is an early event of diabetic nephropathy, can lead to the destruction of the filtration barrier, resulting in proteinuria, eventually aggravate renal injury. But the mechanism caused by foot cell damage is still not completely clear in diabetic nephropathy. Progranulin (progranulin, PGRN) is an autocrine growth factor expression, widely divided The cloth in various tissues in the body and organs, such as nerve cells, cartilage cells, immune cells and epithelial cells,.PGRN has many biological functions, widely involved in the pathological process of many kinds, such as the occurrence and development of tumors, autoimmune diseases, tissue repair and wound healing. Although our previous studies have demonstrated that PGRN can signaling through negative regulation mediated by NOD2 in acute kidney injury plays a protective role in diabetic nephropathy, but the role is not clear. Studies suggest that autophagy plays an important role in the pathological process of diabetic nephropathy, normal physiological conditions of podocyte autophagy maintained higher levels of diabetic nephropathy. Occurs when the podocyte autophagy leading to podocyte damage and reduce the level of renal damage aggravated. Reports indicate that AMPK is the activator of autophagy, AMPK through direct phosphorylation of autophagy Start the ULK1 protein to promote autophagy, which play a protective role in diabetic nephropathy. So this experiment in order to explore the role of PGRN in diabetic nephropathy, and whether autophagy, AMPK signaling pathway. The research methods and results of 1.PGRN in diabetic nephropathy on the expression of selected 8-12 week old male wild type (wild type WT, C57BL/6J) mice, single kidney removed recovery after a week, intraperitoneal injection with streptozotocin (streptozotocin, STZ) 100mg/kg for three days induced diabetic nephropathy. When the successful construction of mouse model, using Western blotting (Western blot, WB), Real-time RT-PCR, immunofluorescence (immunofluorescence, IF) and immunohistochemistry (immunohistochemistry, IHC), the expression and distribution of PGRN was detected in renal cortex, showed that PGRN in the cortex of kidney in diabetic nephropathy model group compared with the control group was significantly decreased. At the same time were stimulated with high glucose in vitro human glomerular podocyte (humanpodocyte, HPC), endothelial cells (glomerular endothelial cell, GEnC) and rat mesangial cells (rat messangial cell, RMC) results showed that glomerular podocytes and endothelial cells in the expression of PGRN was decreased in diabetic nephropathy.2.PGRN injury age of wild type male C57BL/6 mice and Grn knockout mice (B6 (Cg) -Grntml lAidi/J) were randomly divided into diabetic nephropathy model were constructed and the control group. The PAS knockout group staining on paraffin sections of kidney and wild type Grn gene, observe the podocyte injury in mice by transmission electron microscope. The results showed that in the absence of PGRN in diabetic nephropathy group glomerular mesangial matrix hyperplasia in renal cortex increased. TEM results showed that deletion of PGRN increased glomerular podocyte injury, leading to podocyte footprocess fusion disappear podocyte.IF markers Specific protein Nephrin, Podocin results showed that PGRN deletion aggravated the injury of podocyte proteins. At the same time, Real-time RT-PCR detection of wild-type and Grn knockout rat renal cortex changes, inflammatory cytokines and chemokines showed that deletion of PGRN leads to inflammatory factors in renal cortex and chemotactic factor than the wild type diabetic nephropathy was higher in vitro. Flow cytometry was used to detect the apoptosis of podocyte levels, the results showed that the addition of recombinant PGRN can significantly reduce the cell apoptosis induced by high glucose, WB detection of podocyte protein Nephrin, the results showed that Nephrin protein levels with recombinant PGRN can restore glucose induced WB reduction mechanism of.3.PGRN in injury in diabetic nephropathy detection of wild-type and Grn knockout LC3B rats renal cortex autophagy related molecules, the results showed that Grn knockout mice diabetic nephropathy group compared with the wild type diabetic nephropathy The in vitro results showed lower levels of autophagy. Podocyte autophagy under high glucose decreased significantly and the recovery of autophagy with recombinant PGRN, Tandem experimental observation indicated that the addition of recombinant PGRN autophagy flow can restore podocytes under high glucose levels. At the same time WB for the detection of autophagy flow autophagy protein ULK1 phosphorylation level. The results show that the phosphorylation of ULK1 under the condition of high glucose levels decreased with recombinant PGRN can restore the phosphorylation level of WB. Further detection of diabetic nephropathy renal cortical AMPK signaling level, results showed that the diabetic nephropathy group compared to the wild type Grn gene knockout AMPK signal pathway is further reduced. The conclusion and Innovation: PGRN 1. diabetic nephropathy mice decreased significantly, PGRN the lack of increase of podocyte injury in diabetic nephropathy.2.PGRN can activate the AMPK signaling pathway to promote ULK1 phosphorylation and initiation of podocyte Autophagy. This topic provides an important basis for guiding the design of PGRN as a target for the treatment of diabetic nephropathy.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R692.9

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