【作者】 苏蔚；

【导师】 申宗侯； 郝传明； 杨海春； 陈靖； 林善锬；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 第一部分巢蛋白Nestin在正常成人肾组织中的表达背景足细胞是位于肾小球基底膜外具有大量突起的特殊上皮细胞，是肾小球滤过膜的重要组成成分，足细胞依靠其指状足突附着在毛细血管基底膜表面，足突之间形成曲折的滤过裂隙，是肾小球滤过膜的最后一道屏障，足细胞异常是产生蛋白尿的重要机制。它可以通过调整自身形态进而改变细胞之间的滤过间隙从而适应肾小球滤过容积的改变，而这一功能的完成依赖于足细胞强大的细胞骨架系统。nestin(巢蛋白)是一种属于中间丝的细胞骨架蛋白。巢蛋白首先被发现表达于胚胎神经干细胞。随后的研究发现，在多种组织(肌肉、胰岛、皮肤等)的干细胞或前体细胞中也有巢蛋白的表达。巢蛋白在干细胞中的表达通常为一过性，随组织分化成熟，巢蛋白的表达即消失。为此，巢蛋白被广泛用作干细胞的标志性蛋白。最近的研究发现在小鼠肾脏的发育成熟的足细胞中有大量巢蛋白表达。然而Nestin是否也表达于人类足细胞尚不清楚。本实验观察了Nestin在正常成人肾组织中的表达，并且明确了Nestin在足细胞中的定位。方法正常成人肾组织来自于肾癌手术切除肾组织，应用免疫组化和免疫荧光的方法观察Nestin的表达；应用免疫电镜技术检测Nestin在足细胞中的定位。结果正常人肾组织中巢蛋白选择性表达于肾小球，Nestin主要围绕在肾小球毛细血管袢外周，呈线状或点状分布；Nestin与基底膜标志物Laminin和足细胞标志蛋白WT1荧光双染显示：Nestin表达于基底膜外，WT1阳性的细胞胞浆中。免疫电镜进一步确定了Nestin的阳性颗粒主要分布于足细胞胞浆和初级足突，二级足突中少见。结论(1)在正常成人肾组织中Nestin选择性表达于肾小球足细胞中(2) Nestin定位于足细胞的胞浆和一级足突第二部分Nestin在足细胞中作用的探讨(一)足细胞中与Nestin相互作用的蛋白分子背景Nestin蛋白全长约1600个氨基酸，分子量约为240KD，包括一个高度保守的300～330个氨基酸的α—螺旋核心结构和两侧的N-末端，C—末端。由于N末端较短，只有11个氨基酸残基，使之成为细胞内可溶性的非多聚型蛋白，它不能自行组装成中间丝，必需依赖与其共存的其他中间丝分子如vimentin，desmin，α—intemexin等形成异源二聚体，然后参与中间丝的组成。越来越多的研究提示，足细胞中丰富的细胞骨架蛋白在维持足细胞的形态及滤过屏障功能中起重要作用。对Nestin的结构研究提示，Nestin蛋白分子中含有多个可与其他结构蛋白结合的区域，因此很可能在足细胞结构蛋白的组装中起重要的桥梁作用。本研究就足细胞中可与Nestin相互作用的其他结构蛋白进行了研究。方法体外培养条件性永生的小鼠足细胞，应用免疫荧光的方法检测Nestin在足细胞中的表达；免疫荧光双染的方法检测足细胞中Nestin与中间丝蛋白Vimentin、α—internexin的共表达；用羊抗鼠的巢蛋白抗体做免疫共沉淀，以羊抗鼠Vimentin抗体和兔抗鼠α-internexin抗体为一抗进行western blot，分别以细胞裂解蛋白和以小鼠IgG做免疫共沉淀蛋白为阳性对照和阴性对照。结果体外培养的37度分化状态的足细胞，细胞形成树枝状结构，Nestin免疫反应蛋白沿细胞纵轴呈束状排列，无横向排列，并由胞浆延伸至一级足突和二级足突，呈纤维细丝状整齐排列，在核周形成长卷曲状纤维，呈笼状结构围绕细胞核；免疫荧光显示vimentin及α-internexin均呈纤维细丝状分布，由胞浆延伸至足突，荧光双标显示Nestin与vimentin或internexin能够完全吻合。免疫共沉淀结果显示：在Vimentin(58 KD)和α-internexin(66KD)的位置均有清晰条带，提示该两种蛋白与巢蛋白结合在一起。以同样的方法检测α-actinin4，100KD的位置未见到免疫反应阳性信号。结论足细胞中Nestin能够与第Ⅲ类中间丝蛋白Vimentin及第Ⅳ类中间丝蛋白α-internexin直接相互作用，而不与α-actinin4直接相互作用。(二) nestin在维持足细胞形态方面的作用背景自Urban Lendahl发现Nestin表达于CNS干细胞中以来，越来越多的功能在异源细胞中被发现，诸如在细胞骨架蛋白中的连接作用；磷酸化调节其他中间丝蛋白的聚合和解聚，参与有丝分裂过程中细胞骨架蛋白的重排；参与细胞形态的重塑；。但是Nestin在终末分化状态组织中的表达及作用还未有报道。本实验结果首次显示Nestin并不是干细胞和快速分裂细胞的专有蛋白，还可表达于高度分化的足细胞中，而且提示Nestin与足细胞正常形态和功能的维持有关。但是Nestin在足突结构中的作用机制还有待于进一步研究。方法体外培养条件性永生的小鼠足细胞，应用RNA干扰技术使得Nestin在足细胞中的表达被抑制，western blotting和免疫荧光的方法检测抑制效率；观察Nestin蛋白被抑制后足细胞形态的改变；计数每高倍视野转染和未转染nestin siRNA细胞有足突的数目，并作统计学分析。结果Nestin在足细胞中被抑制后，足细胞形态发生改变，失去Nestin的足细胞足突变短或消失。转染nestin siRNA的细胞每高倍视野有足突的细胞数目为16％±4.6％，转染阴性对照质粒的细胞每高倍视野有足突的细胞数目为77％±6.3％，两者有显著差异。结论Nestin在维持足细胞正常形态方面具有一定意义第三部分病理状态Nestin在人肾组织中的表达及其与蛋白尿的关系背景足细胞对维持肾小球功能具有重要意义，足细胞的足突形成曲折的滤过裂隙，是肾小球滤过膜的最后一道屏障，阻止蛋白成分随尿液流失。足突融合是蛋白尿产生的形态学基础。中间丝蛋白是维持细胞和组织的完整性的重要细胞结构蛋白，中间丝蛋白对于足细胞抵抗机械压力、阻止血管壁膨突、保护肾小球毛细血管丛的正常结构等方面具有重要意义。我们的初步细胞实验提示，Nestin可能对足细胞结构蛋白的中间丝的组装以及足突正常结构的维持具有重要作用。本研究观察了Nestin表达水平与蛋白尿的关系，初步探讨Nestin与足细胞损伤的联系。但是关于Nestin在足细胞损伤中的确切特性和作用机制尚未完全清楚。方法病理肾组织标本取自于华山医院肾脏科1997—2005年肾穿刺存档标本，其中微小病变性肾病(MCD)10例、膜性肾病(MN)8例、IgA相关肾病(无蛋白尿，无足突融合)4例、局灶阶段硬化性肾小球肾炎(FSGS)10例、糖尿病肾病(DN)4例。应用免疫组化方法检测Nestin在各种病理状态的表达；应用连续切片分别做Nestin、WT1免疫组化，观察Nestin表达与足细胞丢失的关系；对Nestin表达阳性指数进行半定量分析，与临床蛋白尿水平统计处理，分析两者之间相关关系。结果IgA相关肾病中Nestin的表达水平与正常肾组织没有显著差别；FSGS病例中，硬化区域的Nestin的表达由于足细胞的丢失而减少，因为连续切片在同一区域未见到WT1的表达。MCD、MN中，Nestin在肾小球中的表达比正常肾组织显著降低，连续切片显示WT1表达未见减少；MCD、MN、FSGS病例中Nestin表达水平与24小时蛋白尿呈负相关。结论(1)MCD、MN、FSGS病例中，由于足突融合Nestin表达水平降低，IgA相关肾病中未见足突融合，Nestin表达水平没有显著降低，提示Nestin表达水平与肾小球中足细胞的损伤有关。(2) Nestin表达水平降低与蛋白尿的相关性提示Nestin与足细胞损伤后蛋白尿的形成有关。更多还原

【Abstract】 PART I the expression of Nest in in the normal adult kidneyBackground podocytes are highly specialized cells with a voluminous cell body. It’s location, It’s architecture, and it’s relevance are unique. Their most prominent features are interdigitated foot processes. These foot processes are bridged by the slit diaphragm, which plays a major role in establishing selective permeability of the glomerular filtration barrier .They have adapted themselves to facilitating the bulk flow of the glomerular filtrate through the intercellular spaces or filtration slits. Nestin is a cytoskeleton associated class VI intermediate filament first identified in mitotically active central and peripheral nervous system progenitor cells that give rise to both neurons and glia during early neurogenesis. Subsequently, nestin is detected in progenitor cells of non-neuronal tissues, including muscle, the heart, pancreas and the skin follicle. Recently nestin expression has also been reported in adult tissues, such as skin and central nervous system, but is mainly restricted to areas of regeneration. Owing to its characteristic expression pattern, nestin is generally considered a marker of stem cells or progenitor cell. The present study show in the adult human kidney, Nestin expression is restricted to differentiated podocytes.However, wether Nestin can be expressed in human kidney remains explored.The present studies showed the expression of Nestin in the human kidney and identified the localization in the podocyte.Methods The expression of Nestin was detected by immunofluoresence; Normal kidneys from surgical nephrectomy were obtained during resection of kidneys for renal tumor. The expression of Nestin was examined by the immunohistochemistry and immunofluoresence; With the immunoelectron microscope, the location of Nestin in the podocyte was detected.Results Nestin was selectively expressed in the glomerular podocytes. Co-staining with anti-laminin antibody,which labels glomerular basement membrane, demonstrated that nestinimmunoreactivity was localized outside the glomerular basement membraneCo-staining with WT1 antibody showed that nestin immunoreactive protein waslocalized in the cytosol of the cells that were positive for wt1, a marker of podocyte.Immunoelectron microscopy showed nestin immunoreactivity in the cell body as wellas in primary and secondary processConclusions (1) Nestin was selectively expressed in the adult human glomerularpodocytes.(2) Nestin was localized in the cell body as well as in primary and secondary processPART II The effect of ncstin expression in podocyte1、 The interacting proteins with NestinBackground Nestin is a large protein (>1600 amino acids).As is typical for IF proteins, nestin is characterized by an α -helical ’rod’ domain that contains repeated hydrophobic heptad motif. The 240KD protein nestin contains a short N-terminus and an unusually long C-terminus. Nestin is unable to self-assemble, most likely because of its very short N-terminus(a domain necessary for IF assembly);therefore, nestin requires the presence of other IF proteins,such as vimentin or desmin,to assemble into heterodimers and mixed polymers. Since Nestin is reported to interact with all three major classes of cytoskeletal proteins, Nestin may be involved in the organization of cellular cytoskeletons,and may therefore play an important role in the maintenance normal podocyte function.To determine the portential significance of Nestin in podocyte,we examine the interaction of Nestin with other IF. Methods Immortalized murine podocytes were cultured .Co-expression of nestin with vimentin and α -internexin in the cultured podocye with immunofluoresence double stains. Podocyte protein extract was immunoprecipitated by anti nestin antibody. The immunoprecipitated proteins were separated on SDS-PAGE and blotted for vimentin α -internexin and nestin. The total cell lysate and normal mouse IgG were used as the positive and negative controlsResults. Immortalized mouse podocyte clones(MPC)grown under nonpermissive conditions display characteristics of differentiated arborized-type. Nestin is organized into fibroblast-like stress fibers extending into the processes of the differentiated cells and it forms a cage-like structure surrounding the nuclear. Immunofluoscence showed that nestin and vimentin ,nestin and α -internexin co-localized in the adult human podocyte. In cultured differentiated podocytes, nestin vimentin and α -internexin immunofluorescence demonstrated filamentous staining in cytosol and processes, and nestin co-localized with vimentin and α -internexin. Immunoprecipitation using anti-nestin antibody not only precipitated nestin, but also precipitated vimentin and α-interexin detected by immunoblot via an anti-vimentin and α -internexin antibody. With the same way, α -actinin4 could not be detected . Conclusion Nestin could physically interact with vimentin and α -internexin, but notwith α -actinin42、 The role of Nestin in the maintance the structure of podocyteBackground Two decades ago, Nestin was first identified in a population ofproliferating neuronal cells, which was recognized as neuronal stem cell. Later, moreand more functions were identified in non-neuronal cells. such as the important role incoordinating or bridging cytoskeletal proteins; in regulation of the assembly anddisassembly of IF, participating in remodeling of the cell. But the nestin expressionand function in the differentiated cells have no reports. The present studies show thatnestin is not only a special protein in the stem cell and rapidly dividing progenitor cell,but also can express in differentiated podocyte. It is suggested that nestin may play animportant role in maintaining normal structure of podocyte .But the precise role ofNestin in the processes structure remains to be fully elucidated.Methods The immortalized murine podocytes were cultured. In differentiatedpodocytes,the expression of nestin was knock-down by RNAi. The effect of nestinknock-down was examined by western blotting and immunofluoresence. The cellswith the projections longer than maximal length of the cell body in siRNA or controlvector transfected cells were counted respectively.Results Nestin siRNA markedly reduced or abolished nestin expression .Moreimportantly, siRNA nestin knock-down was associated with significantly reducedprocess formation. In cells transfected with nestin siRNA, the percentage of cells withprocesses was significantly lower than cells transfected with control vector(77% ±6.3% vs 16%±4.6%,n=3,p<0.01).Conclusion nestin may play an important role in maintaining normal function ofpodocyte .part IIIThe expression of nestin in the human disease kidneyBackground The visceral glomerular epitheliar cell,also known as the podocyte, plays an important role in the maintenance of renal glomerular function .This cell type is highly specialized and its foot processes together with the interposed slit diaphragm(SD) form the final barrier to urinary protein loss. Effacement of foot processes is the morphological basement of proteinuria .To maintain an intact glomerular filter barrier ,podocyte-podocyte interactions and podocyte interactions with the glomerular basement membrane are essential. Formation and maintenance of these podocyte processes depend on a well-developed and complex cytoskeleton. The podocyte cytoskeleton is also suggested to play a critical role in counterbalancing the mechanical stretch stress,preventing outward ballooning of the vessel and preserving the normal architecture of the glomerular tuft.Our cellular experiments showed that Nestin may be involved in the intermediate filament arrangement and the processes structure maintanace .The present studies demonstrated the relationship between the podocyte Nestin expression and proteinuria.It is suggested the reduction of Nestin expression is associated with podocyte injury. The mechanism by which Nestin is involved in maintaining normal podocyte function and the role of Nestin in diseased podocyte remains to be explored.Methods Kidney tissues were obtained from needle biopsies of 25 patients with nephritic syndrome and four patients diagnosed with IgA nephropathy with mild mesangial expansion (IgA GN).The pathological diagnosis of these patients with proteinuria was minimal change disease (MCD, n=10),membranous nephropathy(MN, n=8,) focal segmental glomerular sclerosis(FSGS, n=3), diabetic nephropathy (DN, n=4). The expression of nestin in these diseases was detected by immunohistrochemistry .Series sections of renal biopsies were stained with nestin and WT1 respectively. Nestin expression levels were assessed semi-quantitatively using morphometric analysis.Results Semi-quantitative morphometric analysis , which examined both area and signal intensity of staining, showed that glomerular nestin expression levels in IgAGN did not differ from normal control. However, glomerular nestin expression levels in MCD,MN were significantly lower compared to normal kidney and IgAGN. In FSGS ,nestin expression in sclerotic area was substantially reduced or absent.this appears to be due to loss of podocytes in these areas, since WT1was absent in the same sclerotic area in a continuous series of sections. Glomerular nestin expressionlevel was inversely correlated with 24 hour proteinuria.Conclusions Podocyte nestin expression reduced in kidneys with podocyte foot processes effacement and proteinuria. The present studies suggest that nestin may play an important role in maintaining normal function of podocyte in human kidney.更多还原