肝星状细胞中表皮形态发生素表达调控机制及其作用研究

肝星状细胞是肝脏中重要的间质细胞,是肝细胞外基质的主要来源.表皮形态发生素(epimorphin、EPM、syntaxin2)在肝脏发育、再生及癌变过程中发挥了重要的作用,目前其表达变化的调控机制及对肝星状细胞的作用还未有报道.通过对肝组织标本进行检测,发现肝纤维化过程中肝星状细胞表达EPM上调.从表观遗传学的角度对EPM表达变化调控机制进行研究,发现DNA去甲基化促进了EPM的表达.为了研究EPM对肝星状细胞的可能的调节作用,将EPM表达质粒转染肝星状细胞,之后检测了EPM对肝星状细胞增殖及迁移能力的变化.结果证明EPM能够促进肝星状细胞的增殖与迁移.本研究发现,激活的肝星状细胞高表达EPM可能是由于DNA去甲基化引起的,同时,高表达的EPM能够促进肝星状细胞的增殖与迁移,进而促进肝纤维化进展.     

14种木犀榄族植物叶表皮微形态的研究

利用光学显微镜和扫描电子显微镜对木犀榄族（Oleeae）5属14种植物的叶表皮微形态特征进行了观察,结果表明：（1）叶表皮细胞形状有无规则形和多边形两种;下表皮腺点的数目远远大于上表皮,流苏树上表皮细胞没有腺点;叶下表皮有气孔器,形状为圆形、椭圆形;气孔器均为不规则型;气孔器外围角质层有放射状、条状、环状、颗粒状等多种类型;（2）气孔器和叶表皮细胞许多特征具有明显的种间差异,可以作为种间鉴定的重要依据;（3）叶表皮微形态结构上的许多共同特征,表明木犀榄族植物是单独一自然类群。上述特征可为木犀榄族植物属及物种的划分与鉴定、系统演化关系的探讨等提供参考。     

中国羊茅属部分植物叶下表皮微形态研究

通过光学显微镜及电子显微镜对国产羊茅属21个种叶下表皮微形态进行观察。结果显示:(1)羊茅属叶下表皮属于典型的狐茅型。长细胞长筒状或短筒状,细胞壁波状弯曲,少数细胞壁平直;短细胞单生或对生,方形、椭圆形或新月形;气孔器少见或1列至多列,副卫细胞平行形到圆屋顶形;刺细胞常见或少见;脉上硅细胞方形、椭圆形、新月形或长方形边缘波状弯曲。许多结构细胞在羊茅属内存在较丰富的变异。(2)对羊茅属的叶表皮微形态特征进行UPGMA的聚类分析,结果分成了2个分支,其中分支Ⅰ的植物叶下表皮几乎无气孔,刺细胞常见,且分支Ⅰ由羊茅亚属植物组成;分支Ⅱ的植物叶下表皮气孔常见,气孔器1至多列,刺细胞少见,且分支Ⅱ由长花序亚属、宽叶亚属、贫芒亚属植物组成。     

山东飘拂草属叶下表皮微形态的研究

利用光学显微镜观察了飘拂草属12个类群（包括变种和变型）的叶片下表皮微形态。飘拂草属叶下表皮微形态基本特征如下：即长细胞为长筒形、短筒形,少数为近方形或多边形,边缘深波状、浅波状至近平直;无短细胞存在;刺毛仅存在于个别种中;气孔器大多为1～2列,少数2～4列;副卫细胞为圆屋顶形至三角形、圆屋顶形,少数为平行形。根据脉间刺毛的有无,可以分为无刺毛类型和有刺毛类型。根据外部形态和叶下表皮微形态上的区别,矮扁鞘飘拂草,应恢复其种级（Fimbristylis kraussiana Hochst. et Steud.）。矮两歧飘拂草作为变种处理比较好,其新等级种名为：F.dichotoma (L.) Vahl var. depauperata（C. B. Clarke）F. Z. Li et L. N. Liu comb. &; stat. nov.     

RNA干扰技术体外抑制肝星状细胞表皮形态发生素表达及其对肝癌细胞迁移能力的影响

肿瘤微环境中肝星状细胞能够影响肝癌细胞的生物学行为.在肝脏发育过程中,肝星状细胞通过表皮形态发生素(epimorphin,EPM,syntaxin2)与肝干细胞接触而促进肝干细胞的功能性分化.EPM在溃疡性结肠炎、间质性肺炎以及结肠癌中也发挥了重要的作用.发现肝细胞癌(HCC)细胞能够促使星状细胞EPM表达上调.为了研究肝星状细胞的EPM对于肝癌可能的作用,构建了针对EPM基因表达的shRNA干扰载体,并将质粒转染肝星状细胞,获得了两株携带该干扰片段的细胞系.RT-PCR与Westernblot检测结果表明,转基因干扰星状细胞系EPMmRNA和蛋白质表达量明显降低.之后,使用转基因细胞系的条件培养基对于肿瘤细胞进行侵袭能力的检测,并对星状细胞与肝癌细胞三维共培养,证明EPM干涉后肝星状细胞促进肿瘤细胞迁移的能力降低.结果表明通过RNAi可稳定干扰人肝星状细胞EPM基因的表达,并且EPM能够促进肝癌细胞的转移.     

表皮形态发生素对肝细胞癌SK-HEP-1细胞生物学活性的影响

目的：明确表皮形态发生素（EPM）对盱细胞癌SK-HEP-1细胞生物学行为的影响。方法：构建高表达EPM的SK-HEP-1细胞,real-timePCR和Westem印迹检测EPM在肿瘤细胞内的表达,CCK8分析和克隆形成实验检测细胞的增殖能力,Matrigel-transwell实验检测细胞的浸润能力。结果：EPM在肿瘤细胞内的高表达不影响细胞的增殖能力,怛明显增强肿瘤细胞的浸润能力。结论：肝癌肿瘤微环境有可能通过EPM影响肿瘤细胞的生物学活性,对其作用机制的进一步明确,将有助于阐明肝癌发生发展的病理机制,发现新的恶性肿瘤诊断和治疗手段。     

斑马鱼管腔器官空腔形态发生及分子机制

后生动物复杂的体内结构和器官结构多以网络状的管道系统出现。中空的管腔作为这个系统的重要结构单元承担了运输物质、区分器官不同部位功能、分隔机体和外环境等诸多重要的生理功能。管腔的发育障碍将致使相关器官形态发生畸形、功能紊乱。管腔型器官形态发生易被直接观察以及各种相关突变鱼和荧光转基因鱼的出现, 使得斑马鱼(Danio rerio)成为管道器官研究的优秀模式动物。斑马鱼血管、神经管、小肠、胰腺外分泌腺、前肾管等几种重要的器官的形态发生都伴随着典型的腔道发育过程, 是研究管腔形成的重要器官模型。管腔形成由胞外信号诱导、细胞极性化、胞内物质定向运输、腔内液体形成和胞内细胞骨架重构等相关管腔细胞内外发生的结构功能变化过程所构成, 而这些结构与功能的变化过程是通过精确而复杂的分子调控网络来实现, 最终形成管道器官。文章对斑马鱼4种典型管腔型器官的空腔形态发生过程进行了综述, 并总结了此过程中的分子机制, 为今后的相关研究提供了参考。     

小鼠胎肺分支形态发生的分子机制

哺乳动物在早期胚胎发育过程中,肺发育经历了气管分支的形态发生、树样结构上皮管道的形成,并伴随着血管的发育而发生的气体通路和肺泡的分化等过程.肺发生涉及到许多复杂的分子机制.肺形态学的变化受到一系列持家基因、激素、核转录因子、生长因子及其他因素的综合调控.目前已经发现决定肺分支形态发生的许多重要因子.本文根据目前最新研究进展,阐述了小鼠胚胎肺在分支形态发生过程中,上皮与间充质之间诱导的信号通路之间的相互作用及其对呼吸树形态建成的调控机制.     

叉蕨科30种植物叶表皮形态特征研究

利用光镜对叉蕨科7属30种植物叶表皮形态特征进行详细观察研究。结果显示：（1）叉蕨科30种植物的叶上表皮和下表皮细胞形状均为不规则型,垂周壁式样为深波状或浅波状,具单晶或针晶;上表皮细胞的长宽比为1.62~4.0,下表皮细胞的长宽比为1.63~3.06。（2）在30种植物中共观察到7种气孔器类型,分别为：极细胞型、腋下细胞型、聚合极细胞型、聚腋下细胞型、不等细胞型、无规则四细胞型和不规则型,每种植物分别具有4~7种气孔器类型,均为下生型气孔;气孔长宽比为1.22~1.91,气孔密度为8~76个/mm²,气孔指数为3.9%~25.7%。（3）基于气孔器类型组成进行聚类分析,可将30种植物分成3个类群。（4）对叶表皮形态特征分析认为,轴脉蕨属应介于叉蕨属和肋毛蕨属之间,且与叉蕨属关系更近;叉蕨属的范畴还有待进一步研究;支持将肋毛蕨属从叉蕨科中分离出来置于鳞毛蕨科,但不支持黄腺羽蕨属归入鳞毛蕨科。     

15种铁角蕨科植物叶表皮形态特征的研究

利用光学显微镜对铁角蕨科15种植物的叶表皮形态特征进行观察。研究结果表明：（1）15种铁角蕨科植物的叶上、下表皮细胞形状为不规则型,垂周壁为深波状、波状或浅波状;上表皮细胞长宽比1.3~2.6,下表皮细胞长宽比1.3~4.1;（2）在15种铁角蕨科植物中共观察到7种气孔器类型,分别为腋下细胞型、不等细胞型、无规则四细胞型、无规则型、极细胞型、聚合极细胞型和聚腋下细胞型,每种植物具有2~5种气孔器类型,气孔均为下生型,多为椭圆形;气孔的长宽比1.12~2.81,气孔密度16.4~105.1个·mm^-2,气孔指数为5.7%~21.1%;（3）铁角蕨科植物叶表皮形态特征中的上表皮细胞形状、垂周壁形状、上表皮细胞长宽比、主要气孔器类型及衍生类型等具有一定的属内稳定性,可作为铁角蕨科属间分类的依据之一;（4）该研究在一定程度上支持秦仁昌和吴兆洪对铁角蕨科的划分以及铁角蕨属内分组和组内分系,并为铁角蕨科植物的分类鉴定及系统演化研究提供基础资料。     

Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: involvement of small G protein RhoA and C/EBPβ

Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem-like cells (WB-F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67(EPM)) with WB-F344 cells. Here, we showed that EPM could promote WB-F344 cells differentiation into bile duct-like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin-1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA-WB-F344 cell line (WB(RhoA-DN)) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up-regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM-induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase-reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPβ. Taken together, these data demonstrated that EPM regulates bile duct formation of WB-F344 cells through effects on RhoA and C/EBPβ, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.     

Getting to the heart of planar cell polarity signaling

The genes that underpin normal heart development, and which can be disrupted to result in congenital structural malformations, are rapidly being uncovered. However, the specific cellular processes that lie downstream of these genetic cascades, accurately shaping tissues and complex structures within the heart, remain relatively unclear. The noncanonical Wnt planar cell polarity (PCP) signaling pathway is known to have a role in embryonic morphogenesis and as such is an important candidate pathway to carry out these roles in heart development. The pathway regulates the polarization of cells in a variety of contexts, allowing cells to change shape and position and to "know" their orientation within a mass of tissue. PCP signaling has also been shown recently to regulate the cellular position of the primary cilium. This organelle is known to be crucial for the establishment of left-right patterning in the early embryo and may also act as a signaling antenna for other developmental and regulatory pathways. It is not surprising that recent studies have also linked PCP to left-right patterning. In this review, we will examine the current evidence suggesting that PCP signaling has a central role in cardiac development and malformation.     

Epimorphin Regulates Bile Duct Formation via Effects on Mitosis Orientation in Rat Liver Epithelial Stem-Like Cells

Understanding how hepatic precursor cells can generate differentiated bile ducts is crucial for studies on epithelial morphogenesis and for development of cell therapies for hepatobiliary diseases. Epimorphin (EPM) is a key morphogen for duct morphogenesis in various epithelial organs. The role of EPM in bile duct formation (DF) from hepatic precursor cells, however, is not known. To address this issue, we used WB-F344 rat epithelial stem-like cells as model for bile duct formation. A micropattern and a uniaxial static stretch device was used to investigate the effects of EPM and stress fiber bundles on the mitosis orientation (MO) of WB cells. Immunohistochemistry of liver tissue sections demonstrated high EPM expression around bile ducts in vivo. In vitro, recombinant EPM selectively induced DF through upregulation of CK19 expression and suppression of HNF3α and HNF6, with no effects on other hepatocytic genes investigated. Our data provide evidence that EPM guides MO of WB-F344 cells via effects on stress fiber bundles and focal adhesion assembly, as supported by blockade EPM, β1 integrin, and F-actin assembly. These blockers can also inhibit EPM-induced DF. These results demonstrate a new biophysical action of EPM in bile duct formation, during which determination of MO plays a crucial role.     

Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro

Growth factors such as hepatocyte growth factor (HGF) are highly up-regulated during development and following renal injury and are known to induce marked morphogenic actions in cultured tubular epithelial cells, including scattering, migration, single cell branching morphogenesis, and multicellular branching tubulogenesis. In the present study, we demonstrate that HGF stimulates epithelial cells to express neutrophil gelatinase-associated lipocalin (Ngal), a member of the lipocalin family of secreted proteins that has recently been shown to participate in mesenchymal-epithelial transformation via its ability to augment cellular iron uptake. At concentrations below those found to mediate iron transport, purified Ngal can induce a promigratory and probranching effect that is dependent on ERK activation. The suppression of Ngal expression using short hairpin RNA results in increased cyst formation by tubular cells. However, the simultaneous addition of Ngal and HGF leads to direct association of the two proteins, and results in a partial inhibition of HGF-mediated activation of c-Met and the downstream MAPK and phosphatidylinositol 3-kinase signaling pathways. This inhibitory effect down-regulates HGF-stimulated single cell migration, and limits branching morphogenesis at both the single cell and multicellular level. These experiments demonstrate that the local expression of Ngal can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures while simultaneously negatively modulating the branching effects of HGF.     

A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.     

How to make tubes: signaling by the Met receptor tyrosine kinase

Hepatocyte growth factor/scatter factor (HGF/SF), acting through the receptor tyrosine kinase Met, stimulates cells derived from a variety of different organs to form elongated hollow tubules when grown in three-dimensional gels. In vivo data also indicate a role for HGF/SF and Met in tubule formation during liver and kidney regeneration and mammary gland formation. Activation of Met results in the recruitment of a myriad of signal transducers that regulate dissociation of adherens junctions and the stimulation of cellular motility, survival, proliferation and morphogenesis during tubule formation. Among these many signal transducers, the Gab1 adaptor protein and its effector, the SHP2 tyrosine phosphatase, have been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAP kinase pathway. Here, we discuss the contribution of these and other signaling pathways and the role of HGF/SF and Met in the formation of epithelial cell tubules both in vitro in branching-morphogenesis assays and in vivo during organogenesis.