【作者】 林毅；

【导师】 徐虹；

【作者基本信息】 复旦大学 ， 儿科学， 2013， 博士

【摘要】 研究背景大量蛋白尿是肾病综合征的特征性表现,也是直接影响肾脏疾病进展的独立危险因素,其产生与足细胞广泛足突融合有关。拥有大量的足突是足细胞形态学的突出特点,这一结构由足细胞内肌动蛋白细胞骨架所维系。病理情况下的足细胞运动能力增加、肌动蛋白细胞骨架重排是足突广泛融合消失及大量蛋白尿发生的基础。足细胞内肌动蛋白受到诸多分子的精密调控,分布于足细胞基底膜面的整合素家族在其中起重要作用。Rho家族小GTP酶成员,特别是RhoA, Rac1和Cdc42,可以将膜蛋白的分子信号传递到肌动蛋白纤维,被公认为细胞骨架调节过程的分子开关。血管生成素样蛋白3(Angiopoietin-like3, Angptl3)是一种分泌蛋白,正常情况下肾脏仅微量表达,而在多种以蛋白尿为主要表现的肾脏疾病中表达量显著增多。我们的前期研究发现,足细胞分泌的Angptl3参与了蛋白尿的发生,且体外实验证实上调足细胞Angptl3表达可以增强足细胞活动能力。预实验中我们也发现,重组pAngptl3分子干预可使足细胞发生明显的肌动蛋白细胞骨架重排,这一过程的具体分子机制尚不清楚；Angptl3有螺旋样结构域(Coiled-coil domain, CCD)和纤维蛋白原样结构域(Fibrinogen-like domain, FLD)两个不同结构域,这是Angptl3具有多种生物学作用的基础。这两个结构域是否都参与了对足细胞的影响,目前尚不明确。本课题分两部分分别解决以上两个问题。第一部分血管生成素样蛋白3通过小GTP酶影响足细胞肌动蛋白细胞骨架重排的分子机制研究研究目的：探讨Angptl3引起足细胞肌动蛋白纤维重排的主要表现及分子机制。方法：(1)免疫荧光染色检测细胞株WT-1及podocin的表达,鉴定足细胞株；(2)重组Angptl3分子干预培养足细胞,以荧光素标记的鬼笔环肽对足细胞肌动蛋白纤维染色,荧光显微观察肌动蛋白骨架重排情况；(3)重组Angptl3分子干预培养足细胞,以G-LISATM方法检测足细胞小GTP酶的激活情况；(4)阻断剂阻断可被Angptl3活化的小GTP酶,观察Angptl3引起的肌动蛋白骨架重排现象是否消失；(5)阻断整合素αvβ3,然后应用重组Angptl3干预培养足细胞,观察是否可以阻断足细胞骨架重排、检测是否可以阻断小GTP酶的激活；(6)分别阻断整合素αvβ3的下游关键分子FAK和PI3K,观察是否可以阻断Angptl3引起的足细胞肌动蛋白骨架重排及小GTP酶激活,并应用Western Blot检测可能的下游分子的磷酸化水平变化,以明确可能的分子通路；(7)以WesternBlot检测Angptl3干预后小GTP酶的表达水平变化,明确Angptl3是否可以影响小GTP酶的表达。结果：(1)足细胞株WT-1及podocin表达均呈阳性,可用于实验；(2)Angptl3可使足细胞出现明显的肌动蛋白细胞骨架重排,主要表现为板状伪足和细胞棘的生成；(3)Angptl3可激活足细胞中的小GTP酶Racl和RhoA,其中Racl活化程度较强、持续时间长,RhoA仅存在一个瞬时、低水平的活化；(4)阻断Racl可阻断Angptl3引起的足细胞板状伪足的生成；(5)阻断整合素αvβ3可阻断Angptl3引起的足细胞肌动蛋白纤维重排及阻断小GTP酶激活；(6)阻断FAK或PI3K均可阻断Angptl3引起的足细胞板状伪足的生成及并阻断Racl的激活；Angptl3可使足细胞FAK、PI3K的磷酸化水平增高；阻断整合素αvβ3可阻断Angptl2引起的FAK、PI3K磷酸化；阻断FAK可阻断Angpt13引起的PI3K的磷酸化；(7)Angptl3可引起足细胞Rac1表达水平的增加,不影响RhoA及Cdc42表达。结论：(1)Angptl3可以引起足细胞肌动蛋白细胞骨架重排,主要作用为促进板状伪足生成；(2)Rac1和RhoA的激活,特别是Rac1激活,是Angptl3引起足细胞肌动蛋白细胞骨架重排的基础；(3)Angplt3引起足细胞板状伪足生成通过整合素αvβp3-FAK-PI3K-Rac1途径；(4)足细胞可增加Rac1的表达。第二部分血管生成素样蛋白3不同结构域对足细胞肌动蛋白纤维重排及足细胞失黏附损伤的影响研究目的：明确Angpt13不同结构域对足细胞肌动蛋白骨架重排的影响；探讨Angptl3对嘌呤霉素氨基核苷(Puromycin aminonucleoside, PAN)引起的足细胞失黏附的影响,及各个结构域在其中的作用方法：(1)应用重组Aingptl3-CCD分子片段、Angptl3-FLD分子片段以及重组完整Angptl3分子干预培养足细胞,分别观察其对足细胞肌动蛋白纤维重排情况的影响；(2)应用重组Angpt13分子分别按浓度和时间梯度干预培养足细胞,之后给予PAN处理足细胞,应用Hexosaminidase Assay观察Angptl3对PAN引起的足细胞失黏附情况的影响；应用重组Angptl3-CCD分子片段、Angptl3-FLD分子片段预处理足细胞,继给予PAN处理,观察Angptl3各个结构域对PAN引起的足细胞失黏附情况的影响。结果：(1)仅Angptl3-FLD可引起足细胞肌动蛋白细胞骨架重排,且与Aingptl3完整分子处理时等效；(2)Angptl3预处理后,足细胞较对照组失黏附情况明显减轻；且失黏附的减轻程度随处理时间的延长及处理剂量的加大而明显；Angptl3-FLD分子片段干预的结果与完整Angptl3分子干预相类似,随干预时间的延长足细胞失黏附情况较前减轻；Angptl3-CCD分子片段干预时广足细胞表现出失黏附情况先加重后减轻的情况。结论：(1)FLD片段是Angptl3引起足细胞肌动蛋白骨架重排的关键结构域；CCD片段不参与Angptl3对足细胞肌动蛋白骨架重排的影响；(2)Angptl3可减轻PAN引起的足细胞失黏附损伤；FLD和CCD片段都参与了PAN对足细胞失黏附损伤的影响,两者作用效果有所不同,FLD片段起主导作用；CCD片段也可对足细胞产生影响,其受体和具体途径目前尚不明确。更多还原

【Abstract】 BackgroundsSignificant proteinuria is the main character of nephrotic syndrome which is related with diffused podocyte foot process effacement, is an independant risk factor for renal diseases. Foot process is a characteristic structure of glomerular podocytes, which is maintained by actin filaments of podocyte. Diffused foot process effacement and proteinuria is related with the increase of podocyte motility.Actin filament in podocyte is under highly regulation of one than100kinds of moleculi, in which integrin of podocyte basement memebrane area plays an important part. Rho family small GTPases, espacially RhoA, Racl and Cdc42are believed to be the most important moleculi in actin filament regulation for the effect of passing cell signal from membrane molecule to actin filament.Angiopoietin-like3(Angptl3) is a secreted protein weakly expressed in normal kidney, and the expression of which increases greatly in proteinuic diseases. In our previous study, podocyte secreted Angptl3was proved to involve in proteinuria development. And up-regulation of Angptl3increases the motility of podocyte in vitro, the molecular pathway of which is not clear. There are two different domains in Angptl3which is the coiled-coil domain (CCD) and the fibrinogen-like domain (FLD), which is the structural basis for its multiple biological functions. Whether the two domains involve in the effect on podocytes is still not sure. Part I. Study on molecular mechanisms of Angiopoietin-like3on podocytes actin cytoskeleton rearrangement through small GTPasesObjectives To investigate the molecular mechanisms of Angptl3induced podocyte actin filaments rearrangement.Methods1. To identify the cell line by detecting WT-1and podocin expression with immonofluorescence staining.2. To observe the rearrangement of podocytes actin filaments by staining podocyte with FITC-labelled phalloidine following Angptl3treatment.3. To detect the small GTPase activation level in podocyte treated with Angptl3by G-LISATM assay.4. To observe whether blocking the small GTPases could inhibit the effect of Angptl3on actin filament rearrangement.5. To observe whether blocking integrin αvβ3could inhibit the effect of Angptl3on actin filament rearrangement and small GTPases activation.6. To observe whether blocking downstream molecules of integrin αvβ3could inhibit the effect of Angptl3on actin filament rearrangement and small GTPases activation. And to detect key downstream molecule phosphorylation with Western Blot after blocking upstream molecule followed by Angptl3treatment.7. To detect small GTPases expression with Western Blot after Angptl3treatment.Results1. The WT-1and podocin were both expressed in our podocyte cell line, demonatrating the cell line could be used in the following experiment.2. Angptl3treatment could induce podocyte actin filament rearrangement, mainly expressed as lamellipodia and cell spikes formation.3. Angptl3could lead to podocyte small GTPases Racl and RhoA acivation. Among which Racl expressed a stronger and long lasting activation while RhoA only expressed a low level and short term activation.4. Blocking Racl could block the podocyte lamellipodia formation effect induced by Angptl3.5. Blocking integrin αvβ3could block the lamellipodia formation effect and small GTPases activation induced by Angptl3.6. Blocking FAK or PI3K, which are downstream molecules of integrin avP3, could block the lamellipodia formation effect and small GTPases activation induced by Angptl3. Recombinant Angptl3treatment could lead to FAK and PI3K phosphorylation in podocytes. Blocking integrin avP3could block the phosphorylation of both FAK and PI3K induced by Angptl3, while blocking FAK could block the phosphorylation of PI3K induced by Angptl3.7. Recombinant Angptl3treatment could increase the Racl expression in podocyte. Summary1. Angptl3could induce podocyte actin filaments rearrangement, which mainly expressed as lamellipodia formation.2. Small GTPases Racl and RhoA, especially Racl activation were of great important part in Angptl3induced podocyte F-actin rearrangement.3. Angptl3induced podocyte lamellipodia formation through integrin αvβ3-FAK-PI3K-Racl pathway.4. Angptl3could increase Racl expression but not RhoA or Cdc42expression in podocytes. Part Ⅱ. Effects of different domains of Angptl3on podocyte actin filaments rearrangement and podocyte detachmentObjectives1. Study the effect of different domains of Angptl3on podocyte actin filament rearrangement.2. Study the effect of Angptl3and its different domains on podocytes detachment injury induced by Puromycin aminonucleoside (PAN).Methods1. To observe the podocyte actin filaments rearrangement with FITC-labelled phalloidine staining after Angptl3, or its CCD or FLD domain fragments treatment.2. To study whether Angptl3or its different domains have effect on PAN induced podocyte detachment by pretreating podocytes with Angptl3or its CCD or FLD domain fragments followed by PAN treatment.Results1. Angptl3-FLD fragment could induce podocyte F-actin rearrangement, while Angptl3-CCD did not have such effect.2. Angptl3treatment alleviates the PAN induced podocytes detachment, and the anti-detachment effects were time and dose dependent. Angptl3-FLD treatment showed the similar effects as complete Angptl3on PAN induced podocyte detachmen. Angptl3-CCD fragement increased podocyte detachment followed a resistant to PAN induced podocyte detachment.Summary1. FLD fragement was the key domain inducing podocyte F-actin rearrangement.2. Angptl3could resist the PAN induced podocyte detachment. Both FLD domain and CCD domain were involved in this effect but played different part. FLD domain was the most important domain in this process. CCD domain also had its effect of podocyte, the receptor and molecular pathway still needs further study.更多还原