肝/造血共祖细胞的鉴定及Epimorphin调控肝干细胞分化的机制研究
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摘要
胎肝是微环境调控干细胞增殖分化的一个极佳模型,在胎肝微环境中,中胚层起源的造血细胞、间质细胞和内胚层起源的上皮细胞在其中共同发育成熟。本论文主要分为两大部分,在第一部分中,我们尝试建立一个新的研究肝和造血发育关系的单克隆模型;在第二部分中,我们对胎肝发育后期(胎肝造血已经结束,肝上皮细胞开始逐渐发育成熟时)特异性表达在胆板间质表面的一个蛋白——上皮形态发生素(EPM)调控肝干细胞分化的机制进行了研究。
一、肝/造血共祖细胞的鉴定
胎肝是造血和肝脏系统发育的重要器官,在胎肝的不同发育阶段,造血和肝脏系统均具有密切的关联性和相互的调控作用,同时胎肝也是微环境调控干细胞增殖分化的一个极佳模型,对其进行发育生物学的系统研究将有助于了解造血起源、肝脏发育、干细胞多向分化及调控机制等一系列热点问题,并且在肝脏疾病和造血系统疾病的发生发展和治疗新策略领域也具有十分重要的意义。此前有研究者提出过一个假说,即在胎肝发育的早期可能存在着肝和造血的共祖细胞,此类共祖细胞的存在与否,对早期造血起源和肝脏发育具有重要的研究价值。此后的一些研究报道也在一定程度上支持了这个假说,因为这些报道都证实在骨髓或其它一些造血位点存在着具有向肝和造血双向分化能力的细胞群体。
我们研究室之前的工作也表明,在人脐带血中可以分离出一群具有β2m-/c-Met+表面标志特征的细胞亚群,此群细胞也具有双向分化的潜能。但这些研究在组织来源、细胞模型、鉴定技术等方面均存在一定局限,都不能排除这样一种重要的可能性:即在这些细胞群体中存在着独立的肝干/祖细胞和造血干/祖细胞(HS/PCs),只是因为这两类独立存在的干/祖细胞在特定的环境下各自分化形成了肝脏细胞和造血细胞,从而显现出细胞群体的双向分化潜能。因此,为了准确地发现和验证肝/造血共祖细胞的存在,我们建立了小鼠胎肝来源的高增殖潜能集落形成细胞(HPP-CFC)模型,HPP-CFC是在体外不需要基质支持的最早的具有多向分化潜能的造血前体细胞,以往对AGM(主动脉-性腺-中肾区域)区来源的HPP-CFC克隆的研究已经证实其除具有造血分化潜能外,还具有向内皮分化的潜能。因此,我们利用此类单克隆起源的前体细胞,重点研究胎肝来源的单克隆HPP-CFC向肝上皮细胞分化的潜能,本部分研究内容如下:
(一)小鼠胎肝HPP-CFC体系的建立及向肝系细胞分化
我们首先进行了单克隆HPP-CFC的培养以及诱导分化实验。利用造血和肝诱导因子的共同作用,对单克隆来源的HPP集落细胞向造血和肝上皮细胞进行诱导分化,并采用透射电镜、巢式RT-PCR、细胞免疫荧光等方法,从细胞形态、超微结构、上皮细胞分化标志等方面对分化后的细胞进行检测。检测结果显示诱导后的部分细胞具有肝细胞特异性的超微结构,在细胞表面形成微绒毛,在胞浆内具有较大的细胞核,大量的圆形线粒体和糖原颗粒。并在mRNA和蛋白水平以不同比例的表达白蛋白(ALB)、甲胎蛋白(AFP)、细胞角蛋白(CK8,CK18)等肝上皮分化标志。
(二)小鼠胎肝细胞的分选
为进一步研究这群具有肝和造血双向分化潜能的细胞在胎肝中的表型,我们利用免疫磁珠(MACS)进行细胞分选。结果表明:胎肝来源的HPP-CFC主要来自于CD45+细胞,CD45-细胞不具有形成造血克隆的能力。在肝上皮细胞分化潜能上,流式分选(FACS)获得的CD49f+/Sca-1+细胞与未分选细胞无明显差异。究其原因,有两种可能性:(a)CD49f+/Sca-1+并没有富集我们发现的这群具有肝和造血双向分化潜能的前体细胞;(b)这群双阳性细胞在半固体培养体系中对于造血和肝因子刺激的应答不同,可能更倾向于造血分化。
(三)小鼠胎肝HPP-CFC单克隆源性的鉴定
本部分研究通过细胞混合实验,采用巢式PCR联合荧光显微镜观察,对GFP和Sry双遗传标记的HPP克隆进行了检测,证明了本研究采用的单克隆HPP模型具有严格的单克隆源性。
令人惊讶的是,这些诱导后的HPP克隆细胞同时还表达间质标志α-SMA(α-smooth muscle actin)和原始内皮细胞标志Flk-1(fetal liver kianse-1)。我们推测这群CK8+/α-SMA+细胞可能代表了胎肝中的一群具有多向分化潜能的上皮-间质转换细胞(EMT),这群细胞可能起源于中内胚层细胞。
以上结果说明,胎肝来源的HPP-CFC可能代表了一个新的假想中的肝/造血共祖细胞单克隆模型,为研究胎肝中造血和非造血细胞的发育关系提供了一个新的切入点,虽然这个模型本身还有很多需要改进的地方。
二、Epimorphin蛋白调控肝干细胞分化的机制研究
在胎肝微环境中,我们初步验证了肝/造血共祖细胞存在的可能性,实际上,在胎肝发育的后期,胎肝微环境逐渐由支持造血分化转向促进上皮细胞的发育成熟,那么在这个过程中胎肝中的基质微环境必然发生较大的改变。已知在小鼠胎肝发育至E17.5时,此时胎肝造血已经结束,造血细胞开始向骨髓迁移,一个称为“上皮形态发生素”的重要基质蛋白EPM开始随着胚肝细胞上其受体αvβ1整合素的逐渐上调而在胎肝间质上逐渐开始表达,而此时胆板正在进入一个比较大的组织重塑期,在胆板的两层细胞之间逐渐出现局部膨大,最终形成胆管结构,那么我们要问,EPM是否有可能在肝/造血共祖细胞向上皮细胞的分化决定中发挥作用呢?
目前的研究表明,EPM在众多的上皮组织器官包括肺、乳腺、胰腺、胆囊、发毛囊、小肠、皮肤、肾、生殖腺中,调控上皮细胞腺管形态的发生。已有研究表明,EPM在肝再生修复的后期表达上调,而且EPM参与肝干细胞的分化过程。基于上述研究,我们推测EPM可能在胆管形态发生以及肝干细胞向胆管上皮细胞的分化过程中发挥作用。对EPM在干细胞向胆管细胞分化过程中机制的研究,不仅有助于理解胎肝发育过程中上皮细胞特化命运的决定,更有助于指导体外肝组织工程等应用方面的研究。
组织工程化肝单元的构建可能代表了未来以干细胞为种子细胞的肝组织工程发展的方向。目前在肝单元构建的研究中,研究者着眼于种子细胞向肝细胞、血管内皮细胞的诱导分化,但对于胆管的分化和形态发生研究较少,究其原因,可能是因为和肝细胞相比,胆管上皮细胞(BEC)发挥功能除了一般意义上的单个上皮细胞极性的产生外,更加强调细胞群体的极性排列和空间结构的产生和维持,因而增加了研究的难度。但实际上,虽然BEC在肝脏全部细胞中的比例较低,但由BEC构成的肝内胆管(IHBD)对于肝代谢功能的发挥尤其是肝细胞分泌的胆汁的输送和调节至关重要。
因此本研究中,我们将结合体内CCl4肝损伤模型和体外肝干/祖细胞模型——WB-F344细胞系来重点研究EPM和胆管形态发生的关系以及EPM调控WB-F344细胞管腔样形态发生的机制,本部分研究内容如下:
(一) EPM蛋白诱导WB细胞形成胆管样结构
为了证明EPM蛋白和胆管形态的发生和功能的维持密切相关,我们进行了小鼠体内EPM蛋白和胆管的共定位研究。在本研究中,我们采用小鼠CCl4肝损伤模型,首次发现了无论在CCl4肝损伤组织还是正常的肝脏组织中,在胆管周围的间质组织中存在EPM蛋白的高表达。这些结果表明EPM在IHBD正常形态的维持和再生过程中发挥着重要的作用。
既然在肝脏内表明EPM可能与胆管的功能维持和再生有关,为了进一步研究EPM与胆管形态发生的关系,我们在体外以大鼠肝干/祖细胞系WB-F344细胞为模型,进一步开展了EPM在WB细胞向胆管系特异性分化和管腔形态发生过程中的作用研究。
I-EPM对于WB细胞的增殖具有一定的抑制作用,并能够特异性的诱导WB细胞形成管状样结构,无论是EPM抗体还是其受体β1 integrin的抗体均能阻断i-EPM诱导的形态发生过程;对诱导后的细胞进行肝和胆管系相应特异性标志的RT-PCR和Western Blotting分析表明,肝上皮细胞特异性标志包括ALB,CK18和TAT均未被诱导表达,与肝祖细胞和/或肝上皮细胞系相关的基因如AFP,HNF3α和HNF6受到抑制,胆管特异性标志CK19上调,考虑到i-EPM并不上调成熟胆管的功能性标志如GGT、Yp,我们的结果表明i-EPM特异性诱导WB细胞形成的管腔样结构属于幼稚的胆管系细胞。
(二) EPM蛋白具有调控WB细胞有丝分裂方向(MO)的能力
Hirai此前曾经提出EPM蛋白排布信息的不同可能会影响三维培养的乳腺上皮细胞团中细胞的有丝分裂方向的推测,而干细胞在分化和自我更新的过程中必然牵涉到细胞有丝分裂方向的调控,因此从本部分研究开始主要探讨i-EPM蛋白促进WB细胞分化的相关生物力学机制。
在micropattern模型中,绝大多数WB细胞沿着i-EPM蛋白阵列线和细胞膜交界面的切线方向分裂。在上述micropattern体系中加入抗EPM多克隆抗体、抗EPM受体(β1 integrin)抗体、La-A阻断后,这种i-EPM调控的有丝分裂方向的规律性消失。在i-EPM诱导的管状样结构中也观察到了沿着切线方向分裂的WB细胞。总之我们这部分的研究表明,i-EPM具有调控WB细胞有丝方向分裂定位的能力;而不论在i-EPM诱导MO还是管腔形成(DF)的过程中,细胞黏附和应力纤维的排布都至关重要。
(三)外力作用下应力纤维排布方向决定WB细胞有丝分裂方向
Micropattern模型中La-A阻断实验结果表明应力纤维方向(SFO)在i-EPM决定WB细胞MO过程中具有潜在重要作用,为了进一步验证SFO在引导细胞有丝分裂方向过程中的重要作用,我们建立了一个经过改进的静态单轴拉伸模型来研究WB细胞在收到外力刺激后SFO对MO的调控作用,结果表明,在持续外力作用条件下,MO从属于SFO,且二者在方向定位上具有高度相关性。
(四)细胞黏附、SFO、MO决定在i-EPM诱导WB细胞胆管样结构形成中的作用
第一节的工作中我们证明了i-EPM具有诱导WB细胞向幼稚的胆管上皮细胞分化的能力,同时我们发现了一个新的有趣的现象:即具有同样EPM蛋白活性结构域的可溶性EPM(s-EPM)对WB细胞并不具有如同不可溶性EPM蛋白(i-EPM)一样的形态发生效果,究其原因可能和i-EPM蛋白空间分布信息、介导的细胞黏附状态等与s-EPM不同有关,这可能激发的是一条不完全等同于生物化学信号通路的途径。而我们实验室的前期研究也表明Matrigel促进WB细胞向胆管细胞分化过程中需要RhoA蛋白的参与,而RhoA在干细胞分化过程中与细胞骨架的重排和细胞形状的改变等生物力学信号密切相关,那么i-EPM诱导WB细胞向胆管系的分化是否也和i-EPM蛋白空间分布信息调节了细胞骨架有关呢?
我们的结果表明,i-EPM在体外可以诱导WB细胞较快的装配形成黏着斑,并诱导WB细胞的应力纤维成束状集中于细胞边缘按一定方向排列,而不接触EPM和抗EPM阻断组的WB细胞的的应力纤维呈杂乱无章分布,尤其是s-EPM处理的WB细胞更为显著。而且F-actin阻断剂La-A可以扰乱管腔样结构的形成。
最后我们要探讨的是i-EPM诱导WB细胞MO决定和DF形态发生之间是充分还是必要的关系。在我们的研究中,所有能够扰乱MO决定的因素对于DF结构的形成都有抑制性作用。但我们在培养表面涂布此前报道具有调节细胞MO能力的层纤连蛋白(FN),却并不能诱导WB细胞分化形成管状样结构。这些结果表明MO定位的决定对于i-EPM诱导形成DF是必需的,但不是充分的。
综上所述,我们的研究初步表明,在i-EPM诱导肝干细胞(WB细胞)向胆管系分化,产生管腔样形态发生的过程中,除了传统上研究的生物化学信号通路外,还存在着一条相对独立、不完全依赖于生物化学信号的生物物理信号途径: EPM-β1 integrin-SFO-MO-DF。即i-EPM通过介导细胞的FA组装和引导SF的排布,调节MO,从而诱导DF形态发生。
Fetal liver is a good model in which the differentiation and proliferation of stem cells is modulated. Some different germ layer derived cells including mesoderm -originated hematopoietic cells, mesenchymal cells and endoderm-originated epithelial cells can develop into maturation reciprocally in fetal liver. The academic dissertation can be divided into two parts. In part I, we are trying to set up a new clonal model to facilitate the researches on developmental relationship between hematopoietic and hepatic lineage. In part II, we explore the mechanism of epimorphin (EPM) in differentiation of hepatic stem cells. EPM, a mesenchymal associated protein, is not expressed on ductal plate of fetal liver until hematopoiesis is terminated and hepatic maturation is started in fetal liver.
Part I. Identification of a Common Precursor for Hepatic and Hematopoietic Lineage
Fetal liver is a major organ for hematopoietic and hepatic development during ontogenesis. Hematopoietic and hepatic system are intertwined and regulated reciprocally in each stage of fetal liver development. As fetal liver is a good model in which the differentiation and proliferation of stem cells is modulated, it will help to understand a series of key questions such as hematopoiesis origin, hepatic development, multipotential differentiation of stem cells and underlying mechanisms by the way of a systematic study of fetal liver development. It can be expected to benefit from the above studies in the fields of progression and therapeutic strategy in liver and blood diseases. A hypothesis of a common precursor for hematopoietic and hepatic lineage in earlier developmental stage of fetal liver is presented previously. Whether the presentation is right or not will be a founding valuable to hematopoiesis origin and hepatic development. Support for this concept has been provided at some extent, by confirming the existence of the subpopulation with biopotential differentiation capacity for hematopoietic and hepatic lineage in bone marrow or other hematopoietic site.
The previous study in our lab also demonstrated that a subset of umbilical cord bloodβ2m-/c-Met+ cells can differentiate into both hepatocyte-like cells and hematopoietic cells in vitro. However, most of the studies were subjected to the limitations in tissue sources, cell models, identification techniques. Most importantly, it is hard to exclude the possibility that individual hepatic stem/progenitor cells and hematopoietic stem/progenitor cells (HS/PCs) were contained in cell populations, which appear to retain the capacity of differentiation into both lineages due to the existence of two distinct stem cell compartments. To address the issue, a high proliferative potential colony forming cells (HPP-CFC) model of mouse fetal liver was set up. HPP-CFC is well known as the earliest multipotential precursors within the hematopoietic hierarchy that can be cultured in vitro without stromal support. In addition to hematopietic potential, HPP-CFC derived from aorta-gonadal- mesoneohros (AGM) region also has endothelial potential. Here we examined hepatic differentiation capacity of HPP-CFC derived from fetal liver at single colony level.
1. The Establishment of Mouse Fetal Liver-derived HPP-CFC Model and Differentiation into Hepatic Lineage
Some differentiational assays based on individual HPP colonies were performed. Under the condition of combinations of hematopoietic and hepatic factors, some individual HPP colonies were induced into hematopoietic or hepatic cells, which were identified with transmission electron microscope, nested RT-PCR and immuno- fluorescence staining. The results showed that induced HPP colonies cells with a specific ultrastructure similar to hepatic epithelial cells, such as many microvilli on the cell surface, large nucleolus and many round-shaped mitochondria, and lots of glycogen granule in the cytoplasm. They also expressed hepatic markers including albumin (ALB),α-fetoprotein (AFP), and cytokeratins (CK8, CK18) at different extent of percentage at mRNA or protein level.
2. Mouse Fetal Liver Cells Sorting
To further explore the phenotype of the cell populations with hepatic and hematopoietic differentiation potential, we undertook cell sorting experiments. The magnetic activated cell sorting (MACS) results suggested that the fetal liver-derived HPP-CFCs were all from CD45+ cells, while CD45- cells had no capacity to form hematopoietic colonies at all. The fluorescence activated cell sorting (FACS) sorted CD49f+/Sca-1+ cells had no difference of hepatic differentiation potential compared with whole fetal liver cells. One possibility is that CD49f+/Sca-1+ cells can not enrich the hypothesized common precursors. Alternatively, these cell populations have a predominant hematopoietic response to the effects of these combined factors in methylcellulose medium.
3. Identification of the Clonality of Mouse Fetal Liver-derived HPP-CFC
The clonality of HPP-CFC was then confirmed by cell mixing assay with GFP and Sry marker. GFP or Sry positive-HPP colonies were examined by nested RT-PCR combined with fluorescence microscope. The experimental results showing that our single clonal model is highly reliable.
It was surprisingly that these induced clonal cells also expressed mesenchymal markerα-SMA (α-smooth muscle actin) and primary endothelial cell marker Flk-1(fetal liver kianse-1), suggesting that these CK8+/α-SMA+ cells may represent the epithelial-mesenchymal transition (EMT) cells with multipotential capacity, which may originate from mesendoderm in developing fetal liver.
Taken together, the HPP-CFC may represent a novel clonal model of hypothesized common precursors for hepatic and hematopoietic lineage in the mouse feta liver and will shed light on the associations underlying the hepatic and hematopoietic development, although the model still needs further improvement.
Part II. Regulation of Epimorphin (EPM) on Differentiation of Hepatic Stem/Progenitor Cells
We initially verified the concept of a common precursor for hepatic and hematopoietic lineage in the fetal liver microenvironment. In fact, fetal liver is undergoing an developmental process characterized by changes in the microenvironment from hematopoietic supportive functions to enhancing hepatic maturation in the late stage of embryonic development. EPM can be not detected on ductal plate of fetal liver until hematopoiesis is terminated and bilayered ductal plate is profoundly remodeled into focal dilations that are developed into bile ducts eventually. The expression of EPM is reported to be correlated with the up-regulation of its receptor,αvβ1 integrin in hepatoblasts around E17.5 in fetal liver. We then asked whether EPM was possibly involved in the specific differentiation of the common precursors we studied into hepatic lineage.
The current studies show that EPM can direct the key processes of tubulogenesis in many epithelial organs including lung, mammary gland, pancreas, gallbladder, hair follicles, intestine, skin, kidney and sexual gland. Some reports suggested that the expression of EPM is enhanced in the late stage of liver regeneration and EPM is involved in the differentiation of hepatic stem-like cells in vitro. We then conclude that EPM may play an essential role in bile ducts morphogenesis and the differentiation of hepatic stem cells into biliary lineage. It will not only help to understand the process in specification of the developing epithelial cell in fetal liver, but also help to direct the studies in liver tissue engineering in vitro.
Construction of liver units for tissue engineering may be emerging as a new trend in the field of stem cells based-liver tissue engineering in the future. So far, much attention has been paid during the past years to the induction of hepatocyte and endothelial-like cells from stem cells, but little is known about differentiation and morphogenesis of the biliary tract. It may be due to the difficulties in induction and maintenance the polarity of cellular organization and distribution in bile duct structures in addition to the plasma membrane polarity as well as in hepatocytes. Although the percentage of biliary epithelial cells (BEC) is little in total of liver mass, BEC-delineated intrahepatic bile ducts (IHBD) is essential in coordination of liver metabolism function by transitting and modifying biles produced by hepatocytes to gallbladder.
We will focus on the role and underlying mechanisms of EPM in bile ducts morphogenesis and differentiation of hepatic stem cells into biliary lineage in CCl4 injured liver model and WB-F344 stem cell lines in vitro.
1. I-EPM Induced Bile Duct-like Structures of WB Cells in vitro
To confirm the involvement of EPM in morphogenesis and function maintenance of bile ducts, we carried out the spatial co-localization study of EPM and bile ducts. Our findings showed a strong and previously undocumented expression of EPM in the mesenchyme around the bile ducts in CCl4-treated and normal adult liver. These results suggested that EPM may be involved in maintaining normal morphogenesis and/or regeneration of IHBD in vivo.
Considering that EPM may be involved in maintaining normal morphogenesis and/or regeneration of IHBD in vivo, we will further investigate the role of EPM in differentiation and duct formation (DF) of hepatic stem cells into biliary lineage in WB-F344 cell model in vitro.
Insoluble EPM (i-EPM) had a slight inhibition effect on proliferation of WB cells. WB cells contact with i-EPM could be differentiated into duct-like structures, a specific morphogenesis which could be blocked by anti-EPM antibody and anti-EPM receptor (β1 integrin) antibody. RT-PCR and Western Blotting analysis of i-EPM-treated WB cells showed that hepatocytic markers including ALB, CK18 and TAT were not induced, hepatoblast and/or hepatocyte associated markers including AFP, HNF3α, and HNF6 were suppressed, and bile duct marker CK19 was up-regulated. Taken together, these results indicated that the duct-like structures induced by i-EPM in our system are mostly immature biliary ones, since i-EPM did not elevate the expression level of some other functional biliary genes such as GGT and Yp.
2. I-EPM Guided the Mitosis Orientation (MO) of WB cells
A hypothesis that orientation of EPM presentation might in turn control the orientation of the mitotic spindle axis of 3D collagen containing-mammary epithelial cells clusters was once proposed by Hirai. Regulation of MO is essential in the self-renewal and differentiation of stem cells. Here, we will investigate the bio-mechanical role of i-EPM in the differentiation of WB cells.
In micropattern model, most of the WB cells divide along the tangential direction of the interface between the cell membrane and i-EPM, which can be blocked by antiβ1-integrin antibody, anti-EPM antibody, and La-A. Guidance of MO can also be observed in the duct-like structures formed on the 2D substrata. Together, these results suggested that i-EPM has the ability to guide MO determination of the WB cells by the way of mediating focal adhesion (FA) assembly and F-actin bundles alignment, a way as important as in DF.
3. MO is Secondary to SFO under the Stimulation of Stress
The La-A blocking results in micropattern experiments demonstrated a potential role of SF in i-EPM guiding MO determination. To further confirm the effects of SFO on guiding MO, we established a modified system of static uniaxial stretch to study the regulation of SFO on MO. The results showed MO is secondary to SFO and had a high correlation of orientation with SFO under the stimulation of the lasting stress.
4. The Role of FAs, SFO, MO Determination in i-EPM-induced Bile Duct-like Formation of WB Cells
In section I, we confirmed that i-EPM had the ability to differentiate WB cells into DF, assumed to be immature biliary ones. Meanwhile, we found an interesting phenomenon that soluble EPM (s-EPM) sharing the same active EPM domain with i-EPM had no morphological effects on the WB cells. It may be due to the differences between i-EPM and s-EPM in spatial distribution and focal adhesions formation etc., which triggereda new pathway different from biochemical signaling pathway. Our previous study demonstrated that Matigel-induced biliary differentiation of WB cells is required for RhoA activity, which plays an independent bio-mechanical role in stem cells differentiation via mediating cell shape changes and cytoskeletal arrangement. We ask whether i-EPM also promotes WB DF via a biomechanical signaling pathway.
Our results demonstrated that i-EPM induced a quick assembly of focal adhesions of WB cells. F-actin microfilament bundles (i.e. stress fibers, SF) were organized into cortical bundles which predominantly arranged along the long axis of the i-EPM-treated cells. SF appeared to be randomly oriented in the cells on i-EPM free or blocked substrata, especially for s-EPM. An actin polymerization inhibitor (La-A) can disrupt the DF of WB cells.
Lastly, we asked whether MO determination is necessary or sufficient in i-EPM -induced DF of WB cells. All of the factors (antiβ1-integrin antibody, anti-EPM antibody, and La-A) which could disrupt MO determination in our experiments had a suppression impact on the duct-like structure formation. However, fibronectin (FN), which was reported to have the ability to regulate MO of the cells, failed to induce DF of WB cells. Together, these results suggest that the guidance of MO is necessary but not sufficient for the i-EPM-induced DF of the WB cells.
Above all, in addition to traditional biochemical signaling pathway, a new biophysical signaling pathway (EPM-β1 integrin-SFO-MO-DF) independent of biochemical signals, was revealed in i-EPM-inducedbile duct formation of WB cells. Briefly, i-EPM has the ability to guide MO determination of the WB cells along the tangential direction of cell-EPM contact surface via mediating focal adhesion assembly and F-actin bundles alignment, which may be vital to bile duct-like formation of the WB cells.
一、肝/造血共祖细胞的鉴定
胎肝是造血和肝脏系统发育的重要器官,在胎肝的不同发育阶段,造血和肝脏系统均具有密切的关联性和相互的调控作用,同时胎肝也是微环境调控干细胞增殖分化的一个极佳模型,对其进行发育生物学的系统研究将有助于了解造血起源、肝脏发育、干细胞多向分化及调控机制等一系列热点问题,并且在肝脏疾病和造血系统疾病的发生发展和治疗新策略领域也具有十分重要的意义。此前有研究者提出过一个假说,即在胎肝发育的早期可能存在着肝和造血的共祖细胞,此类共祖细胞的存在与否,对早期造血起源和肝脏发育具有重要的研究价值。此后的一些研究报道也在一定程度上支持了这个假说,因为这些报道都证实在骨髓或其它一些造血位点存在着具有向肝和造血双向分化能力的细胞群体。
我们研究室之前的工作也表明,在人脐带血中可以分离出一群具有β2m-/c-Met+表面标志特征的细胞亚群,此群细胞也具有双向分化的潜能。但这些研究在组织来源、细胞模型、鉴定技术等方面均存在一定局限,都不能排除这样一种重要的可能性:即在这些细胞群体中存在着独立的肝干/祖细胞和造血干/祖细胞(HS/PCs),只是因为这两类独立存在的干/祖细胞在特定的环境下各自分化形成了肝脏细胞和造血细胞,从而显现出细胞群体的双向分化潜能。因此,为了准确地发现和验证肝/造血共祖细胞的存在,我们建立了小鼠胎肝来源的高增殖潜能集落形成细胞(HPP-CFC)模型,HPP-CFC是在体外不需要基质支持的最早的具有多向分化潜能的造血前体细胞,以往对AGM(主动脉-性腺-中肾区域)区来源的HPP-CFC克隆的研究已经证实其除具有造血分化潜能外,还具有向内皮分化的潜能。因此,我们利用此类单克隆起源的前体细胞,重点研究胎肝来源的单克隆HPP-CFC向肝上皮细胞分化的潜能,本部分研究内容如下:
(一)小鼠胎肝HPP-CFC体系的建立及向肝系细胞分化
我们首先进行了单克隆HPP-CFC的培养以及诱导分化实验。利用造血和肝诱导因子的共同作用,对单克隆来源的HPP集落细胞向造血和肝上皮细胞进行诱导分化,并采用透射电镜、巢式RT-PCR、细胞免疫荧光等方法,从细胞形态、超微结构、上皮细胞分化标志等方面对分化后的细胞进行检测。检测结果显示诱导后的部分细胞具有肝细胞特异性的超微结构,在细胞表面形成微绒毛,在胞浆内具有较大的细胞核,大量的圆形线粒体和糖原颗粒。并在mRNA和蛋白水平以不同比例的表达白蛋白(ALB)、甲胎蛋白(AFP)、细胞角蛋白(CK8,CK18)等肝上皮分化标志。
(二)小鼠胎肝细胞的分选
为进一步研究这群具有肝和造血双向分化潜能的细胞在胎肝中的表型,我们利用免疫磁珠(MACS)进行细胞分选。结果表明:胎肝来源的HPP-CFC主要来自于CD45+细胞,CD45-细胞不具有形成造血克隆的能力。在肝上皮细胞分化潜能上,流式分选(FACS)获得的CD49f+/Sca-1+细胞与未分选细胞无明显差异。究其原因,有两种可能性:(a)CD49f+/Sca-1+并没有富集我们发现的这群具有肝和造血双向分化潜能的前体细胞;(b)这群双阳性细胞在半固体培养体系中对于造血和肝因子刺激的应答不同,可能更倾向于造血分化。
(三)小鼠胎肝HPP-CFC单克隆源性的鉴定
本部分研究通过细胞混合实验,采用巢式PCR联合荧光显微镜观察,对GFP和Sry双遗传标记的HPP克隆进行了检测,证明了本研究采用的单克隆HPP模型具有严格的单克隆源性。
令人惊讶的是,这些诱导后的HPP克隆细胞同时还表达间质标志α-SMA(α-smooth muscle actin)和原始内皮细胞标志Flk-1(fetal liver kianse-1)。我们推测这群CK8+/α-SMA+细胞可能代表了胎肝中的一群具有多向分化潜能的上皮-间质转换细胞(EMT),这群细胞可能起源于中内胚层细胞。
以上结果说明,胎肝来源的HPP-CFC可能代表了一个新的假想中的肝/造血共祖细胞单克隆模型,为研究胎肝中造血和非造血细胞的发育关系提供了一个新的切入点,虽然这个模型本身还有很多需要改进的地方。
二、Epimorphin蛋白调控肝干细胞分化的机制研究
在胎肝微环境中,我们初步验证了肝/造血共祖细胞存在的可能性,实际上,在胎肝发育的后期,胎肝微环境逐渐由支持造血分化转向促进上皮细胞的发育成熟,那么在这个过程中胎肝中的基质微环境必然发生较大的改变。已知在小鼠胎肝发育至E17.5时,此时胎肝造血已经结束,造血细胞开始向骨髓迁移,一个称为“上皮形态发生素”的重要基质蛋白EPM开始随着胚肝细胞上其受体αvβ1整合素的逐渐上调而在胎肝间质上逐渐开始表达,而此时胆板正在进入一个比较大的组织重塑期,在胆板的两层细胞之间逐渐出现局部膨大,最终形成胆管结构,那么我们要问,EPM是否有可能在肝/造血共祖细胞向上皮细胞的分化决定中发挥作用呢?
目前的研究表明,EPM在众多的上皮组织器官包括肺、乳腺、胰腺、胆囊、发毛囊、小肠、皮肤、肾、生殖腺中,调控上皮细胞腺管形态的发生。已有研究表明,EPM在肝再生修复的后期表达上调,而且EPM参与肝干细胞的分化过程。基于上述研究,我们推测EPM可能在胆管形态发生以及肝干细胞向胆管上皮细胞的分化过程中发挥作用。对EPM在干细胞向胆管细胞分化过程中机制的研究,不仅有助于理解胎肝发育过程中上皮细胞特化命运的决定,更有助于指导体外肝组织工程等应用方面的研究。
组织工程化肝单元的构建可能代表了未来以干细胞为种子细胞的肝组织工程发展的方向。目前在肝单元构建的研究中,研究者着眼于种子细胞向肝细胞、血管内皮细胞的诱导分化,但对于胆管的分化和形态发生研究较少,究其原因,可能是因为和肝细胞相比,胆管上皮细胞(BEC)发挥功能除了一般意义上的单个上皮细胞极性的产生外,更加强调细胞群体的极性排列和空间结构的产生和维持,因而增加了研究的难度。但实际上,虽然BEC在肝脏全部细胞中的比例较低,但由BEC构成的肝内胆管(IHBD)对于肝代谢功能的发挥尤其是肝细胞分泌的胆汁的输送和调节至关重要。
因此本研究中,我们将结合体内CCl4肝损伤模型和体外肝干/祖细胞模型——WB-F344细胞系来重点研究EPM和胆管形态发生的关系以及EPM调控WB-F344细胞管腔样形态发生的机制,本部分研究内容如下:
(一) EPM蛋白诱导WB细胞形成胆管样结构
为了证明EPM蛋白和胆管形态的发生和功能的维持密切相关,我们进行了小鼠体内EPM蛋白和胆管的共定位研究。在本研究中,我们采用小鼠CCl4肝损伤模型,首次发现了无论在CCl4肝损伤组织还是正常的肝脏组织中,在胆管周围的间质组织中存在EPM蛋白的高表达。这些结果表明EPM在IHBD正常形态的维持和再生过程中发挥着重要的作用。
既然在肝脏内表明EPM可能与胆管的功能维持和再生有关,为了进一步研究EPM与胆管形态发生的关系,我们在体外以大鼠肝干/祖细胞系WB-F344细胞为模型,进一步开展了EPM在WB细胞向胆管系特异性分化和管腔形态发生过程中的作用研究。
I-EPM对于WB细胞的增殖具有一定的抑制作用,并能够特异性的诱导WB细胞形成管状样结构,无论是EPM抗体还是其受体β1 integrin的抗体均能阻断i-EPM诱导的形态发生过程;对诱导后的细胞进行肝和胆管系相应特异性标志的RT-PCR和Western Blotting分析表明,肝上皮细胞特异性标志包括ALB,CK18和TAT均未被诱导表达,与肝祖细胞和/或肝上皮细胞系相关的基因如AFP,HNF3α和HNF6受到抑制,胆管特异性标志CK19上调,考虑到i-EPM并不上调成熟胆管的功能性标志如GGT、Yp,我们的结果表明i-EPM特异性诱导WB细胞形成的管腔样结构属于幼稚的胆管系细胞。
(二) EPM蛋白具有调控WB细胞有丝分裂方向(MO)的能力
Hirai此前曾经提出EPM蛋白排布信息的不同可能会影响三维培养的乳腺上皮细胞团中细胞的有丝分裂方向的推测,而干细胞在分化和自我更新的过程中必然牵涉到细胞有丝分裂方向的调控,因此从本部分研究开始主要探讨i-EPM蛋白促进WB细胞分化的相关生物力学机制。
在micropattern模型中,绝大多数WB细胞沿着i-EPM蛋白阵列线和细胞膜交界面的切线方向分裂。在上述micropattern体系中加入抗EPM多克隆抗体、抗EPM受体(β1 integrin)抗体、La-A阻断后,这种i-EPM调控的有丝分裂方向的规律性消失。在i-EPM诱导的管状样结构中也观察到了沿着切线方向分裂的WB细胞。总之我们这部分的研究表明,i-EPM具有调控WB细胞有丝方向分裂定位的能力;而不论在i-EPM诱导MO还是管腔形成(DF)的过程中,细胞黏附和应力纤维的排布都至关重要。
(三)外力作用下应力纤维排布方向决定WB细胞有丝分裂方向
Micropattern模型中La-A阻断实验结果表明应力纤维方向(SFO)在i-EPM决定WB细胞MO过程中具有潜在重要作用,为了进一步验证SFO在引导细胞有丝分裂方向过程中的重要作用,我们建立了一个经过改进的静态单轴拉伸模型来研究WB细胞在收到外力刺激后SFO对MO的调控作用,结果表明,在持续外力作用条件下,MO从属于SFO,且二者在方向定位上具有高度相关性。
(四)细胞黏附、SFO、MO决定在i-EPM诱导WB细胞胆管样结构形成中的作用
第一节的工作中我们证明了i-EPM具有诱导WB细胞向幼稚的胆管上皮细胞分化的能力,同时我们发现了一个新的有趣的现象:即具有同样EPM蛋白活性结构域的可溶性EPM(s-EPM)对WB细胞并不具有如同不可溶性EPM蛋白(i-EPM)一样的形态发生效果,究其原因可能和i-EPM蛋白空间分布信息、介导的细胞黏附状态等与s-EPM不同有关,这可能激发的是一条不完全等同于生物化学信号通路的途径。而我们实验室的前期研究也表明Matrigel促进WB细胞向胆管细胞分化过程中需要RhoA蛋白的参与,而RhoA在干细胞分化过程中与细胞骨架的重排和细胞形状的改变等生物力学信号密切相关,那么i-EPM诱导WB细胞向胆管系的分化是否也和i-EPM蛋白空间分布信息调节了细胞骨架有关呢?
我们的结果表明,i-EPM在体外可以诱导WB细胞较快的装配形成黏着斑,并诱导WB细胞的应力纤维成束状集中于细胞边缘按一定方向排列,而不接触EPM和抗EPM阻断组的WB细胞的的应力纤维呈杂乱无章分布,尤其是s-EPM处理的WB细胞更为显著。而且F-actin阻断剂La-A可以扰乱管腔样结构的形成。
最后我们要探讨的是i-EPM诱导WB细胞MO决定和DF形态发生之间是充分还是必要的关系。在我们的研究中,所有能够扰乱MO决定的因素对于DF结构的形成都有抑制性作用。但我们在培养表面涂布此前报道具有调节细胞MO能力的层纤连蛋白(FN),却并不能诱导WB细胞分化形成管状样结构。这些结果表明MO定位的决定对于i-EPM诱导形成DF是必需的,但不是充分的。
综上所述,我们的研究初步表明,在i-EPM诱导肝干细胞(WB细胞)向胆管系分化,产生管腔样形态发生的过程中,除了传统上研究的生物化学信号通路外,还存在着一条相对独立、不完全依赖于生物化学信号的生物物理信号途径: EPM-β1 integrin-SFO-MO-DF。即i-EPM通过介导细胞的FA组装和引导SF的排布,调节MO,从而诱导DF形态发生。
Fetal liver is a good model in which the differentiation and proliferation of stem cells is modulated. Some different germ layer derived cells including mesoderm -originated hematopoietic cells, mesenchymal cells and endoderm-originated epithelial cells can develop into maturation reciprocally in fetal liver. The academic dissertation can be divided into two parts. In part I, we are trying to set up a new clonal model to facilitate the researches on developmental relationship between hematopoietic and hepatic lineage. In part II, we explore the mechanism of epimorphin (EPM) in differentiation of hepatic stem cells. EPM, a mesenchymal associated protein, is not expressed on ductal plate of fetal liver until hematopoiesis is terminated and hepatic maturation is started in fetal liver.
Part I. Identification of a Common Precursor for Hepatic and Hematopoietic Lineage
Fetal liver is a major organ for hematopoietic and hepatic development during ontogenesis. Hematopoietic and hepatic system are intertwined and regulated reciprocally in each stage of fetal liver development. As fetal liver is a good model in which the differentiation and proliferation of stem cells is modulated, it will help to understand a series of key questions such as hematopoiesis origin, hepatic development, multipotential differentiation of stem cells and underlying mechanisms by the way of a systematic study of fetal liver development. It can be expected to benefit from the above studies in the fields of progression and therapeutic strategy in liver and blood diseases. A hypothesis of a common precursor for hematopoietic and hepatic lineage in earlier developmental stage of fetal liver is presented previously. Whether the presentation is right or not will be a founding valuable to hematopoiesis origin and hepatic development. Support for this concept has been provided at some extent, by confirming the existence of the subpopulation with biopotential differentiation capacity for hematopoietic and hepatic lineage in bone marrow or other hematopoietic site.
The previous study in our lab also demonstrated that a subset of umbilical cord bloodβ2m-/c-Met+ cells can differentiate into both hepatocyte-like cells and hematopoietic cells in vitro. However, most of the studies were subjected to the limitations in tissue sources, cell models, identification techniques. Most importantly, it is hard to exclude the possibility that individual hepatic stem/progenitor cells and hematopoietic stem/progenitor cells (HS/PCs) were contained in cell populations, which appear to retain the capacity of differentiation into both lineages due to the existence of two distinct stem cell compartments. To address the issue, a high proliferative potential colony forming cells (HPP-CFC) model of mouse fetal liver was set up. HPP-CFC is well known as the earliest multipotential precursors within the hematopoietic hierarchy that can be cultured in vitro without stromal support. In addition to hematopietic potential, HPP-CFC derived from aorta-gonadal- mesoneohros (AGM) region also has endothelial potential. Here we examined hepatic differentiation capacity of HPP-CFC derived from fetal liver at single colony level.
1. The Establishment of Mouse Fetal Liver-derived HPP-CFC Model and Differentiation into Hepatic Lineage
Some differentiational assays based on individual HPP colonies were performed. Under the condition of combinations of hematopoietic and hepatic factors, some individual HPP colonies were induced into hematopoietic or hepatic cells, which were identified with transmission electron microscope, nested RT-PCR and immuno- fluorescence staining. The results showed that induced HPP colonies cells with a specific ultrastructure similar to hepatic epithelial cells, such as many microvilli on the cell surface, large nucleolus and many round-shaped mitochondria, and lots of glycogen granule in the cytoplasm. They also expressed hepatic markers including albumin (ALB),α-fetoprotein (AFP), and cytokeratins (CK8, CK18) at different extent of percentage at mRNA or protein level.
2. Mouse Fetal Liver Cells Sorting
To further explore the phenotype of the cell populations with hepatic and hematopoietic differentiation potential, we undertook cell sorting experiments. The magnetic activated cell sorting (MACS) results suggested that the fetal liver-derived HPP-CFCs were all from CD45+ cells, while CD45- cells had no capacity to form hematopoietic colonies at all. The fluorescence activated cell sorting (FACS) sorted CD49f+/Sca-1+ cells had no difference of hepatic differentiation potential compared with whole fetal liver cells. One possibility is that CD49f+/Sca-1+ cells can not enrich the hypothesized common precursors. Alternatively, these cell populations have a predominant hematopoietic response to the effects of these combined factors in methylcellulose medium.
3. Identification of the Clonality of Mouse Fetal Liver-derived HPP-CFC
The clonality of HPP-CFC was then confirmed by cell mixing assay with GFP and Sry marker. GFP or Sry positive-HPP colonies were examined by nested RT-PCR combined with fluorescence microscope. The experimental results showing that our single clonal model is highly reliable.
It was surprisingly that these induced clonal cells also expressed mesenchymal markerα-SMA (α-smooth muscle actin) and primary endothelial cell marker Flk-1(fetal liver kianse-1), suggesting that these CK8+/α-SMA+ cells may represent the epithelial-mesenchymal transition (EMT) cells with multipotential capacity, which may originate from mesendoderm in developing fetal liver.
Taken together, the HPP-CFC may represent a novel clonal model of hypothesized common precursors for hepatic and hematopoietic lineage in the mouse feta liver and will shed light on the associations underlying the hepatic and hematopoietic development, although the model still needs further improvement.
Part II. Regulation of Epimorphin (EPM) on Differentiation of Hepatic Stem/Progenitor Cells
We initially verified the concept of a common precursor for hepatic and hematopoietic lineage in the fetal liver microenvironment. In fact, fetal liver is undergoing an developmental process characterized by changes in the microenvironment from hematopoietic supportive functions to enhancing hepatic maturation in the late stage of embryonic development. EPM can be not detected on ductal plate of fetal liver until hematopoiesis is terminated and bilayered ductal plate is profoundly remodeled into focal dilations that are developed into bile ducts eventually. The expression of EPM is reported to be correlated with the up-regulation of its receptor,αvβ1 integrin in hepatoblasts around E17.5 in fetal liver. We then asked whether EPM was possibly involved in the specific differentiation of the common precursors we studied into hepatic lineage.
The current studies show that EPM can direct the key processes of tubulogenesis in many epithelial organs including lung, mammary gland, pancreas, gallbladder, hair follicles, intestine, skin, kidney and sexual gland. Some reports suggested that the expression of EPM is enhanced in the late stage of liver regeneration and EPM is involved in the differentiation of hepatic stem-like cells in vitro. We then conclude that EPM may play an essential role in bile ducts morphogenesis and the differentiation of hepatic stem cells into biliary lineage. It will not only help to understand the process in specification of the developing epithelial cell in fetal liver, but also help to direct the studies in liver tissue engineering in vitro.
Construction of liver units for tissue engineering may be emerging as a new trend in the field of stem cells based-liver tissue engineering in the future. So far, much attention has been paid during the past years to the induction of hepatocyte and endothelial-like cells from stem cells, but little is known about differentiation and morphogenesis of the biliary tract. It may be due to the difficulties in induction and maintenance the polarity of cellular organization and distribution in bile duct structures in addition to the plasma membrane polarity as well as in hepatocytes. Although the percentage of biliary epithelial cells (BEC) is little in total of liver mass, BEC-delineated intrahepatic bile ducts (IHBD) is essential in coordination of liver metabolism function by transitting and modifying biles produced by hepatocytes to gallbladder.
We will focus on the role and underlying mechanisms of EPM in bile ducts morphogenesis and differentiation of hepatic stem cells into biliary lineage in CCl4 injured liver model and WB-F344 stem cell lines in vitro.
1. I-EPM Induced Bile Duct-like Structures of WB Cells in vitro
To confirm the involvement of EPM in morphogenesis and function maintenance of bile ducts, we carried out the spatial co-localization study of EPM and bile ducts. Our findings showed a strong and previously undocumented expression of EPM in the mesenchyme around the bile ducts in CCl4-treated and normal adult liver. These results suggested that EPM may be involved in maintaining normal morphogenesis and/or regeneration of IHBD in vivo.
Considering that EPM may be involved in maintaining normal morphogenesis and/or regeneration of IHBD in vivo, we will further investigate the role of EPM in differentiation and duct formation (DF) of hepatic stem cells into biliary lineage in WB-F344 cell model in vitro.
Insoluble EPM (i-EPM) had a slight inhibition effect on proliferation of WB cells. WB cells contact with i-EPM could be differentiated into duct-like structures, a specific morphogenesis which could be blocked by anti-EPM antibody and anti-EPM receptor (β1 integrin) antibody. RT-PCR and Western Blotting analysis of i-EPM-treated WB cells showed that hepatocytic markers including ALB, CK18 and TAT were not induced, hepatoblast and/or hepatocyte associated markers including AFP, HNF3α, and HNF6 were suppressed, and bile duct marker CK19 was up-regulated. Taken together, these results indicated that the duct-like structures induced by i-EPM in our system are mostly immature biliary ones, since i-EPM did not elevate the expression level of some other functional biliary genes such as GGT and Yp.
2. I-EPM Guided the Mitosis Orientation (MO) of WB cells
A hypothesis that orientation of EPM presentation might in turn control the orientation of the mitotic spindle axis of 3D collagen containing-mammary epithelial cells clusters was once proposed by Hirai. Regulation of MO is essential in the self-renewal and differentiation of stem cells. Here, we will investigate the bio-mechanical role of i-EPM in the differentiation of WB cells.
In micropattern model, most of the WB cells divide along the tangential direction of the interface between the cell membrane and i-EPM, which can be blocked by antiβ1-integrin antibody, anti-EPM antibody, and La-A. Guidance of MO can also be observed in the duct-like structures formed on the 2D substrata. Together, these results suggested that i-EPM has the ability to guide MO determination of the WB cells by the way of mediating focal adhesion (FA) assembly and F-actin bundles alignment, a way as important as in DF.
3. MO is Secondary to SFO under the Stimulation of Stress
The La-A blocking results in micropattern experiments demonstrated a potential role of SF in i-EPM guiding MO determination. To further confirm the effects of SFO on guiding MO, we established a modified system of static uniaxial stretch to study the regulation of SFO on MO. The results showed MO is secondary to SFO and had a high correlation of orientation with SFO under the stimulation of the lasting stress.
4. The Role of FAs, SFO, MO Determination in i-EPM-induced Bile Duct-like Formation of WB Cells
In section I, we confirmed that i-EPM had the ability to differentiate WB cells into DF, assumed to be immature biliary ones. Meanwhile, we found an interesting phenomenon that soluble EPM (s-EPM) sharing the same active EPM domain with i-EPM had no morphological effects on the WB cells. It may be due to the differences between i-EPM and s-EPM in spatial distribution and focal adhesions formation etc., which triggereda new pathway different from biochemical signaling pathway. Our previous study demonstrated that Matigel-induced biliary differentiation of WB cells is required for RhoA activity, which plays an independent bio-mechanical role in stem cells differentiation via mediating cell shape changes and cytoskeletal arrangement. We ask whether i-EPM also promotes WB DF via a biomechanical signaling pathway.
Our results demonstrated that i-EPM induced a quick assembly of focal adhesions of WB cells. F-actin microfilament bundles (i.e. stress fibers, SF) were organized into cortical bundles which predominantly arranged along the long axis of the i-EPM-treated cells. SF appeared to be randomly oriented in the cells on i-EPM free or blocked substrata, especially for s-EPM. An actin polymerization inhibitor (La-A) can disrupt the DF of WB cells.
Lastly, we asked whether MO determination is necessary or sufficient in i-EPM -induced DF of WB cells. All of the factors (antiβ1-integrin antibody, anti-EPM antibody, and La-A) which could disrupt MO determination in our experiments had a suppression impact on the duct-like structure formation. However, fibronectin (FN), which was reported to have the ability to regulate MO of the cells, failed to induce DF of WB cells. Together, these results suggest that the guidance of MO is necessary but not sufficient for the i-EPM-induced DF of the WB cells.
Above all, in addition to traditional biochemical signaling pathway, a new biophysical signaling pathway (EPM-β1 integrin-SFO-MO-DF) independent of biochemical signals, was revealed in i-EPM-inducedbile duct formation of WB cells. Briefly, i-EPM has the ability to guide MO determination of the WB cells along the tangential direction of cell-EPM contact surface via mediating focal adhesion assembly and F-actin bundles alignment, which may be vital to bile duct-like formation of the WB cells.
引文
[1]、Udani VM. The continuum of stem cell transdifferentiation: possibility of hematopoietic stem cell plasticity with concurrent CD45 expression. Stem Cells Dev, 2006,15(1):1-3.
[2]、Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science, 1999,284(5417):1168-1170.
[3]、Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med, 2000,6(11):1229-1234.
[4]、Almeida-Porada G, Porada CD, Chamberlain J, et al. Formation of human hepatocytes by human hematopoietic cells in sheep. Blood, 2004,104(8): 2582-2590.
[5]、Jang Y, Collector MI, Baylin SB, et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nature Cell Biol, 2004,6(6):532-539.
[6]、Wang X, Willenberg H, Akkari Y, et al. Cell fusion is the principal source of bone marrow derived hepatocytes. Nature, 2003,422(6934):897-901.
[7]、Harris RG, Herzog EL, Bruscia EM, et al. Lack of a fusion requirement for development of bone marrow-derived epithelia. Science, 2004,305 (5680): 90-93.
[8]、Snorri S, Thorgeirsson, Joe W. Grisham. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence. Hepatology, 2006,43(1):2-8.
[9]、Danet GH, Luongo JL, Butler G, et al. C1qRp defines a new human stem cell population with hematopoietic and hepatic potential. Proc Natl Acad Sci USA, 2002,99(16): 10441-10445.
[10]、Wang Y, Nan X, Li Y, et al. Induction of umbilical cord blood-derivedβ2m–c-Met+ cells into hepatocyte-like cells by coculture with CFSC/HGF cells. Liver Transplantation, 2005,11(6):635-643.
[11]、Petersen BE, Grossbard B, Hatch H, et al. Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology, 2003,37(3):632-640.
[12]、Petersen BE, Goff JP, Greenberger JS, et al. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology, 1998,(2): 433-445.
[13]、Masson NM, Currie IS, Terrace JD, et al. Hepatic progenitor cells in human fetalliver express the oval cell marker Thy-1. Am J Physiol Gastrointest Liver Physiol, 2006,291(1):G45-G54.
[14]、Krause DS, Theise ND, Collector MI, et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell, 2001,105(3): 369-377.
[15]、Wagers AJ, Sherwood RI, Christensen JL, et al. Little evidence for develop- mental plasticity of adult hematopoietic stem cells. Science, 2002,297 (5590):2256-2259.
[16]、Stadtfeld M, Graf T. Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing. Development, 2004,132:203-213.
[17]、Baron MH. Embryonic origins of mammalian hematopoiesis. Experimental Hematology,2003, 31: 1160~1169.
[18]、Zhao R, Duncan SA. Embryonic development of the liver. Hepatology, 2005, 41(5):956-967.
[19]、Miyajima A, Kinoshita T, Tanaka M, et al. Role of Oncostatin M in hematopoiesis and liver development.Cytokine Growth Factor Rev, 2000,11 (3):177-183.
[20]、Cardier JE, Guillem EB. Extramedullary hematopoiesis in the adult mouse liver is associated with specific hepatic sinusoidal endothelial cells. Hepatology, 1997,26(1):165-175.
[21]、Kotton DN, Fabian1 AJ, Mulligan RC. A novel stem cell population in adult liver with potent hematopoietic reconstitution activity. Blood, 2005,106(5): 1574-1580.
[22]、Morrison SJ, Hemmati HD, Wandycz AM et al. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci USA, 1995,92(22):10302-10306.
[23]、Yao H, Liu B, Wang X, et al. Identification of high proliferative potential precursors with hemangioblastic activity in the mouse aorta-gonad- mesonephros region. Stem Cells, 2007,25(6):1423-1430.
[24]、Stuart H, Orkin, Leonard I. Zon. Hematopoiesis: an evolving paradigm for stem cell biology. Cell, 2008,132(4):631-644.
[25]、Rogers I, Yamanaka N, Bielecki R, et al. Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation. Exp CellRes, 2007,313(9):1839-1852.
[26]、Suzuki A, Zheng YW, Kaneko S, et al. Clonal identification and character- rization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol, 2002,156(1):173-184.
[27]、Qian H, Tryggvason K, Jacobsen SE, et al. Contribution of alpha6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with alpha4 integrins. Blood, 2006,107(9):3503-3510..
[28]、Nierhoff D, Ogawa A, Oertel M, et al. Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity. Hepatology, 2005,42(1):130-139.
[29]、Choi K, Kennedy M, Kazarov A, et al. A common precursor for hematopoietic and endothelial cells. Development, 1998,125(4):725-732.
[30]、Huber TL, Kouskoff V, Fehling HJ, et al. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature, 2004, 432 (7017):625-630.
[31]、Kennedy M, D'Souza SL, Lynch-Kattman M, et al. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differen- tiation cultures. Blood, 2007,109(7):2679-2687.
[32]、LaRue AC, Masuya M, Ebihara Y, et al. Hematopoietic origins of fibroblasts: I. In vivo studies of fibroblasts associated with solid tumors. Exp Hematol, 2006,34(2):208-218.
[33]、Ebihara Y, Masuya M, Larue AC, et al. Hematopoietic origins of fibroblasts: II. In vitro studies of fibroblasts, CFU-F, and fibrocytes. Exp Hematol, 2006,34 (2):219-229.
[34]、Baba S, Fujii H, Hirose T, et al. Commitment of bone marrow cells to hepatic stellate cells in mouse. J Hepatol, 2004,40(2):255-260.
[35]、Miyata E, Masuya M, Yoshida S, et al. Hematopoietic origin of hepatic stellate cells in the adult liver. Blood, 2008,111(4):2427-2435.
[36]、Dominici M, Pritchard C, Garlits JE, et al. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplant- tation. Proc Natl Acad Sci USA, 2004, 101(32):11761-11766.
[37]、Waller E K, Olweus J, Lund-Johansen F, et al. The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate popu- lations of hematopoietic and stromal progenitors. Blood, 1995,85(9): 2422-2435.
[38]、Koide Y, Morikawa S, Mabuchi Y, et al. Two Distinct Stem Cell Lineages in Murine Bone Marrow. Stem Cells, 2007,25(5):1213-1221.
[39]、de Bruijn MF, Ma X, Robin C, et al. Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity, 2002,16(5):673-683.
[40]、Bertrand JY, Giroux S, Golub R, et al. Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin. Proc Natl Acad Sci U S A, 2005,102(1):134-139.
[41]、Zovein AC, Hofmann JJ, Lynch M, et al. Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell, 2008,3(6):625-636.
[42]、Rodaway A, Patient R. Mesendoderm: an ancient germ layer? Cell, 2001,105(2):169-172.
[43]、Zhao Y, Glesne D, Huberman E. A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA, 2003,100 (5):2426-2431.
[44]、Palis J, Chan RJ, Koniski A, et al. Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis. Proc Natl Acad Sci USA, 2001,98(8):4528-4533.
[45]、Inderbitzin D, Avital I, Keogh A, et al. Interleukin-3 induces hepatocyte- specific metabolic activity in bone marrow-derived liver stem cells. J Gastrointest Surg, 2005,9(1):69-74.
[46]、Nishino T, Hisha H, Nishino N, et al. Hepatocyte growth factor as a hematopoietic regulator. Blood, 1995,85(11):3093-3100.
[47]、Kubo A, Shinozaki K, Shannon J M, et al. Development of definitive endoderm from embryonic stem cells in culture. Development, 2004,131(7): 1651-1662.
[48]、Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev, 2005,19(10):1129-1155.
[49]、Dzierzak E, Speck NA. Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol, 2008,9(2):129-136.
[50]、Mikkola HK, Fujiwara Y, Schlaeger TM, et al. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood, 2003,101(2):508-516.
[51]、Mitjavila-Garcia MT, Cailleret M, Godin I, et al. Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.Development, 2002,129(8):2003-2013.
[52]、Ferkowicz MJ, Starr M, Xie X, et al. CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo. Development, 2003,130(18):4393-4403.
[53]、Bordoni V, Alonzi T, Zanetta L, et al. Hepatocyte-conditioned medium sustains endothelial differentiation of human hematopoietic-endothelial progenitors. Hepatology, 2007,45(5):1218-1228.
[54]、Koenig S, Krause P, Drabent B, et al. The expression of mesenchymal, neural and haematopoietic stem cell markers in adult hepatocytes proliferating in vitro. J Hepatol, 2006,44(6):1115-1124.
[55]、Wulf GG, Luo KL, Jackson K A, et al. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica, 2003,88(4):368-378.
[56]、Suskind DL, Muench MO. Searching for common stem cells of the hepatic and hematopoietic systems in the human fetal liver: CD34+/cytokeratin 7/8+ cells express markers for stellate cells. J Hepatol, 2004,40(2):261-268.
[57]、Khurana S, Mukhopadhyay A. Hematopoietic progenitors from early murine fetal liver possess hepatic differentiation potential. Am J Pathol, 2008,173(6): 1818-1827.
[58]、Dan YY, Riehle KJ, Lazaro C, et al. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci USA, 2006,103(26):9912-9917.
[59]、Inada M, Follenzi A, Cheng K, et al. Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation. J Cell Sci, 2008,121(Pt7):1002-1013.
[60]、Chagraoui J, Lepage-Noll A, Anjo A, et al. Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition. Blood, 2003,101(8):2973-2982.
[61]、Kubota H, Reid LM. Clonogenic hepatoblasts, common precursors for hepato- cytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc Natl Acad Sci U S A, 2000,97(22):12132-12137.
[62]、Lemaigre FP. Development of the biliary tract. Mech Dev, 2003,120(1):81-87.
[63]、Couchie D, Holic N, Chobert MN, et al. In vitro differentiation of WB-F344 rat liver epithelial cells into the biliary lineage. Differentiation, 2002,69(4-6): 209-215.
[64]、Hirai Y, Takebe K, Takashina M, et al. Epimorphin: a mesenchymal protein essential for epithelial morphogenesis. Cell, 1992,69(3):471-481.
[65]、Hirai Y. Molecular cloning of human epimorphin: identification of isoforms and their unique properties. Biochem Biophys Res Commun, 1993,191(3): 1332-1337.
[66]、Zha H, Remmers EF, Szpirer C, et al. The epimorphin gene is highly conserved among humans, mice, and rats and maps to human chromosome 7, mouse chromosome 5, and rat chromosome 12. Genomics, 1996,37(3):386- 389.
[67]、Chen D, Bernstein AM, Lemons PP, et al. Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release. Blood, 2000,95(3):921-929.
[68]、Radisky DC, Hirai Y, Bissell MJ, et al. Delivering the message: epimorphin and mammary epithelial morphogenesis. Trends Cell Biol, 2003,13(8): 426-434.
[69]、Hirai Y. Epimorphin as a morphogen: does a protein for intracellular vesicular targeting act as an extracellular signaling molecule? Cell Biol Int, 2001,25(3): 193-195.
[70]、Hirai Y, Nelson CM, Yamazaki K, et al. Non-classical export of epimorphin and its adhesion toαv-integrin in regulation of epithelial morphogenesis. J Cell Sci, 2007,120( Pt12):2032-2043.
[71]、Zhang L, Ishikawa O, Takeuchi Y, et al. Immunohistochemical distribution of epimorphin in human and mouse tissues. Histochem J, 1998,30(12):903-908.
[72]、Hirai Y, Lochter A, Galosy S, et al. Epimorphin functions as a key morphoregulator for mammary epithelial cells. J Cell Biol, 1998,140(1):159- 169.
[73]、Simian M, Hirai Y, Navre M, et al. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development, 2001,128(16):3117-3131.
[74]、Hirai Y, Radisky D, Boudreau R, et al. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPβ. J Cell Biol, 2001,153 (4):785-794.
[75]、Lehnert L, Lerch MM, Hirai Y, et al. Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro. J Cell Biol, 2001,152(5):911-922.
[76]、Tulachan SS, Doi R, Hirai Y, et al. Mesenchymal epimorphin is important for pancreatic duct morphogenesis. Dev Growth Differ, 2006,48(2):65-72.
[77]、Mori M, Miyazaki K. Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels. Cell Tissue Res, 2000,300(2): 331-344.
[78]、Takebe K, Oka Y, Radisky D, et al. Epimorphin acts to induce hair follicle anagen in C57BL/6 mice. FASEB J, 2003,17(14):2037-2047.
[79]、Hirai Y, Takebe K, Nakajima K. Structural optimization of pep7, a small peptide extracted from epimorphin, for effective induction of hair follicle anagen. Exp Dermatol, 2005,14(9):692-699.
[80]、Fritsch C, Swietlicki EA, Lefebvre O, et al. Epimorphin expression in intestinal myofibroblasts induces epithelial morphogenesis. J Clin Invest, 2002,110(11):1629-1641.
[81]、Zhang L, Ishikawa O, Takeuchi Y, et al. Influences of keratinocyte-fibroblast interaction on the expression of epimorphin by fibroblasts in vitro. J Dermatol Sci, 1999,20: 191-196.
[82]、Horikoshi S, Yoshikawa M, Shibata T, et al. Protein localization and mRNA expression of epimorphin in mouse and human kidneys. Exp Nephrol. 2001,9(6):412-419.
[83]、von Schalburg KR, McCarthy SP, Rise ML, et al. Expression of morphogenic genes in mature ovarian and testicular tissues: potential stem-cell niche markers and patterning factors. Mol Reprod Dev, 2006,73(2):142-152.
[84]、Oka Y, Hirai Y. Inductive influences of epimorphin on endothelial cells in vitro. Exp Cell Res, 1996,222(1):189-198.
[85]、Miura K, Nagai H, Ueno Y , et al. Epimorphin is involved in differentiation of rat hepatic stem-like cells through cell-cell contact. Biochem Biophys Res Commun, 2003,311(2):415-423.
[86]、Van Eyken P, Sciot R, Callea F, et al. The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study. Hepatology, 1988,8(6): 1586-1595.
[87]、Miura K, Goto T, Oshima S, et al. Epimorphin is involved in morphogenesis of endothelial cells and hepatocytes in developing and regenerative liver in mice. Hepatology, 2004,40(Suppl):614A.
[88]、Tsao MS, Smith JD, Nelson KG, et al. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of“oval”cells. Exp Cell Res, 1984,154(1):38-52.
[89]、Coleman WB, Grisham JW. Epithelial stem-like cells of the rodent liver. LiverGrowth and Repair: From Basic Science to Clinical Practice. Edited by AJ Strain, AM Diehl. New York, Chapman and Hall. 1998,pp50-99.
[90]、Grisham JW, Coleman WB, Smith GJ. Isolation, culture, and transplantation of rat hepatocytic precursor (stemlike) cells. Proc Soc Exp Biol Med, 1993, 204:270-279.
[91]、Zvibel I, Fiorino AS, Brill S, et al. Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents. Differentiation, 1998,63(4):215-223.
[92]、Coleman WB, Wennerberg AE, Smith GJ, et al. Regulation of the differentiation of diploid and some aneuploid rat liver epithelial (stemlike) cells by the hepatic microenvironment. Am J Pathol, 1993,142(5):1373-1382.
[93]、Tsao MS, Grisham JW. Hepatocarcinomas, cholangiocarcinomas, and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells. A light-and electron-microscopic analysis. Am J Pathol, 1987, 127(1):168-181.
[94]、Fan J, Shen H, Dai Q, et al. Bone morphogenetic protein-4 induced Rat hepatic progenitor cell (WB-F344 cell) differentiation toward hepatocyte lineage. J Cell Physiol, 2009,220(1):72-81.
[95]、Watanabe S, Hirose M, Wang XE, et al. A novel hepatic stellate (Ito) cell-derived protein, epimorphin, plays a key role in the late stages of liver regeneration. Biochem Biophys Res Commun, 1998,250(2):486-490.
[96]、Segawa D, Miura K, Goto T, et al. Distribution and isoforms of epimorphin in carbon tetrachloride-induced acute liver injury in mice. J Gastroenterol Hepatol, 2005,20(11):1769-1780.
[97]、Yoshino R, Miura K, Segawa D, et al. Epimorphin expression and stellate cell status in mouse liver injury. Hepatol Res, 2006,34(4):238-249.
[98]、Miura K, Yoshino R, Hirai Y, et al. Epimorphin, a morphogenic protein, induces proteases in rodent hepatocytes through NF-kappaB. J Hepatol, 2007,47(6):834-843.
[99]、Hirai Y. Sodium-dodecyl-sulfate-resistant complex formation of epimorphin monomers and interaction of the 150-kDa complex with the cell surface. Eur J Biochem, 1994,225(3):1133-1139.
[100]、Jang YY, Collector MI, Baylin SB, et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol, 2004,6(6):532-539.
[101]、Sicklick JK, Choi SS, Bustamante M, et al. Evidence for epithelial- mesenchymal transitions in adult liver cells. Am J Physiol Gastrointest Liver Physiol, 2006,291(4):G575-G1583.
[102]、Wang Y, Wang L, Iordanov H, et al. Epimorphin–/– mice have increased intestinal growth, decreased susceptibility to dextran sodium sulfate colitis, and impaired spermatogenesis. J Clin Invest, 2006,116(6):1535-1546.
[103]、Zaret K. Developmental competence of the gut endoderm: genetic potentiation by GATA and HNF3/fork head proteins. Dev Biol, 1999,209 (1):1-10.
[104]、Coffinier C, Gresh L, Fiette L, et al. Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1β. Development, 2002,129(8):1829-1838.
[105]、Kalinichenko VV, Zhou Y, Bhattacharyya D, et al. Haploinsufficiency of the mouse Forkhead box F1 gene causes defects in gall bladder development. J Biol Chem, 2002,277(14):12369-12374.
[106]、Akiyama K, Akimaru S, Asano Y, et al. A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene. J Reprod Dev, 2008,54(2):122-128.
[107]、Tang DQ, Lu S, Sun YP, et al. Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors. Lab Invest, 2006,86(1):83-93.
[108]、Romagnani S, Amadori A, Giudizi MG, et al. Different mitogenic activity of soluble and insoluble staphylococcal protein A (SPA). Immunology, 1978, 35(3):471-478.
[109]、Lubarsky B, Krasnow MA. Tube morphogenesis: making and shaping biological tubes. Cell, 2003(1),112:19-28.
[110]、Hammarback JA, Letourneau PC. Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding. Dev Biol, 1986,117(2):655-671.
[111]、Matsuda T, Inoue K, Sugawara T. Development of micropatterning technology for cultured cells. ASAIO Transactions, 1990,36(3):559-562.
[112]、Clark P, Britland S, Connolly P. Growth cone guidance and neuron morphology on micropatterned laminin. J Cell Sci, 1993,105(Pt 1):203-212.
[113]、Lom B, Healy KE, Hockberger PE. A versatile technique for patterning biomolecules onto glass coverslips. J Neurosci Methods, 1993,50(3):385-397.
[114]、Ranieri JP, Bellamkonda R, Jacob J, et al. Selective neuronal cell attachment to a covalently patterned monoamine on fluorinated ethylene propylene films. J Biomed Mater Res, 1993,27(7):917-925.
[115]、Théry M, Racine V, Pepin A, et al. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol, 2005,7(10):947-953.
[116]、Singhvi R, Kumar A, Lopez GP, et al. Engineering cell shape and function. Science, 1994,264(5159):696-698.
[117]、Chen CS, Mrksich M, Huang S, et al. Geometric control of cell life and death. Science, 1997,276(5317):1425-1428.
[118]、O'Brien LE, Zegers MM, Mostov KE. Building epithelial architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol, 2002, 3(7):531-537.
[119]、Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell, 2002,110(6):673-687.
[120]、B?kel C, Brown NH. Integrins in development: moving on, responding to, and sticking to the extracellular matrix. Dev Cell, 2002,(3):311-321.
[121]、Lee AA, Delhaas T, Waldman LK, et al. An equibiaxial strain system for cultured cells. Am J Physiol, 1996,271(4 Pt1):C1400-1408.
[122]、王婧,胡青华,米强等.静态单轴拉伸应变刺激对细胞有丝分裂方向的影响.生物化学与生物物理进展, 2008,35(3): 297-303.
[123]、Cayouette M, Raff M. The orientation of cell division influences cell-fate choice in the developing mammalian retina. Development, 2003,130(11): 2329-2339.
[124]、Egger B, Boone JQ, Stevens NR, et al. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe. Neural Dev, 2007,2:1.
[125]、Wilcock AC, Swedlow JR, Storey KG. Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord. Development, 2007,134(10):1943-1954.
[126]、Yingling J, Youn YH, Darling D, et al. Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division. Cell, 2008,132(3):474-486.
[127]、Hwang E, Kusch J, Barral Y, et al. Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables. J Cell Biol, 2003,161(3):483-488.
[128]、Kaunas R, Nguyen P, Usami S, et al. Cooperative effects of Rho and mechanical stretch on stress fiber organization. Proc Natl Acad Sci USA, 2005,102(44):15895–15900.
[129]、Yao H, Jia Y, Zhou J, et al. RhoA promotes differentiation of WB-F344 cells into the biliary lineage. Differentiation, 2009,77(2):154-161.
[130]、Sordella R, Jiang W, Chen GC, et al. Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis. Cell, 2003,113 (2):147-158.
[131]、Pacary E, Tixier E, Coulet F, et al. Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth. Mol Cell Neurosci, 2007,35(3):409-423.
[132]、Couvelard A, Bringuier AF, Dauge MC, et al. Expression of integrins during liver organogenesis in humans. Hepatology, 1998,27(3):839-1847.
[133]、Terada T, Okada Y, Nakanuma Y. Expression of matrix proteinases during human intrahepatic bile duct development. A possible role in biliary cell migration. Am J Pathol, 1995,(147):1207-1213.
[134]、Bossard P, McPherson CE, Zaret KS. In vivo footprinting with limiting amounts of embryo tissues: a role for C/EBP beta in early hepatic development. Methods, 1997,11(2):180-188.
[135]、Rahman MM, Kukita A, Kukita T, et al. Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages. Blood, 2003,101(9):3451-3459.
[136]、Fernández-Mi?án A, Martín-Bermudo MD, González-Reyes A. Integrin Signaling Regulates Spindle Orientation in Drosophila to Preserve the Follicular-Epithelium Monolayer. Curr Biol, 2007,17(8):683-688.
[137]、Toyoshima F, Nishida E. Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner. EMBO J, 2007,26(6):1487-1498.
[138]、Toyoshima F, Nishida E. Spindle orientation in animal cell mitosis: roles of integrin in the control of spindle axis. J Cell Physiol 2007;213(2):407-411.
[139]、Woolner S, O'Brien LL, Wiese C, et al. Myosin-10 and actin filaments are essential for mitotic spindle function. J Cell Biol, 2008,182(1):77-88.
[2]、Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science, 1999,284(5417):1168-1170.
[3]、Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med, 2000,6(11):1229-1234.
[4]、Almeida-Porada G, Porada CD, Chamberlain J, et al. Formation of human hepatocytes by human hematopoietic cells in sheep. Blood, 2004,104(8): 2582-2590.
[5]、Jang Y, Collector MI, Baylin SB, et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nature Cell Biol, 2004,6(6):532-539.
[6]、Wang X, Willenberg H, Akkari Y, et al. Cell fusion is the principal source of bone marrow derived hepatocytes. Nature, 2003,422(6934):897-901.
[7]、Harris RG, Herzog EL, Bruscia EM, et al. Lack of a fusion requirement for development of bone marrow-derived epithelia. Science, 2004,305 (5680): 90-93.
[8]、Snorri S, Thorgeirsson, Joe W. Grisham. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence. Hepatology, 2006,43(1):2-8.
[9]、Danet GH, Luongo JL, Butler G, et al. C1qRp defines a new human stem cell population with hematopoietic and hepatic potential. Proc Natl Acad Sci USA, 2002,99(16): 10441-10445.
[10]、Wang Y, Nan X, Li Y, et al. Induction of umbilical cord blood-derivedβ2m–c-Met+ cells into hepatocyte-like cells by coculture with CFSC/HGF cells. Liver Transplantation, 2005,11(6):635-643.
[11]、Petersen BE, Grossbard B, Hatch H, et al. Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology, 2003,37(3):632-640.
[12]、Petersen BE, Goff JP, Greenberger JS, et al. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology, 1998,(2): 433-445.
[13]、Masson NM, Currie IS, Terrace JD, et al. Hepatic progenitor cells in human fetalliver express the oval cell marker Thy-1. Am J Physiol Gastrointest Liver Physiol, 2006,291(1):G45-G54.
[14]、Krause DS, Theise ND, Collector MI, et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell, 2001,105(3): 369-377.
[15]、Wagers AJ, Sherwood RI, Christensen JL, et al. Little evidence for develop- mental plasticity of adult hematopoietic stem cells. Science, 2002,297 (5590):2256-2259.
[16]、Stadtfeld M, Graf T. Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing. Development, 2004,132:203-213.
[17]、Baron MH. Embryonic origins of mammalian hematopoiesis. Experimental Hematology,2003, 31: 1160~1169.
[18]、Zhao R, Duncan SA. Embryonic development of the liver. Hepatology, 2005, 41(5):956-967.
[19]、Miyajima A, Kinoshita T, Tanaka M, et al. Role of Oncostatin M in hematopoiesis and liver development.Cytokine Growth Factor Rev, 2000,11 (3):177-183.
[20]、Cardier JE, Guillem EB. Extramedullary hematopoiesis in the adult mouse liver is associated with specific hepatic sinusoidal endothelial cells. Hepatology, 1997,26(1):165-175.
[21]、Kotton DN, Fabian1 AJ, Mulligan RC. A novel stem cell population in adult liver with potent hematopoietic reconstitution activity. Blood, 2005,106(5): 1574-1580.
[22]、Morrison SJ, Hemmati HD, Wandycz AM et al. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci USA, 1995,92(22):10302-10306.
[23]、Yao H, Liu B, Wang X, et al. Identification of high proliferative potential precursors with hemangioblastic activity in the mouse aorta-gonad- mesonephros region. Stem Cells, 2007,25(6):1423-1430.
[24]、Stuart H, Orkin, Leonard I. Zon. Hematopoiesis: an evolving paradigm for stem cell biology. Cell, 2008,132(4):631-644.
[25]、Rogers I, Yamanaka N, Bielecki R, et al. Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation. Exp CellRes, 2007,313(9):1839-1852.
[26]、Suzuki A, Zheng YW, Kaneko S, et al. Clonal identification and character- rization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol, 2002,156(1):173-184.
[27]、Qian H, Tryggvason K, Jacobsen SE, et al. Contribution of alpha6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with alpha4 integrins. Blood, 2006,107(9):3503-3510..
[28]、Nierhoff D, Ogawa A, Oertel M, et al. Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity. Hepatology, 2005,42(1):130-139.
[29]、Choi K, Kennedy M, Kazarov A, et al. A common precursor for hematopoietic and endothelial cells. Development, 1998,125(4):725-732.
[30]、Huber TL, Kouskoff V, Fehling HJ, et al. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature, 2004, 432 (7017):625-630.
[31]、Kennedy M, D'Souza SL, Lynch-Kattman M, et al. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differen- tiation cultures. Blood, 2007,109(7):2679-2687.
[32]、LaRue AC, Masuya M, Ebihara Y, et al. Hematopoietic origins of fibroblasts: I. In vivo studies of fibroblasts associated with solid tumors. Exp Hematol, 2006,34(2):208-218.
[33]、Ebihara Y, Masuya M, Larue AC, et al. Hematopoietic origins of fibroblasts: II. In vitro studies of fibroblasts, CFU-F, and fibrocytes. Exp Hematol, 2006,34 (2):219-229.
[34]、Baba S, Fujii H, Hirose T, et al. Commitment of bone marrow cells to hepatic stellate cells in mouse. J Hepatol, 2004,40(2):255-260.
[35]、Miyata E, Masuya M, Yoshida S, et al. Hematopoietic origin of hepatic stellate cells in the adult liver. Blood, 2008,111(4):2427-2435.
[36]、Dominici M, Pritchard C, Garlits JE, et al. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplant- tation. Proc Natl Acad Sci USA, 2004, 101(32):11761-11766.
[37]、Waller E K, Olweus J, Lund-Johansen F, et al. The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate popu- lations of hematopoietic and stromal progenitors. Blood, 1995,85(9): 2422-2435.
[38]、Koide Y, Morikawa S, Mabuchi Y, et al. Two Distinct Stem Cell Lineages in Murine Bone Marrow. Stem Cells, 2007,25(5):1213-1221.
[39]、de Bruijn MF, Ma X, Robin C, et al. Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity, 2002,16(5):673-683.
[40]、Bertrand JY, Giroux S, Golub R, et al. Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin. Proc Natl Acad Sci U S A, 2005,102(1):134-139.
[41]、Zovein AC, Hofmann JJ, Lynch M, et al. Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell, 2008,3(6):625-636.
[42]、Rodaway A, Patient R. Mesendoderm: an ancient germ layer? Cell, 2001,105(2):169-172.
[43]、Zhao Y, Glesne D, Huberman E. A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA, 2003,100 (5):2426-2431.
[44]、Palis J, Chan RJ, Koniski A, et al. Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis. Proc Natl Acad Sci USA, 2001,98(8):4528-4533.
[45]、Inderbitzin D, Avital I, Keogh A, et al. Interleukin-3 induces hepatocyte- specific metabolic activity in bone marrow-derived liver stem cells. J Gastrointest Surg, 2005,9(1):69-74.
[46]、Nishino T, Hisha H, Nishino N, et al. Hepatocyte growth factor as a hematopoietic regulator. Blood, 1995,85(11):3093-3100.
[47]、Kubo A, Shinozaki K, Shannon J M, et al. Development of definitive endoderm from embryonic stem cells in culture. Development, 2004,131(7): 1651-1662.
[48]、Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev, 2005,19(10):1129-1155.
[49]、Dzierzak E, Speck NA. Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol, 2008,9(2):129-136.
[50]、Mikkola HK, Fujiwara Y, Schlaeger TM, et al. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood, 2003,101(2):508-516.
[51]、Mitjavila-Garcia MT, Cailleret M, Godin I, et al. Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.Development, 2002,129(8):2003-2013.
[52]、Ferkowicz MJ, Starr M, Xie X, et al. CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo. Development, 2003,130(18):4393-4403.
[53]、Bordoni V, Alonzi T, Zanetta L, et al. Hepatocyte-conditioned medium sustains endothelial differentiation of human hematopoietic-endothelial progenitors. Hepatology, 2007,45(5):1218-1228.
[54]、Koenig S, Krause P, Drabent B, et al. The expression of mesenchymal, neural and haematopoietic stem cell markers in adult hepatocytes proliferating in vitro. J Hepatol, 2006,44(6):1115-1124.
[55]、Wulf GG, Luo KL, Jackson K A, et al. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica, 2003,88(4):368-378.
[56]、Suskind DL, Muench MO. Searching for common stem cells of the hepatic and hematopoietic systems in the human fetal liver: CD34+/cytokeratin 7/8+ cells express markers for stellate cells. J Hepatol, 2004,40(2):261-268.
[57]、Khurana S, Mukhopadhyay A. Hematopoietic progenitors from early murine fetal liver possess hepatic differentiation potential. Am J Pathol, 2008,173(6): 1818-1827.
[58]、Dan YY, Riehle KJ, Lazaro C, et al. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci USA, 2006,103(26):9912-9917.
[59]、Inada M, Follenzi A, Cheng K, et al. Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation. J Cell Sci, 2008,121(Pt7):1002-1013.
[60]、Chagraoui J, Lepage-Noll A, Anjo A, et al. Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition. Blood, 2003,101(8):2973-2982.
[61]、Kubota H, Reid LM. Clonogenic hepatoblasts, common precursors for hepato- cytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc Natl Acad Sci U S A, 2000,97(22):12132-12137.
[62]、Lemaigre FP. Development of the biliary tract. Mech Dev, 2003,120(1):81-87.
[63]、Couchie D, Holic N, Chobert MN, et al. In vitro differentiation of WB-F344 rat liver epithelial cells into the biliary lineage. Differentiation, 2002,69(4-6): 209-215.
[64]、Hirai Y, Takebe K, Takashina M, et al. Epimorphin: a mesenchymal protein essential for epithelial morphogenesis. Cell, 1992,69(3):471-481.
[65]、Hirai Y. Molecular cloning of human epimorphin: identification of isoforms and their unique properties. Biochem Biophys Res Commun, 1993,191(3): 1332-1337.
[66]、Zha H, Remmers EF, Szpirer C, et al. The epimorphin gene is highly conserved among humans, mice, and rats and maps to human chromosome 7, mouse chromosome 5, and rat chromosome 12. Genomics, 1996,37(3):386- 389.
[67]、Chen D, Bernstein AM, Lemons PP, et al. Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release. Blood, 2000,95(3):921-929.
[68]、Radisky DC, Hirai Y, Bissell MJ, et al. Delivering the message: epimorphin and mammary epithelial morphogenesis. Trends Cell Biol, 2003,13(8): 426-434.
[69]、Hirai Y. Epimorphin as a morphogen: does a protein for intracellular vesicular targeting act as an extracellular signaling molecule? Cell Biol Int, 2001,25(3): 193-195.
[70]、Hirai Y, Nelson CM, Yamazaki K, et al. Non-classical export of epimorphin and its adhesion toαv-integrin in regulation of epithelial morphogenesis. J Cell Sci, 2007,120( Pt12):2032-2043.
[71]、Zhang L, Ishikawa O, Takeuchi Y, et al. Immunohistochemical distribution of epimorphin in human and mouse tissues. Histochem J, 1998,30(12):903-908.
[72]、Hirai Y, Lochter A, Galosy S, et al. Epimorphin functions as a key morphoregulator for mammary epithelial cells. J Cell Biol, 1998,140(1):159- 169.
[73]、Simian M, Hirai Y, Navre M, et al. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development, 2001,128(16):3117-3131.
[74]、Hirai Y, Radisky D, Boudreau R, et al. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPβ. J Cell Biol, 2001,153 (4):785-794.
[75]、Lehnert L, Lerch MM, Hirai Y, et al. Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro. J Cell Biol, 2001,152(5):911-922.
[76]、Tulachan SS, Doi R, Hirai Y, et al. Mesenchymal epimorphin is important for pancreatic duct morphogenesis. Dev Growth Differ, 2006,48(2):65-72.
[77]、Mori M, Miyazaki K. Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels. Cell Tissue Res, 2000,300(2): 331-344.
[78]、Takebe K, Oka Y, Radisky D, et al. Epimorphin acts to induce hair follicle anagen in C57BL/6 mice. FASEB J, 2003,17(14):2037-2047.
[79]、Hirai Y, Takebe K, Nakajima K. Structural optimization of pep7, a small peptide extracted from epimorphin, for effective induction of hair follicle anagen. Exp Dermatol, 2005,14(9):692-699.
[80]、Fritsch C, Swietlicki EA, Lefebvre O, et al. Epimorphin expression in intestinal myofibroblasts induces epithelial morphogenesis. J Clin Invest, 2002,110(11):1629-1641.
[81]、Zhang L, Ishikawa O, Takeuchi Y, et al. Influences of keratinocyte-fibroblast interaction on the expression of epimorphin by fibroblasts in vitro. J Dermatol Sci, 1999,20: 191-196.
[82]、Horikoshi S, Yoshikawa M, Shibata T, et al. Protein localization and mRNA expression of epimorphin in mouse and human kidneys. Exp Nephrol. 2001,9(6):412-419.
[83]、von Schalburg KR, McCarthy SP, Rise ML, et al. Expression of morphogenic genes in mature ovarian and testicular tissues: potential stem-cell niche markers and patterning factors. Mol Reprod Dev, 2006,73(2):142-152.
[84]、Oka Y, Hirai Y. Inductive influences of epimorphin on endothelial cells in vitro. Exp Cell Res, 1996,222(1):189-198.
[85]、Miura K, Nagai H, Ueno Y , et al. Epimorphin is involved in differentiation of rat hepatic stem-like cells through cell-cell contact. Biochem Biophys Res Commun, 2003,311(2):415-423.
[86]、Van Eyken P, Sciot R, Callea F, et al. The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study. Hepatology, 1988,8(6): 1586-1595.
[87]、Miura K, Goto T, Oshima S, et al. Epimorphin is involved in morphogenesis of endothelial cells and hepatocytes in developing and regenerative liver in mice. Hepatology, 2004,40(Suppl):614A.
[88]、Tsao MS, Smith JD, Nelson KG, et al. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of“oval”cells. Exp Cell Res, 1984,154(1):38-52.
[89]、Coleman WB, Grisham JW. Epithelial stem-like cells of the rodent liver. LiverGrowth and Repair: From Basic Science to Clinical Practice. Edited by AJ Strain, AM Diehl. New York, Chapman and Hall. 1998,pp50-99.
[90]、Grisham JW, Coleman WB, Smith GJ. Isolation, culture, and transplantation of rat hepatocytic precursor (stemlike) cells. Proc Soc Exp Biol Med, 1993, 204:270-279.
[91]、Zvibel I, Fiorino AS, Brill S, et al. Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents. Differentiation, 1998,63(4):215-223.
[92]、Coleman WB, Wennerberg AE, Smith GJ, et al. Regulation of the differentiation of diploid and some aneuploid rat liver epithelial (stemlike) cells by the hepatic microenvironment. Am J Pathol, 1993,142(5):1373-1382.
[93]、Tsao MS, Grisham JW. Hepatocarcinomas, cholangiocarcinomas, and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells. A light-and electron-microscopic analysis. Am J Pathol, 1987, 127(1):168-181.
[94]、Fan J, Shen H, Dai Q, et al. Bone morphogenetic protein-4 induced Rat hepatic progenitor cell (WB-F344 cell) differentiation toward hepatocyte lineage. J Cell Physiol, 2009,220(1):72-81.
[95]、Watanabe S, Hirose M, Wang XE, et al. A novel hepatic stellate (Ito) cell-derived protein, epimorphin, plays a key role in the late stages of liver regeneration. Biochem Biophys Res Commun, 1998,250(2):486-490.
[96]、Segawa D, Miura K, Goto T, et al. Distribution and isoforms of epimorphin in carbon tetrachloride-induced acute liver injury in mice. J Gastroenterol Hepatol, 2005,20(11):1769-1780.
[97]、Yoshino R, Miura K, Segawa D, et al. Epimorphin expression and stellate cell status in mouse liver injury. Hepatol Res, 2006,34(4):238-249.
[98]、Miura K, Yoshino R, Hirai Y, et al. Epimorphin, a morphogenic protein, induces proteases in rodent hepatocytes through NF-kappaB. J Hepatol, 2007,47(6):834-843.
[99]、Hirai Y. Sodium-dodecyl-sulfate-resistant complex formation of epimorphin monomers and interaction of the 150-kDa complex with the cell surface. Eur J Biochem, 1994,225(3):1133-1139.
[100]、Jang YY, Collector MI, Baylin SB, et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol, 2004,6(6):532-539.
[101]、Sicklick JK, Choi SS, Bustamante M, et al. Evidence for epithelial- mesenchymal transitions in adult liver cells. Am J Physiol Gastrointest Liver Physiol, 2006,291(4):G575-G1583.
[102]、Wang Y, Wang L, Iordanov H, et al. Epimorphin–/– mice have increased intestinal growth, decreased susceptibility to dextran sodium sulfate colitis, and impaired spermatogenesis. J Clin Invest, 2006,116(6):1535-1546.
[103]、Zaret K. Developmental competence of the gut endoderm: genetic potentiation by GATA and HNF3/fork head proteins. Dev Biol, 1999,209 (1):1-10.
[104]、Coffinier C, Gresh L, Fiette L, et al. Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1β. Development, 2002,129(8):1829-1838.
[105]、Kalinichenko VV, Zhou Y, Bhattacharyya D, et al. Haploinsufficiency of the mouse Forkhead box F1 gene causes defects in gall bladder development. J Biol Chem, 2002,277(14):12369-12374.
[106]、Akiyama K, Akimaru S, Asano Y, et al. A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene. J Reprod Dev, 2008,54(2):122-128.
[107]、Tang DQ, Lu S, Sun YP, et al. Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors. Lab Invest, 2006,86(1):83-93.
[108]、Romagnani S, Amadori A, Giudizi MG, et al. Different mitogenic activity of soluble and insoluble staphylococcal protein A (SPA). Immunology, 1978, 35(3):471-478.
[109]、Lubarsky B, Krasnow MA. Tube morphogenesis: making and shaping biological tubes. Cell, 2003(1),112:19-28.
[110]、Hammarback JA, Letourneau PC. Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding. Dev Biol, 1986,117(2):655-671.
[111]、Matsuda T, Inoue K, Sugawara T. Development of micropatterning technology for cultured cells. ASAIO Transactions, 1990,36(3):559-562.
[112]、Clark P, Britland S, Connolly P. Growth cone guidance and neuron morphology on micropatterned laminin. J Cell Sci, 1993,105(Pt 1):203-212.
[113]、Lom B, Healy KE, Hockberger PE. A versatile technique for patterning biomolecules onto glass coverslips. J Neurosci Methods, 1993,50(3):385-397.
[114]、Ranieri JP, Bellamkonda R, Jacob J, et al. Selective neuronal cell attachment to a covalently patterned monoamine on fluorinated ethylene propylene films. J Biomed Mater Res, 1993,27(7):917-925.
[115]、Théry M, Racine V, Pepin A, et al. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol, 2005,7(10):947-953.
[116]、Singhvi R, Kumar A, Lopez GP, et al. Engineering cell shape and function. Science, 1994,264(5159):696-698.
[117]、Chen CS, Mrksich M, Huang S, et al. Geometric control of cell life and death. Science, 1997,276(5317):1425-1428.
[118]、O'Brien LE, Zegers MM, Mostov KE. Building epithelial architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol, 2002, 3(7):531-537.
[119]、Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell, 2002,110(6):673-687.
[120]、B?kel C, Brown NH. Integrins in development: moving on, responding to, and sticking to the extracellular matrix. Dev Cell, 2002,(3):311-321.
[121]、Lee AA, Delhaas T, Waldman LK, et al. An equibiaxial strain system for cultured cells. Am J Physiol, 1996,271(4 Pt1):C1400-1408.
[122]、王婧,胡青华,米强等.静态单轴拉伸应变刺激对细胞有丝分裂方向的影响.生物化学与生物物理进展, 2008,35(3): 297-303.
[123]、Cayouette M, Raff M. The orientation of cell division influences cell-fate choice in the developing mammalian retina. Development, 2003,130(11): 2329-2339.
[124]、Egger B, Boone JQ, Stevens NR, et al. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe. Neural Dev, 2007,2:1.
[125]、Wilcock AC, Swedlow JR, Storey KG. Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord. Development, 2007,134(10):1943-1954.
[126]、Yingling J, Youn YH, Darling D, et al. Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division. Cell, 2008,132(3):474-486.
[127]、Hwang E, Kusch J, Barral Y, et al. Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables. J Cell Biol, 2003,161(3):483-488.
[128]、Kaunas R, Nguyen P, Usami S, et al. Cooperative effects of Rho and mechanical stretch on stress fiber organization. Proc Natl Acad Sci USA, 2005,102(44):15895–15900.
[129]、Yao H, Jia Y, Zhou J, et al. RhoA promotes differentiation of WB-F344 cells into the biliary lineage. Differentiation, 2009,77(2):154-161.
[130]、Sordella R, Jiang W, Chen GC, et al. Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis. Cell, 2003,113 (2):147-158.
[131]、Pacary E, Tixier E, Coulet F, et al. Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth. Mol Cell Neurosci, 2007,35(3):409-423.
[132]、Couvelard A, Bringuier AF, Dauge MC, et al. Expression of integrins during liver organogenesis in humans. Hepatology, 1998,27(3):839-1847.
[133]、Terada T, Okada Y, Nakanuma Y. Expression of matrix proteinases during human intrahepatic bile duct development. A possible role in biliary cell migration. Am J Pathol, 1995,(147):1207-1213.
[134]、Bossard P, McPherson CE, Zaret KS. In vivo footprinting with limiting amounts of embryo tissues: a role for C/EBP beta in early hepatic development. Methods, 1997,11(2):180-188.
[135]、Rahman MM, Kukita A, Kukita T, et al. Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages. Blood, 2003,101(9):3451-3459.
[136]、Fernández-Mi?án A, Martín-Bermudo MD, González-Reyes A. Integrin Signaling Regulates Spindle Orientation in Drosophila to Preserve the Follicular-Epithelium Monolayer. Curr Biol, 2007,17(8):683-688.
[137]、Toyoshima F, Nishida E. Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner. EMBO J, 2007,26(6):1487-1498.
[138]、Toyoshima F, Nishida E. Spindle orientation in animal cell mitosis: roles of integrin in the control of spindle axis. J Cell Physiol 2007;213(2):407-411.
[139]、Woolner S, O'Brien LL, Wiese C, et al. Myosin-10 and actin filaments are essential for mitotic spindle function. J Cell Biol, 2008,182(1):77-88.