上皮间充质转化机制在胆道闭锁纤维化中研究

上皮间充质转化机制在胆道闭锁纤维化中的研究
ABSTRACT就BA的病因学的最新研究进展作一综述。

INTRODUCTION
胆道闭锁（biliary atresia, BA）是新生儿时期原因不明的一类胆汁淤积性疾病，出生一个月后逐渐出现体重增长缓慢、皮肤黄染、无胆色粪和肝脏肿大等典型的胆道梗阻表现。

患者肝脏病理特点为不同程度的炎症浸润伴有毛细胆管增生，胆汁淤积及间充质组织增多。

如果在2-3个月时早期诊断，可以通过Kasai术重建胆汁引流系统，成功率可达80% (1–3). 尽管目前手术成功率较高，在胆道重建术(Kasai术)后患儿的肝脏病变也将持续进展，主要表现为不同程度的毛细胆管炎症、纤维化并进行性加重，最终导致肝硬化。

数据统计, 术后2年70–80% 患者需要肝脏移植, 因此，目前BA是儿童期肝移植的最常见原因，50%以上的儿童肝移植原发病变为胆道闭锁[5, 6]. 就胆道闭锁疾病的发病机制而言，现在更倾向于解释为，是由多种因素的综合作用而导致的胆管系统的进行性病变(1,7)。

需要强调的是胆道闭锁并不是单纯的肝外胆管纤维化梗阻性疾病，与之伴随的肝内胆道系统进行性的病变决定着患者的预后和最终结果。

所以Kasai术的成功后，接踵而来的便是如何提高自体肝存活率的问题；因此需要对胆道闭锁中自体肝纤维化的病因及发病机制进行深入的探讨。

对大量历史文献阅读后，提出肝内中小型胆管上皮细胞发生了上皮间充质转化，并最终导致肝内胆管增生和纤维化的假设。

Without a better understanding of the etiology and pathogenesis发病机制of this intrahepatic 胆管硬化sclerosing cholangitic process in BA, little progress can be expected in improving the nontransplantation outcome of patients. Therefore, there has been a renewed interest in recent years in understanding the underlying pathogenetic mechanisms of BA.It is now apparent that BA is a phenotype resulting from several pathogenic processes that culminate in obstruction of the biliary tree
(1,7). The majority (80%) of cases of BA in Western countries are of the perinatal or acquired form. These most commonly involving abdominal situs, and has been dubbed the embryonic or fetal form.
Defective morphogenesis, caused by mutations in genes regulating biliary development, has been proposed in these cases. In otherwise normal infants are presumably born with a patent biliary system which undergoes progressive inflammation and fibro-obliteration initiated by a perinatal insult. Although the etiology of this form is not completely understood, proposed precipitating factors include infectious, toxic, vascular, and immune mediators. The second form of BA is associated with other congenital anomalies, most this review, we will examine recent data supporting the proposed viral and immunologic pathogenesis of the perinatal form of BA, and the potential role for defective genetic regulation of bile duct development in the embryonic form.
From significant histologic evidence, the
BECs forming small- and medium-sized bile ducts undergoing
EMT may account for prominent bile ductular
proliferation and directly contribute to fibrogenesis in BA.
.GENERAL CHARACTERISTICS OF EMT
上皮-间充质转化是指上皮细胞通过特定程序转化为具有间质表型细胞的生物学过程。

在胚胎发育、慢性炎症、组织重建、癌症转移和多种纤维化疾病中发挥了重要作用，其主要的特征有细胞黏附分子(如E-钙黏蛋白)表达的减少、细胞角蛋白细胞骨架转化为波形蛋白（Vimentin）为主的细胞骨架及形态上具有间充质细胞的特征等。

通过EMT，上皮细胞失去了细胞极性，失去与基底膜的连接等上皮表型，获得了较高的迁移与侵袭、抗凋亡和降解细胞外基质的能力等间质表型。

EMT是上皮细胞来源的恶性肿瘤细胞获得迁移和侵袭能力的重要生物学过程。

阐明调控恶性肿瘤细胞发生EMT过程的分子机制，明确其在恶性肿瘤的发生、发展、转移中的病理意义，并探索基于EMT关键分子的诊断方法及靶向EMT关键分子的治疗手段是肿瘤转移中EMT机制研究的关键科学问题。

上皮-间质表型转变(Epi恤elial-Mescnchymad Transition，EMT)是指上
皮细胞获得了成纤维细胞样表型．出现间叶细胞表面标志物，如纤维母细胞
特异蛋白．1(fibroblast specific protein 1，FSPI，又称为S100A4)、纤维连接
蛋白(fibronectin)、波形蛋白(vimentin)等；增加or-smooth muscIe actin
(a-SMA)、转化生长园子一B(transforming growth factor-J3，TGF-p)、基质金
属蛋白酶．2和9(matrix mctalloproteinases 2 and 9，MMP-2／9)、I型／lII型胶原
表达。

同时上皮标志物表达下调或丢失，如细胞角蛋白(oytokeratin)，上皮
钙粘素(E-cadherin)，oJpl．r-连环蛋白(“[3／y-catcnin)、紧密连接蛋白(zonula cccludcn 1．ZO．1)等。

细胞功能变化特点是上皮细胞损失极性和细胞．细胞问
通讯，获得迁移和侵袭细胞学行为[16]。

EMT和间质-上皮转变(MET)是胚胎发育时期的生理现象[17]。

在
早期胚胎发育过程中，外胚层的原始上皮细胞经过EMT形成原亚顶极胚层
(仃opobla嘶c germ layers)中的原始间充质细胞．在中胚层期这些间充质细
胞在白血病抑制因子(LIF)和骨形态形成蛋白-7(BoneMorphogenicprotein
7，BMP-7，亦称为成骨蛋白)等因子的作用下经历MET生成次级上皮
(Secondary epithelium)，形成新的上皮组织。

然后经过第二轮EIVlT分化彤
成各种组织细胞，如脂肪细胞、成骨细胞、肌细胞等。

而成熟组织的次级上
皮在应激(如创伤炎症、过氧化应激等)或形成肿瘤时，可出现EMT被重新
“激活”的“返祖现象”，形成FSPI+的成纤维细胞，再在TGF．B等作用下
形成肌成纤维细胞，导致纤维化发生，或使肿瘤细胞侵袭转移能力增强。

在2007年举行的国际EMT会议和2008年冷泉港（Cold Spring Harbor）实验室会议上，以EMT发生的特定生物学环境为依据将其分为3种亚型
1型MET
与胚胎植入、发育和器官形成相关的EMT称为1型MET。

其主要生物学功能是通过间充质细胞上皮化（MET）过程产生次级上皮细胞，实现胚胎形成过程中的细胞类型多样化。

2型MET
与损伤修复、组织再生和器官纤维化相关的EMT被定义为2型EMT。

其主要生物学作用是通过产生纤维细胞以修复由创伤和炎症反应造成的组织损伤。

生理状态下，当炎症反应缓解后该转化过程即自行停止；然而在炎症反应持续活化的情况下，EMT过程也将持续存在，并最终造成器官纤维化。

3型MET
3型MET是指与上皮细胞恶性肿瘤相关的表型转化。

原发性上皮组织肿瘤细胞通过3型EMT形成具有迁移能力的间充质细胞，随血流转移至不同部位，进而通过MET过程形成上皮细胞的肿瘤转移灶。

与1型和2型EMT所形成的
完全丧失上皮细胞表型的间充质细胞不同的是，3型EMT形成的转移性肿瘤细胞在获得间充质表型的同时保持一定的上皮细胞的特性。

The ability of cells to alter their phenotypic and morphological characteristics, known as cellular plasticity, is critical in normal embryonic development and adult tissue repair and contributes to the pathogenesis of diseases, such as organ fibrosis and cancer. The epithelial-to-mesenchymal transition (EMT) is a type of cellular plasticity. This tran-sition involves genetic and epigenetic changes as well as alterations in protein expression and post-translational modifications. These changes result in reduced cell-cell adhesion, enhanced cell adhesion to the extracellular matrix, and altered organization of the cytoskele-ton and of cell polarity. Among these modifications, loss of cell polarity represents the nearly invariable, distinguishing feature of EMT that frequently precedes the other traits or might even occur in their absence. EMT transforms cell morphology and physiology, and hence cell identity, from one typical of cells that form a tight barrier, like epithelial and endothelial cells, to one characterized by a highly motile mesenchymal phenotype. Time-resolved proteomic and phosphoproteomic analyses of cells undergoing EMT recently identified thousands of changes in proteins involved in many cellular processes, including cell proliferation and motility, DNA repair, and –unexpectedly – membrane trafficking (1). These results have highlighted a picture of great complexity. First, the EMT transition is not an all-or-none response but rather a gradual process that develops over time. Second, EMT events are highly dynamic and frequently reversible, involving both cell-autonomous and non-autonomous mechanisms. The net results is that EMT generates populations of mixed cells, with partial or full phenotypes, possibly accounting (at least in part) for the physiological as well as pathological cellular heterogeneity of some tissues. Endocytic circuitries have emerged as complex connectivity infrastructures for numerous cellular networks required for the execution of
different biological processes, with a primary role in the control of polarized functions. Thus, they may be relevant for controlling EMT or certain aspects of it. Here, by discussing a few paradigmatic cases, we will outline how endocytosis may be harnessed by the EMT process to promote dynamic changes in cellu-lar identity, and to increase cellular flexibility and adaptation to
micro-environmental cues, ultimately impacting on physiological and pathological processes, first and foremost cancer progression.
The epithelial-to-mesenchymal transition (EMT) is a fundamen-tal process in embryonic development and tissue repair.EMTis key also for the progression of diseases, including organ fibrosis and cancer (2–4). The pioneering work in the 1980 of Elizabeth Hay first described an “epithelial-mesenchymal transformation” using a model of chick primitive streak formation (5). Subsequently, the term“transformation” was replaced with “transition,”reflect-ing in part the reversibility of the process and the fact that it is distinct from neoplastic transformation (6, 7). The phenotypic plasticity associated with EMT is revealed by the occurrence of the reverse process, the mesenchymal-epithelial transition (MET), which involves the conversion of mesenchymal cells to epithelial derivatives. Epithelial cells form polarized sheets that are held together by various cell adhesion molecules. Beneath this cell layer, the base-ment membrane anchors epithelial cells to the underlying matrix and maintains apical-basal polarity. Adhesion to both the base-ment membrane and adjacent cells is critical for maintaining the epithelial phenotype (8). During EMT, cells lose these epithelial characteristics, acquir-ing instead an invasive and migratory mesenchymal phenotype, which allows them to leave the tissue parenchyma, undergo mor- phogenetic programs, generate new tissues during development or repair wounded ones, and to enter the blood circulation during cancer metastasis .
EMT IN in liver fibrosis
Liver fibrosis results from continuous injury to the liver, including viral hepatitis, alcohol abuse, metabolic diseases, autoimmune diseases,and cholestatic liver diseases. In other words, fibrosis is a consequence of the excessive healing response triggered by chronic liver injury. The end stage of liver fibrosis, cirrhosis, is histologically characterized by increased deposition and altered composition of the extracellular matrix (ECM) and the appearance of regenerative nodules再生性小结.
肝脏纤维化是由长期肝脏损伤导致，病因繁多：病毒性肝炎、酒精中毒、代谢性疾病、自身免疫疾病和胆汁淤积性疾病等。

也就是说，慢性肝损伤性因素持续刺激，肝脏内自我修复过度，肝脏纤维化逐步发生。

肝硬化是肝脏纤维化的最终结局，病理上以细胞外基质沉积和再生性小结出现为特征，临床上出现腹水、门脉高压等症状。

Chronic liver injury of many etiologies produces liver fibrosis and may eventually lead to the formation of cirrhosis. Fibrosis is part of a dynamic process associated with the continuous deposition and resorption of extracellular matrix, mainly fibrillar collagen纤维胶原蛋白. Studies of fibrogenesis conducted in many organs including the liver demonstrate that the primary source of the extracellular matrix in fibrosis is the myofibroblast肌成纤维细胞. Hepatic myofibroblasts are not present in the normal liver but transdifferentiate from heterogeneous异种的cell populations in response to a variety of fibrogenic stimuli. Debate still exists regarding the origin of hepatic myofibroblasts. It is considered that hepatic stellate cells and portal fibroblasts have fibrogenic potential and are the major origin of hepatic myofibroblasts.
各种病因导致的肝脏慢性损伤激发肝脏纤维化，并最终导致肝硬化的形成。

纤维化是由细胞外基质，主要为纤维胶原蛋白，再生并沉积的一个动态过程。

在对各种器官（包括肝脏）纤维化的研究中，至纤维化的细胞外基质主要有肌成纤维细胞产生。

在正常的肝脏组织中并没有这种细胞，但病变组织中却出现，因此推断致病因素可以诱导异种细胞向肌成纤维细胞转化。

Depending on the primary site of injury the fibrosis may be present in the hepatic parenchyma实质as seen in chronic hepatitis or may be restricted to the portal areas as in most biliary diseases. It is suggested that hepatic injury of different etiology triggers the transdifferentiation to myofibroblasts from distinct cell populations. Here we discuss the origin and fate of myofibroblast in liver fibrosis.
In all clinical and experimental liver fibrosis, myofibroblasts are the source of the ECM constituting the fibrous scar. Myofibroblasts are only found in the injured, but not the normal, liver. Thus,the activated myofibroblast is a pivotal player in development of liver cirrhosis, and has recently attracted interest as a therapeutic
target. However, the origin of the hepatic myofibroblast is still unclear, and perhaps the fibrosis induced by different types of liver injury results from different fibrogenic cells. Hepatic myofibroblasts may originate from bone marrow-derived mesenchymal cells and fibrocytes,5 but only a small contribution of BM derived cells to the myofibroblast population has been detected in experimental liver fibrosis. Another mechanism implicated in fibrogenesis is the epithelial-to-mesenchymal transition (EMT), in which epithelial cells acquire features of mesenchymal cells and may give rise to fully differentiated myofibroblasts.6,7 However, recent cell fate mapping studies have failed to detect any hepatic myofibroblasts originating from hepatocytes, cholangiocytes, or epithelial progenitor cells. Endothelial-to-mesenchymal transition (EndMT), when endothelial cells undergo a similar phenotypic change to myofibroblasts8,9 is a theoretically, but not yet assessed source of liver myofibroblasts. Thus, the major sources of myofibroblasts in liver fibrosis appear to be the endogenous liver mesenchymal cells, the hepatic stellate cells and the portal fibroblasts.
ROLE OF EMT IN BA
Evidence for the epithelial to mesenchymal transition in
biliary atresia fibrosis
We therefore obtained liver tissue from patients with biliary atresia as well as a variety of other pediatric and adult liver diseases. Tissues were immunostained with antibodies against the biliary epithelial cell marker CK19 as well as with antibodies against proteins characteristically expressed by cells undergoing the epithelial to mesenchymal transition, including fibroblast-specific protein 1, the collagen chaperone heat shock protein 47, the intermediate filament protein vimentin, and the transcription factor Snail. The degree of colocalization was quantified using a multispectral imaging system. We observed significant colocalization between CK19 and other markers of the epithelial to mesenchymal transition in biliary atresia as well as other liver diseases associated with significant bile ductular proliferation, including primary biliary cirrhosis. There was minimal colocalization seen in healthy adult and pediatric livers, or in livers not also demonstrating bile ductular
proliferation. Multispectral imaging confirmed significant colocalization of the different markers in biliary atresia. In conclusion, we present significant histologic evidence suggesting that the epithelial to mesenchymal transition occurs in human liver fibrosis, particularly in diseases such as biliary atresia and primary biliary cirrhosis with prominent bile ductular proliferation
Epithelial–mesenchymal transition induced by biliary innate immunity contributes to the sclerosing cholangiopathy of biliary atresia Infections of Reoviridae consisting of a double-stranded RNA (dsRNA) genome and the biliary innate immune response to dsRNA are implicated in the aetiopathogenesis of biliary atresia (BA). Epithelial–mesenchymal transition (EMT) has recently been proposed as a mechanism behind the sclerosing cholangitis in BA. We hypothesized that the innate immune response to dsRNA in biliary epithelial cells plays an important role in peribiliary fibrosis via biliary EMT. Experiments using cultured human biliary epithelial cells revealed that stimulation with poly(I : C) (a synthetic analogue of viral dsRNA) increased the expression of basic fibroblast growth factor (bFGF, an EMT-inducer), S100A4 (a mesenchymal marker) and Snail (a transcriptional factor), and decreased that of epithelial markers (biliarytype cytokeratin 19 and E-cadherin) and Bambi (TGF-β1 pseudoreceptor). The expression
of TGF-β1 (EMT-inducer) and vimentin (a mesenchymal marker) was not affected by poly(I :C). Both EMT-inducers, bFGF and TGF-β1, evoked a decrease and increase in
the expression of the epithelial markers and of vimentin respectively, and the expression of
Bambi was down-regulated on stimulation with bFGF. Combined treatment with bFGF and TGF-β1 quickly and completely induced a transformation of morphology as well
as change from epithelial to mesenchymal features in cultured biliary epithelial cells. Immunohistochemistry revealed that biliary epithelial cells lining extrahepatic bile ducts and peribiliary glands in BA frequently show a lack of epithelial markers and an aberrant expression of vimentin. Moreover, the biliary epithelium showing sclerosing cholangitis expressed bFGF accompanied by bFGF-positive mononuclear cells. In conclusion, the EMT may contribute to the histogenesis of sclerosing cholangiopathy, and the biliary innate immune response to dsRNA viruses induces biliary epithelial cells to undergo EMT via
the production of bFGF and the increased susceptibility to TGF-β1 caused by the downregulation of Bambi expression.
Analysis of Biliary Epithelial-Mesenchymal Transition in Portal Tract Fibrogenesis in Biliary Atresia
Background The cellular origin of myofibroblast in the liver fibrosis remains unclear. This study was designed to investigate whether biliary epithelial cells (BECs) undergoing epithelial–mesenchymal transition (EMT) might be found in patients with biliary atresia, thereby serving as a source of fibrotic myofibroblasts. Methods Liver sections from patients with biliary atresia were evaluated to detect antigen for the BECs marker 4 and cytokeratin-7 (CK-7), proteins (fibroblast-specific protein 1, also known S100A4; the collagen chaperone heat shock protein 47, HSP47) characteristically expressed by cells undergoing EMT, as
well as myofibroblasts marker a-smooth muscle actin (a-SMA).Results Normal bile ducts BECs could express CK-7 and low levels of a-SMA; they did not express
S100A4 and HSP47. However, BECs from biliary atresia resulted in increased expression of a-SMA, S100A4, with concurrent transition to a fibroblast-like morphology and decreased expression of AK-7. Furthermore, BECs in biliary atresia were associated with significant bile ductular proliferation and coexpressed both epithelial and mesenchymal markers.Conclusions From significant histologic evidence, the BECs forming small- and medium-sized bile ducts undergoing EMT may account for prominent bile ductular proliferation and directly contribute to fibrogenesis in BA.
Hedgehog Activity,
Epithelial-Mesenchymal Transitions, and Biliary Dysmorphogenesis in Biliary Atresia
Biliary atresia (BA) is notable for marked ductular reaction and rapid development of fibrosis. Activation of the Hedgehog (Hh) pathway promotes the expansion of populations of immature epithelial cells that coexpress mesenchymal markers and may be profibrogenic.We examined the hypothesis that in BA excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Livers and remnant extrahepatic ducts from BA patients were evaluated by quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and immunostaining for Hh ligands, target genes, and markers of mesenchymal
cells or ductular progenitors. Findings were compared to children with genetic cholestatic disease, age-matched deceased donor controls, and adult controls. Ductular cells isolated from adult rats with and without bile duct ligation were incubated with Hh ligand-enriched medium 6 Hh-neutralizing antibody to determine direct effects of Hh ligands on epithelial to mesenchymal transition (EMT) marker expression. Livers from pediatric controls showed greater innate Hh activation than adult controls. In children with BA, both intra- and extrahepatic ductular
cells demonstrated striking up-regulation of Hh ligand production and increased expression of Hh target genes. Excessive accumulation of Hh-producing cells and Hh-responsive cells also occurred in other infantile cholestatic diseases. Further analysis of the BA samples demonstrated that immature ductular cells with a mesenchymal phenotype were Hh-responsive. Treating immature ductular cells with Hh ligand-enriched medium induced mesenchymal genes; neutralizing Hh ligands inhibited this. Conclusion: BA is characterized by excessive Hh pathway activity, which stimulates biliary EMT and may contribute to biliary dysmorphogenesis. Other cholestatic diseases show similar activation, suggesting that this is a common response to cholestatic injury in infancy
Liver fi brosis during the development of biliary atresia:
Proof of principle in the murine model\
Background: Themurinemodel of biliary atresia (BA) is used for
examining the pathogenesis of BA. The aimof the
study was description of the morphological features and illustrating
the detailed development of fi brosis using
the Biliary Atresia Research Consortium (BARC) system.
Methods: Neonatal mice were injected intraperitoneally with rhesus
rotavirus (RRV) strain (N = 17). Healthy
mice were the control group (N = 29). All mice were sacri fi ced at 7
or 14 days after birth. Two pathologists
examined the morphological features using the BARC system; CK19, αSMA and collagen type I were assessed
by immunohistochemistry.
Results: In RRV mice, portal fi brous expansionwith focal bile duct
proliferation and strong portal cellular in fi ltrate
was found in contrast to healthy mice. In RRV mice, CK19 bile duct
staining was signi fi cantly less or absent
(p b 0.01), with stronger portal staining of collagen type I (p = 0.02). Expansion of staining for αSMA was
more in RRV mice (p b 0.01), but αSMA portal staining was stronger in healthy mice (p= 0.02).
Conclusions: The morphological features observed in the murine model of BA correspond with the BA characteristics
according to the BARC criteria. Fibrosis is an important feature of themodel. Therefore, thismurine model is
useful for investigating the pathogenesis of BA.
前期研究中，我们对人类胆道闭锁肝内外胆道和MMU18006病毒致畸胆道闭锁动物模型肝内外胆道进行了系列研究[11-13]，我们发现在人类及动物模型胆道闭锁早期肝内外胆道上皮细胞形态学上发生了不同程度的上皮细胞向间充质细胞
的转化，即胆管上皮细胞形态上发生变化、形成突起，不仅表达上皮细胞特异性标志物，而且同时表达间充质细胞特异性标志物，随着发病时间的延长，这种转化日趋明显，最终导致整个胆管均被间充质细胞及胶原纤维取代；在胆道闭锁发病早期，毛细胆管及胆管上皮细胞呈无序性增生、代表上皮细胞特征的细胞因子CK19、E-cadherin等表达水平增高、参与胚胎期胆管发育的一些重要发育相关基因如SHH、Notch等重新激活，这提示我们胆系在发生上皮细胞向间充质细胞转化、胆汁排泄能力降低的同时，也在进行着相应的自我修复，而这种无序的自我修复也是胆道闭锁发生的一个重要环节；除此之外，胆管上皮基底膜破坏、胆管周围有不同程度炎症细胞浸润、各种炎症相关基因相继激活（TGF-beta、Smad 等）、表达增强，尤其是TGF-β1表达显著增强，说明在肝内外胆道在受到某种外界致病因素的刺激下，发生了上皮细胞基底膜的破坏、自身抗原物质的产生、炎症细胞的浸润及活化、细胞结构破坏、增生、上皮向间充质转化、进而纤维化
等一系列反应，这种反应伴随着炎症相关基因的激活及一些胚胎发育相关基因的重新激活，最终导致了肝内外胆管上皮细胞被分泌胶原蛋白的间充质细胞所代替，继而消失，这也是胆道闭锁肝纤维化进展迅速的重要原因之一。

因而根治胆道闭锁仅仅手术重建肝外胆道显然是不够的，必须针对胆道闭锁胆管纤维化、消失过程的不同环节确定不同的治疗靶点，包括降低胆管上皮细胞损伤和炎症、干预细胞内信号和转录途径、减轻胆管上皮向间充质细胞的转化、拮抗使上皮发生转化的细胞因子和生长因子、促进发生转化的胆管上皮细胞发生逆向转化、加快细胞外基质的降解等。

SIGNAL PATHWAYS ASSOCIATED WITH EMT IN BA
EMT信号转导通路及相关标志物
1型MET
Wnts和Notch信号通路。

细胞表面蛋白（E-cadherin↓ N-cadherin↑）
2型MET
炎症发硬造成的肾脏损伤由其募集的多种类型细胞所介导，其中主要包括巨噬细胞和活化的成纤维细胞。

上述细胞通过释放大量生长因子、趋化因子和基质
蛋白酶，活化包括Smads通路、ERK-MAPK通路、PI3K-AKT通路在内的多条
信号转导径路启动2型EMT。

细胞骨架蛋白（成纤维特异性蛋白1，FSP1），细胞表面蛋白（E-cadherin↓ OB-cadherin↑），细胞外基质蛋白（层连蛋白↓ 纤连蛋白↑）
3型MET
在原发肿瘤的形成过程中，主要存在5条信号通路，包括酪氨酸激酶受体径路、整合素径路、Wnts径路、NF-kB径路和TGF-b径路（涉及
smads/AKT/GSK3/Rho-GTP酶等信号分子）。

转录因子：Snail因子，特别是Snail1和Snail2的上调表达可以作为3性EMT 的共同生物标志物。

Twist在早期的胚胎形成、组织纤维化和肿瘤转移过程中也存在上调表达。

在3型EMT形成的转移性恶性肿瘤细胞中，Twist具有独立的抑制E钙粘蛋白
表达并上调纤连蛋白和N钙粘蛋白的作用。

MicroRNA（miR200s，miR205等），细胞表面蛋白（E-cadherin↓ N-cadherin↑）Dysregulation of upstream and downstream transforming
growth factor-βtranscripts in livers of children with biliary atresia and fibrogenic gene signatures
Introduction: Our previous work demonstrated that the transforming-growth factor (TGF) βpathway plays a central role in the liver fibrosis associated with experimental biliary atresia (BA). To confirm these findings in humans, we performed an in silico analysis of publicly available microarray data from liver specimens from children with BA, with the
hypothesis that the TGF-βpathway would be dysregulated. Methods: We analyzed publicly available liver gene expression microarray data from 47 infants with BA. We re-analyzed the microarray image files and clinical data to compare gene expression differences between the fibrogenic and inflammatory cohorts identified in the initial study. Targets from the microarray analysis were confirmed using the animal model of BA. Results: Analysis of variance (ANOVA) detected 6903 transcripts (2822 distinct genes) differentially regulated between groups (p b 0.01; fold change N 1.2). We used a targeted approach to identified a subgroup of 24 TGF-β-related transcripts. Expressions for procollagen transcripts were increased in the fibrogenic group (1.2-fold to 1.4-fold); expression of matrix metalloproteinase (MMP)-7 was similarly increased 2-fold, while MMP-9 and plasminogen activator inhibitor-1 were decreased 2-fold and 3-fold respectively. Integrins β5 (1.18-fold) and β8 (1.84-fold) also demonstrated increased expression in the fibrogenic group. Increased expression of β5 (3-fold) and β8 (5-fold) as well as Smad-3 (4-fold) and Smad interacting protein (SIP)-1 (3.5-fold) mRNA was confirmed in experimental BA. Phosphorylated Smad-3 protein in the experimental group was also nearly twice that of the control group, further implicating the TGF-βpathway. Conclusion: Gene transcripts for known upstream and downstream TGF-βmediators are differentially expressed in liver specimens from children with BA and a fibrogenic gene signature. The same integrins that were dysregulated in the human specimens were also found to be up-regulated in our animal BA model, as were other intermediaries in the TGF-βpathway. Further investigation into whether these mediators may be attractive targets for future therapy in children with BA is warranted.
Liver fi brosis during the development of biliary atresia: Proof of principle in the murine model
Background: Themurinemodel of biliary atresia (BA) is used for examining the pathogenesis of BA. The aimof the study was description of the morphological features and illustrating the detailed development of fi brosis using the Biliary Atresia Research Consortium (BARC) system. Methods: Neonatal mice were injected intraperitoneally with rhesus rotavirus (RRV) strain (N = 17). Healthy mice were the control group (N = 29). All mice were sacri fi ced at 7 or 14 days after birth. Two pathologists examined the morphological features using the BARC system; CK19, αSMA and collagen type I were assessed by immunohistochemistry. Results: In RRV mice, portal fi brous expansionwith focal bile duct proliferation and strong portal cellular in fi ltrate was found in contrast to healthy mice. In RRV mice, CK19 bile duct staining was signi fi cantly less or absent (p b 0.01), with stronger portal staining of collagen type I (p = 0.02). Expansion of staining for αSMA was more in RRV mice (p b 0.01), but αSMA portal staining was stronger in healthy mice (p= 0.02).Conclusions: The morphological features observed in the murine model of BA correspond with the BA characteristics according to the BARC criteria. Fibrosis is an important feature of themodel. Therefore, thismurine model is useful for investigating the pathogenesis of BA.
CONCLUSION。