成纤维细胞生长因子家族在肾脏疾病中的作用和机制研究

发布时间：2018-05-28 22:44

本文选题：成纤维细胞生长因子 + 急性肾损伤　；参考：《南京医科大学》2014年博士论文

【摘要】：急性肾损伤(Acute Kidney Injury, AKI)是一系列因素引起肾功能迅速恶化的临床综合征。虽然近年来人们在临床实践和实验研究中对AKI进行了大量的研究,但目前对于AKI患者仍以透析及对症支持治疗为主。成纤维细胞生长因子(Fibroblast Growth Factors, FGFs)广泛参与包括细胞增殖、分化、迁移、胚胎发育、血管生成调节以及损伤修复等在内的多种生物学过程。然而其在AKI中的作用特别是对损伤后肾小管上皮细胞存活的作用还有待研究。本部分研究首先通过缺血再灌注损伤(Ischemia/Reperfusion Injury, IR/I)以及腹腔注射顺铂建立AKI小鼠模型,研究损伤后肾组织中FGFs的表达变化以及细胞内下游信号通路的激活情况。实验发现：IR/I肾损伤后肾组织内FGF2、 FGF7、FGF10、FGF12、FGF13、FGF18以及FGF22表达上调；而顺铂注射后肾组织中仅FGF2和FGF 18表达增加。上述两种AKI模型肾组织中Erkl,2磷酸化水平均增加。为进一步研究FGFs-FGFR信号在AKI中的作用,我们通过Cre-LoxP系统建立肾小管上皮细胞FGFR2基因敲除的小鼠模型。肾小管上皮细胞FGFR2基因敲除小鼠在出生后16w内未出现明显肾功能异常,但该基因敲除能够加重IR/I或顺铂诱导的肾功能损伤、增加肾小管上皮细胞凋亡以及抑制Erk1,2信号的激活。在体外培养的大鼠肾小管上皮细胞(NRK-52E)系中,重组FGF2蛋白可以激活Erkl,2信号以及抑制顺铂诱导的细胞凋亡。MEK1/2抑制剂PD98059可以阻断FGF2引起的Erkl,2磷酸化并且减弱FGF2对细胞凋亡的保护作用。因此,本部分研究证明在IR/I和顺铂诱导的急性肾损伤中,FGF2-FGFR2信号的激活可以通过抑制肾小管上皮细胞凋亡从而发挥肾脏保护作用,并且这个作用部分依赖于Erk1,2信号途径的激活。慢性肾脏病(Chronic Kidney Diseases, CKD)在我国有高达10%的发病率且呈逐年增加的趋势。肾小管间质纤维化的程度是决定CKD进展的关键性病理因素。肾间质中的肌成纤维细胞作为纤维化过程中最主要效应细胞,其数目决定肾脏纤维化的程度和疾病的进展。成纤维生长因子7 (Fibroblast Growth Factor 7, FGF7)是一类旁分泌型FGF家族蛋白,在肾脏发育过程中起着关键作用,然而其在肾脏纤维化中作用特别是对间质成纤维细胞作用还有待研究。本部分研究首先通过尾静脉注射FGF7全长表达质粒建立FGF7高表达小鼠模型以及建立缺血再灌注后肾脏纤维化模型,研究FGF7对肾脏间质纤维化的影响；利用Cre-LoxP细胞建立成纤维细胞FGFR2基因缺失的小鼠模型,进一步研究FGF7-FGFR2信号对肾脏纤维化的作用以及间质损伤后成纤维细胞增殖、活化以及对细胞外基质合成的影响；最后在培养的大鼠成纤维细胞系(NRK-49F)细胞中研究体外FGF7高表达对成纤维细胞增殖的影响。实验发现：体内FGF7高表达可以加重IR／I后肾间质纤维化的程度；成纤维细胞FGFR2基因缺失可以显著抑制肾间质胶原的沉积、细胞外基质成分—纤维连接蛋白(Fibronectin, FN)的合成以及肾间质成纤维细胞增殖和活化；并且FGFR2基因敲除抑制纤维化模型中肾组织内Erk1,2的活化；最后,在NRK-49F细胞中,转染FGF7质粒可以促进FN的合成以及诱导细胞发生增殖。因此,本部分研究证明在肾脏纤维化过程中,FGF7信号高表达可以通过促进成纤维细胞的增殖和活化而加剧肾间质纤维的程度；成纤维细胞FGFR2基因敲除可以显著抑制肾间质成纤维细胞增殖和活化及减轻肾纤维化的程度。肾脏纤维化以肾小球硬化和间质纤维化为主要特征,是各种原因所致ESRD的共同病理学特征。间质巨噬细胞浸润以及表型转化是影响慢性肾脏病转归和肾间质纤维化的关键因素。成纤维细胞生长因子23(FGF23)在CKD患者循环中浓度显著增高,然而其在肾脏病变进展中具体病理生理意义仍不清楚。本研究首先通过尾静脉注射FGF23全长表达质粒使体内FGF23高表达,然后建立阿霉素(ADR)肾病模型,研究FGF23高表达对ADR诱导的早期足细胞损伤和后期。肾间质纤维化以及肾小球硬化的影响,并观察体内FGF23高表达对肾脏内巨噬细胞极性的影响；在体外培养的小鼠巨噬细胞系(RAW264.7)中研究FGF23高表达对巨噬细胞表型改变的作用以及具体分子机制；最后,在单侧输尿管梗阻(UUO)模型中验证FGF23高表达对肾脏间质纤维化的作用。实验发现,FGF23高表达加重ADR肾病模型中小鼠肾间质胶原沉积以及肾小球硬化程度；FGF23高表达可以促进纤维化的肾组织内巨噬细胞趋化因子TNF-α、MCP-1以及Rantes mRNA表达增加以及M2型巨噬细胞聚集；在体外培养的RAW264.7细胞中,FGF23高表达促使巨噬细胞表型向M2转化；最后在UUO模型中,FGF23高表达同样能够加剧肾间质纤维化的程度。因此,本部分研究证明在ADR肾病模型中,循环FGF23信号高表达可以诱导肾组织内M2型巨噬细胞聚集以及加剧肾脏纤维化程度。
[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 - 伪 , 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 .
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

【相似文献】