本文選題：成纖維細(xì)胞生長因子 + 急性腎損傷��； 參考：《南京醫(yī)科大學(xué)》2014年博士論文

【摘要】：急性腎損傷(Acute Kidney Injury, AKI)是一系列因素引起腎功能迅速惡化的臨床綜合征。雖然近年來人們在臨床實踐和實驗研究中對AKI進行了大量的研究,但目前對于AKI患者仍以透析及對癥支持治療為主。成纖維細(xì)胞生長因子(Fibroblast Growth Factors, FGFs)廣泛參與包括細(xì)胞增殖、分化、遷移、胚胎發(fā)育、血管生成調(diào)節(jié)以及損傷修復(fù)等在內(nèi)的多種生物學(xué)過程。然而其在AKI中的作用特別是對損傷后腎小管上皮細(xì)胞存活的作用還有待研究。本部分研究首先通過缺血再灌注損傷(Ischemia/Reperfusion Injury, IR/I)以及腹腔注射順鉑建立AKI小鼠模型,研究損傷后腎組織中FGFs的表達變化以及細(xì)胞內(nèi)下游信號通路的激活情況。實驗發(fā)現(xiàn)：IR/I腎損傷后腎組織內(nèi)FGF2、 FGF7、FGF10、FGF12、FGF13、FGF18以及FGF22表達上調(diào)；而順鉑注射后腎組織中僅FGF2和FGF 18表達增加。上述兩種AKI模型腎組織中Erkl,2磷酸化水平均增加。為進一步研究FGFs-FGFR信號在AKI中的作用,我們通過Cre-LoxP系統(tǒng)建立腎小管上皮細(xì)胞FGFR2基因敲除的小鼠模型。腎小管上皮細(xì)胞FGFR2基因敲除小鼠在出生后16w內(nèi)未出現(xiàn)明顯腎功能異常,但該基因敲除能夠加重IR/I或順鉑誘導(dǎo)的腎功能損傷、增加腎小管上皮細(xì)胞凋亡以及抑制Erk1,2信號的激活。在體外培養(yǎng)的大鼠腎小管上皮細(xì)胞(NRK-52E)系中,重組FGF2蛋白可以激活Erkl,2信號以及抑制順鉑誘導(dǎo)的細(xì)胞凋亡。MEK1/2抑制劑PD98059可以阻斷FGF2引起的Erkl,2磷酸化并且減弱FGF2對細(xì)胞凋亡的保護作用。因此,本部分研究證明在IR/I和順鉑誘導(dǎo)的急性腎損傷中,FGF2-FGFR2信號的激活可以通過抑制腎小管上皮細(xì)胞凋亡從而發(fā)揮腎臟保護作用,并且這個作用部分依賴于Erk1,2信號途徑的激活。慢性腎臟病(Chronic Kidney Diseases, CKD)在我國有高達10%的發(fā)病率且呈逐年增加的趨勢。腎小管間質(zhì)纖維化的程度是決定CKD進展的關(guān)鍵性病理因素。腎間質(zhì)中的肌成纖維細(xì)胞作為纖維化過程中最主要效應(yīng)細(xì)胞,其數(shù)目決定腎臟纖維化的程度和疾病的進展。成纖維生長因子7 (Fibroblast Growth Factor 7, FGF7)是一類旁分泌型FGF家族蛋白,在腎臟發(fā)育過程中起著關(guān)鍵作用,然而其在腎臟纖維化中作用特別是對間質(zhì)成纖維細(xì)胞作用還有待研究。本部分研究首先通過尾靜脈注射FGF7全長表達質(zhì)粒建立FGF7高表達小鼠模型以及建立缺血再灌注后腎臟纖維化模型,研究FGF7對腎臟間質(zhì)纖維化的影響；利用Cre-LoxP細(xì)胞建立成纖維細(xì)胞FGFR2基因缺失的小鼠模型,進一步研究FGF7-FGFR2信號對腎臟纖維化的作用以及間質(zhì)損傷后成纖維細(xì)胞增殖、活化以及對細(xì)胞外基質(zhì)合成的影響；最后在培養(yǎng)的大鼠成纖維細(xì)胞系(NRK-49F)細(xì)胞中研究體外FGF7高表達對成纖維細(xì)胞增殖的影響。實驗發(fā)現(xiàn)：體內(nèi)FGF7高表達可以加重IR／I后腎間質(zhì)纖維化的程度；成纖維細(xì)胞FGFR2基因缺失可以顯著抑制腎間質(zhì)膠原的沉積、細(xì)胞外基質(zhì)成分—纖維連接蛋白(Fibronectin, FN)的合成以及腎間質(zhì)成纖維細(xì)胞增殖和活化；并且FGFR2基因敲除抑制纖維化模型中腎組織內(nèi)Erk1,2的活化；最后,在NRK-49F細(xì)胞中,轉(zhuǎn)染FGF7質(zhì)�？梢源龠MFN的合成以及誘導(dǎo)細(xì)胞發(fā)生增殖。因此,本部分研究證明在腎臟纖維化過程中,FGF7信號高表達可以通過促進成纖維細(xì)胞的增殖和活化而加劇腎間質(zhì)纖維的程度；成纖維細(xì)胞FGFR2基因敲除可以顯著抑制腎間質(zhì)成纖維細(xì)胞增殖和活化及減輕腎纖維化的程度。腎臟纖維化以腎小球硬化和間質(zhì)纖維化為主要特征,是各種原因所致ESRD的共同病理學(xué)特征。間質(zhì)巨噬細(xì)胞浸潤以及表型轉(zhuǎn)化是影響慢性腎臟病轉(zhuǎn)歸和腎間質(zhì)纖維化的關(guān)鍵因素。成纖維細(xì)胞生長因子23(FGF23)在CKD患者循環(huán)中濃度顯著增高,然而其在腎臟病變進展中具體病理生理意義仍不清楚。本研究首先通過尾靜脈注射FGF23全長表達質(zhì)粒使體內(nèi)FGF23高表達,然后建立阿霉素(ADR)腎病模型,研究FGF23高表達對ADR誘導(dǎo)的早期足細(xì)胞損傷和后期。腎間質(zhì)纖維化以及腎小球硬化的影響,并觀察體內(nèi)FGF23高表達對腎臟內(nèi)巨噬細(xì)胞極性的影響；在體外培養(yǎng)的小鼠巨噬細(xì)胞系(RAW264.7)中研究FGF23高表達對巨噬細(xì)胞表型改變的作用以及具體分子機制；最后,在單側(cè)輸尿管梗阻(UUO)模型中驗證FGF23高表達對腎臟間質(zhì)纖維化的作用。實驗發(fā)現(xiàn),FGF23高表達加重ADR腎病模型中小鼠腎間質(zhì)膠原沉積以及腎小球硬化程度；FGF23高表達可以促進纖維化的腎組織內(nèi)巨噬細(xì)胞趨化因子TNF-α、MCP-1以及Rantes mRNA表達增加以及M2型巨噬細(xì)胞聚集；在體外培養(yǎng)的RAW264.7細(xì)胞中,FGF23高表達促使巨噬細(xì)胞表型向M2轉(zhuǎn)化；最后在UUO模型中,FGF23高表達同樣能夠加劇腎間質(zhì)纖維化的程度。因此,本部分研究證明在ADR腎病模型中,循環(huán)FGF23信號高表達可以誘導(dǎo)腎組織內(nèi)M2型巨噬細(xì)胞聚集以及加劇腎臟纖維化程度。
[Abstract]:Acute kidney injury ( AKI ) is a series of clinical syndromes which cause rapid deterioration of renal function . Although AKI has been studied in recent years in clinical practice and experimental studies , the role of fibroblast growth factor ( FGFs ) in AKI has been studied .
In vitro cultured rat renal tubular epithelial cells ( NRK - 52E ) , the effect of FGF2 - FGFR2 gene on apoptosis of renal tubular epithelial cells was studied .
The effects of FGF7 - FGFR2 signal on renal fibrosis and fibroblast proliferation , activation and the synthesis of extracellular matrix were further investigated by using mouse model of fibroblast growth FGFR2 gene deletion by using the mouse model .
Finally , the effects of high expression of FGF7 in vitro on the proliferation of fibroblasts were studied in cultured rat fibroblast cell line ( NRK - 49F ) .
The absence of FGFR2 gene in fibroblasts can significantly inhibit the deposition of renal interstitial collagen , the synthesis of extracellular matrix component - Fibronectin ( FN ) and the proliferation and activation of renal interstitial fibroblasts ;
and FGFR2 gene knockout inhibits activation of Erk1 , 2 in renal tissue in fibrosis model ;
Finally , in NRK - 49F cells , the transfection of FGF7 plasmid can promote FN synthesis and induce cell proliferation . Therefore , in the course of renal fibrosis , the high expression of FGF7 signal can increase the extent of renal interstitial fibrosis by promoting the proliferation and activation of fibroblasts ;
The effects of FGF23 overexpression on renal interstitial fibrosis and renal interstitial fibrosis were studied . The effects of FGF23 overexpression on renal interstitial fibrosis and renal interstitial fibrosis were studied .
In vitro cultured mouse macrophage system ( RAW264.7 ) , the effect of high expression of FGF23 on the phenotypic change of macrophages and the specific molecular mechanism were studied .
Finally , the effect of high expression of FGF23 on renal interstitial fibrosis was demonstrated in unilateral ureteral obstruction ( UUO ) model .
The high expression of FGF23 can promote the expression of TNF - 偽 , MCP - 1 and Rantes mRNA in the renal tissue of fibrosis and the accumulation of M2 - type macrophages .
In the RAW264.7 cells cultured in vitro , the high expression of FGF23 caused the phenotype of macrophages to be transformed to M2 ;
Finally , in UUO model , high expression of FGF23 can also aggravate the degree of renal interstitial fibrosis . Therefore , in ADR nephropathy model , high expression of circulating FGF23 signal can induce the accumulation of M2 - type macrophages in renal tissue and increase the degree of renal fibrosis .
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R692

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