Pdx1与NeuroD1联合诱导L02细胞Nkx6.1和GLUT2的表达

目的：应用分子生物学技术,构建胰腺十二指肠同源框蛋白1(Pdx1)和神经分化因子1(NeuroD1)转录因子真核表达质粒,检测其能否在真核细胞中有效表达并诱导人肝细胞转分化的能力。方法:以人胚胎胰腺组织mRNA为模板经RT-PCR扩增获得Pdx1和NeuroD1基因,分别克隆到真核双表达载体pIRES质粒的多克隆位点A(MCSA)和B(MCSB)中,构建pI/Pdx1/NeuroD1真核表达质粒,并转染L02细胞。用RT-PCR、免疫组化、间接荧光法和Western blotting检测目的基因及NK转录因子相关的Nkx6.1(Nkx6.1)和葡萄糖运载体2(GLUT2)的表达情况。结果:目的基因克隆正确且在真核细胞中有效表达;并可诱导肝细胞表达胰腺β细胞功能相关的Nkx6.1和GLUT2因子。结论:pI/Pdx1/NeuroD1质粒成功构建和在人真核细胞表达,并诱导人肝细胞表达β细胞功能相关的Nkx6.1转录因子和GLUT2,提示Pdx1与NeuroD1可诱导肝细胞向胰腺内分泌细胞分化。     

Follicular cholangitis and pancreatitis - clinicopathological features and differential diagnosis of an under-recognized entity

Zen Y, Ishikawa A, Ogiso S, Heaton N & Portmann B
(2012) Histopathology  60, 261–269
Follicular cholangitis and pancreatitis – clinicopathological features and differential diagnosis of an under‐recognized entity Aims: Biliary and pancreatic ductal systems can be involved in several lymphoplasmacytic inflammatory conditions, including primary sclerosing cholangitis, immunoglobulin G (IgG) 4‐related cholangitis and autoimmune pancreatitis. Here in we describe an unusual pancreatocholangitis whose features suggest a distinct disease entity. Methods and results: The study group consists of five adult patients, three with predominantly hilar bile duct stricture and two with a bulky pancreatic head. Four patients were treated surgically for suspected malignancy and one patient underwent liver transplantation with a clinical diagnosis of primary sclerosing cholangitis. Histological examination revealed extensive lymphoplasmacytic inflammation centred on large biliary or pancreatic ducts. Many lymphoid follicles with germinal centres were noted around the affected ducts. Whipple specimens from two patients with a pancreatic head mass showed similar follicular inflammation histologically around bile ducts. In contrast to autoimmune pancreatitis, diffuse infiltration of IgG4⁺ plasma cells, granulocytic epithelial lesions and obliterative phlebitis were not identified. The postoperative course was uneventful, without evidence of recurrence (follow‐up period 17–65 months). Conclusions: This study suggests that a disease entity which can be named follicular cholangitis and pancreatitis exists and may be under‐recognized. The disease mainly affects the hilar bile ducts and pancreatic head in adults.     

The MUC6 secretory mucin gene is expressed in a wide variety of epithelial tissues

Secretory mucins play an important role in the cytoprotection of epithelial surfaces and are used as tumour markers in a variety of cancers. The MUC6 secretory mucin was originally isolated from a gastric cDNA library. The aim was to determine the specific type and location of MUC6 mucin gene expression in a wide range of human adult and fetal epithelial tissues. In situ hybridization, RNA analysis, and immunohistochemistry were used to quantify and localize mucin gene expression. The data obtained show that MUC6 is highly expressed in gastric mucosa, duodenal Brunner's glands, gall bladder, seminal vesicle, pancreatic centroacinar cells and ducts, and periductal glands of the common bile duct; focal expression is seen in basal endometrial and endocervical glands. MUC6 epitopes were also highly expressed in 7/10 pancreatic cancers and 7/10 cholangiocarcinomas and focally expressed in 4/10 endocervical adenocarcinomas. Expression of MUC6 occurs early in fetal development and was observed in Brunner's glands and pancreatic ducts at 18-19 weeks and in gastric glands at 20 weeks' gestation. The tissue distribution of the MUC6 secretory mucin indicates that it may function to protect epithelial tissues from a wide range of substances. Expression of MUC6 is frequently preserved in pancreatic and bile duct adenocarcinomas, but it is only sparsely expressed in endocervical carcinomas.     

Are hepatolithiasis and cholangiocarcinoma aetiologically related?

Summary A few cases of cholangiocarcinoma (CC) related to hepatolithiasis have been reported to date, but the aetiological relationship remains unclear. In an attempt to clarify the relationship between two phenomena, we examined morphologically 12 cases of hepatolithiasis associated with CC and 26 cases of hepatolithiasis without CC, with the aid of immunohistochemical staining for carcinoembryonic antigen (CEA). In the livers where both hepatolithiasis and CC were found, the carcinoma spread along the lumenal surface of the stone-containing bile ducts and invaded the ductal walls. Features of chronic proliferative cholangitis which was a basic feature of hepatolithiasis, were found within the bile duct walls where carcinoma was invading. In some cases of chronic proliferative cholangitis with hepatolithiasis in the absence of CC, atypical epithelial hyperplasia was noted. Atypical epithelial hyperplasia was also found in bile ducts adjacent to and remote from CC. Atypical epithelial hyperplasia was positive for CEA. The data lead us to speculate that chronic proliferative cholangitis in the presence of hepatolithiasis can undergo progressive changes to atypical epithelial hyperplasia which may in turn progress to CC.     

Duodenal window revisited: A histological study using human fetuses

To assess the development of the duodenal window in fetuses, we examined semiserial histological sections of 59 human fetuses with a crown‐rump length of 27–156 mm (～4–18 weeks of gestation). In 44 of the 54 fetuses with horizontal sections, the duodenal window was formed by interdigitation of the anterior and posterior muscle slips from the proper duodenal circular muscle coat. The anterior slips approached the common bile duct from the anterior side and wound around the bile duct from the right aspect, whereas the posterior slips approached the main pancreatic duct from the posterior side, reaching the left or outer aspect of the duct without winding. These slips may become longitudinal muscles in the ampulla after birth. Six specimens showed variations in this typical pattern, in that the posterior muscle slips as well as the duodenal longitudinal muscle coat wound around the bile duct. In the remaining four specimens, we observed an abnormal union of the bile and pancreatic ducts, with the duodenal circular muscles suddenly ending along the window or slightly inserted into the right side of the common duct after joining. In all later‐stage fetuses, the common sphincter surrounded both the bile and pancreatic ducts in the ampulla. Consequently, at and along the duodenal window, the proper duodenal circular muscle seemed to contribute to fetal sphincter formation. The window was not a simple hiatus but a functional interface between the sphincter and the duodenal wall. Clin. Anat. 26:598–609, 2013. © 2012 Wiley Periodicals, Inc.     

Histological and immunohistological findings in biliary intraepithelial neoplasia arising from a background of chronic biliary disease compared with liver cirrhosis of non-biliary aetiology

Aishima S, Iguchi T, Fujita N, Taketomi A, Maehara Y, Tsuneyoshi M & Oda Y (2011) Histopathology 59 , 867–875 Histological and immunohistological findings in biliary intraepithelial neoplasia arising from a background of chronic biliary disease compared with liver cirrhosis of non‐biliary aetiology Aims: Hitherto, biliary intraepithelial neoplasia (BilIN) has been described in chronic biliary disease but rarely in non‐biliary liver cirrhosis (LC). Intraepithelial neoplasia of the pancreas shows alterations in the expression of cell cycle and mucin core proteins. The aim of this study was to evaluate BilIN and reactive biliary lesions in biliary disease and non‐biliary LC. Methods and results: BilIN was found in 51% (33 of 65) of liver tissue cases of biliary disease, and in 11% (34 of 310) of the LC group. Immunohistologically, MUC5AC, an ‘early phase’ protein, and Ki67, reflecting ‘late phase’ expression, were identified with increasing degrees of dysplasia in both groups, but that expression was significantly higher in the biliary disease group. ‘Early phase’ cell cycle proteins, p16 (decrease) and p21 (increase) altered in both biliary and LC groups with increasing degrees of dysplasia. Conclusions: We found BilIN in the large bile ducts of hepatitis B virus‐ and hepatitis C virus‐related LC as well as in cases related to a biliary aetiology. The LC group was significantly less likely to show changes in the expression of MUC5AC and proliferative activity than the biliary group. Alterations in p16 and p21 reflected increasing degrees of dysplasia in both groups.     

Pancreaticobiliary maljunction and choledochal cysts: from embryogenesis to therapeutics aspects

Pancreaticobiliary maljunction (PBM) and choledochal cysts (CC) are rare and little-known diseases. Several definitions have been proposed for the PBM, but the most widely accepted is an excessive length of the common pancreaticobiliary duct due to the abnormal convergence of the pancreatic and biliary ducts out of the duodenal wall. This anomaly, thought to develop during embryogenesis, is associated with a loss of regulation of the Oddi’s sphincter leading to a pancreaticobiliary or biliopancreatic backflow. This reflux could be responsible, or associated with cystic dilatation of the bile ducts and biliary tract cancers, to various biliary or pancreatic events such as cholangitis or pancreatitis. For the diagnosis of PBM, magnetic resonance cholangiopancreatography has now become the gold standard as a noninvasive imaging tool. However, the main risk of PBM is the development of bile duct cancer, most often on a distended area. PBM without CC increase the occurrence of gallbladder cancer and require a preventive cholecystectomy. Surgical treatment of PBM with concomitant CC is more complex and depends on localization of the dilatation(s) as reported in the Todani’s classification. This review describes the pathogenesis, embryogenesis, clinical features, investigation and management of PBM and CC.     

The role of 64‐slice CT following perfusion with iohexol via the hepatopancreatic ampulla in assessing pancreaticobiliary junctions

The aim of this study was to delineate the structure of the pancreatic and biliary ducts in premature infants using a novel imaging method. The duodenal papillae of 30 premature infant cadavers were dissected. The pancreatic and biliary ducts were visualized using 64‐detector multislice spiral computed tomography (MSCT). Contrast agent was injected into the duodenal papilla via the hepatopancreatic ampulla of Vater. MSCT scanning revealed both the pancreatic and biliary ducts as well as the common channel in 18 cases. The bile duct was visualized in the remaining 12 cases. Four patterns of the pancreaticobiliary ductal junction were noted: Y‐type (73.3%), U‐type (13.3%), V‐type (6.7%), and II‐type (6.7%). The results showed that MSCT and three‐dimensional reconstruction can be used to visualize the junction pattern and common channel of the pancreatic and biliary ducts, and the structure of the surrounding tissue, in premature infants. Clin. Anat. 28:645–648, 2015. © 2015 Wiley Periodicals, Inc.     

Intrahepatic cholangiocarcinoma arising in chronic advanced liver disease and the cholangiocarcinomatous component of hepatocellular cholangiocarcinoma share common phenotypes and cholangiocarcinogenesis

Xu J, Sasaki M, Harada K, Sato Y, Ikeda H, Kim J‐H, Yu E & Nakanuma Y
(2011) Histopathology  59 , 1090–1099
Intrahepatic cholangiocarcinoma arising in chronic advanced liver disease and the cholangiocarcinomatous component of hepatocellular cholangiocarcinoma share common phenotypes and cholangiocarcinogenesis Aims: Intrahepatic cholangiocarcinomas (ICCs) are known to arise in cases of non‐biliary, chronic advanced liver disease (CALD), but their clinicopathological features remain unexplored. The aim of this study was to compare the histological and immunohistochemical ICCs arising inCALD with those arising in livers with non‐specific reactive (NSR) changes. Methods and results: Seventy‐one cases of ICC arising in CALD were compared with ICCs arising in livers with NSR changes, including normal livers (72 cases) and the cholangiocarcinomatous (CC) component of hepatocellular cholangioncarcinomas (HC‐CCs) (30 cases). The expression of mucin was higher in ICC with NSR changes, whereas it was relatively low in ICC with CALD and the CC component of HC‐CC. The expression of biliary markers [cytokeratin (CK)7, CK19, epithelial membrane antigen, and epithelial cell adhesion molecule (EpCAM)] was lower in CC with CALD and in the CC component of HC‐CC than in CC with NSR changes. The expression of hepatic progenitor cell markers [neural cell adhesion molecule (NCAM) and c‐kit] was higher in ICC with CALD and the CC component of HC‐CC than in ICC with NSR changes. EpCAM and CK19 were constantly expressed in cultured CC cells, whereas NCAM was infrequently expressed in cultured CC cells. Conclusions: The carcinogenesis of ICC arising in CALD and the ICC component of HC‐CC, each showing similar features, may involve hepatic progenitor cells.     

Progenitor cell niches in the human pancreatic duct system and associated pancreatic duct glands: an anatomical and immunophenotyping study

Pancreatic duct glands (PDGs) are tubule‐alveolar glands associated with the pancreatic duct system and can be considered the anatomical counterpart of peribiliary glands (PBGs) found within the biliary tree. Recently, we demonstrated that endodermal precursor niches exist fetally and postnatally and are composed functionally of stem cells and progenitors within PBGs and of committed progenitors within PDGs. Here we have characterized more extensively the anatomy of human PDGs as novel niches containing cells with multiple phenotypes of committed progenitors. Human pancreata (n = 15) were obtained from cadaveric adult donors. Specimens were processed for histology, immunohistochemistry and immunofluorescence. PDGs were found in the walls of larger pancreatic ducts (diameters > 300 μm) and constituted nearly 4% of the duct wall area. All of the cells identified were negative for nuclear expression of Oct4, a pluripotency gene, and so are presumably committed progenitors and not stem cells. In the main pancreatic duct and in large interlobular ducts, Sox9⁺ cells represented 5–30% of the cells within PDGs and were located primarily at the bottom of PDGs, whereas rare and scattered Sox9⁺ cells were present within the surface epithelium. The expression of PCNA, a marker of cell proliferation, paralleled the distribution of Sox9 expression. Sox9⁺ PDG cells proved to be Pdx1⁺/Ngn3^+/–/Oct4A^?. Nearly 10% of PDG cells were positive for insulin or glucagon. Intercalated ducts contained Sox9⁺/Pdx1⁺/Ngn3⁺ cells, a phenotype that is presumptive of committed endocrine progenitors. Some intercalated ducts appeared in continuity with clusters of insulin‐positive cells organized in small pancreatic islet‐like structures. In summary, PDGs represent niches of a population of Sox9⁺ cells exhibiting a pattern of phenotypic traits implicating a radial axis of maturation from the bottoms of the PDGs to the surface of pancreatic ducts. Our results complete the anatomical background that links biliary and pancreatic tracts and could have important implications for the common patho‐physiology of biliary tract and pancreas.     

Neuroendocrine carcinoma arising in a hepatitis C virus‐infected liver: Mechanism of the tumor development may be similar to that of development of pancreatic neuroendocrine cells

We experienced a case of neuroendocrine carcinoma (NC). The tumor developed in the cirrhotic liver of a 62‐year‐old Japanese man who had been infected with hepatitis C virus. The tumor cells showed high N/C ratio, formed many rosettes, and expressed CD56, synaptophysin, HepPar1 and pancreatic and duodenal homeobox 1. MIB1 expression was 65%. Because both liver and pancreas are derived from a common endodermal layer during fetal development, we speculated that the tumor may have formed via the interaction of neurogenin 3, insulinoma‐associated 1 gene and NeuroD/beta2, which are involved in the stage at which some pancreatic cells commit to becoming endocrine cells. Molecular analysis revealed that the NC had higher relative expression levels of mRNA of the three molecules than did the nontumorous liver. The results indicate that the NC in this patient may have formed via the same mechanism that acts in the development of pancreatic neuroendocrine cells.     

Marked diffuse dilations of the biliary tree associated with intrahepatic calculi,biliary sludges and a mucinous cyst of the pancreatic head in a 99-year-old woman

A 99-year-old woman was admitted to Shizuoka Shimizu Municipal Hospital because of fever and anasarca. Imaging and laboratory tests showed pneumonia, urinary tract infection, and cardiac failure. The patient died 20 days after admission. An autopsy revealed marked diffuse dilations of the biliary tree ranging from the lower common bile duct to intrahepatic bile ducts. Intrahepatic calcium bilirubinate stones and biliary sludges were recognized within the dilated bile ducts. A unilocular cyst (2 cm in diameter) was present in the pancreatic head adjacent to the lower common bile duct, and it appeared to compress the common bile duct. Histologically, the walls of the dilated biliary tree showed proliferation of peribiliary glands, fibrosis, and infiltration of lymphocytes and neutrophils (cholangitis). The lumens of the dilated biliary ducts contained neutral and acidic mucins, fibrinous materials, bacteria, neutrophils, and Aspergillus fungi, in addition to the calculi and sludges. The background liver showed atrophy (400 g). The pancreatic unilocular cyst was composed of mucous columnar cells with a few infoldings, and the pancreas also showed foci of mucinous duct hyperplasia and ectasia; the pathological diagnosis of the cyst was cystic dilations of a pancreatic duct branch (mucinous ductal ectasia or mucinous cyst). Other lesions included aspiration pneumonia, emaciation, atrophy of systemic organs, gastric leiomyoma, serous cystadenoma of the right ovary, and arteriosclerotic nephrosclerosis. The present case suggests that a mucinous cyst of the pancreas may compress the biliary tree and lead to marked diffuse dilations of the biliary tree. Alternatively, the dilations of the bile ducts may be associated with aging or may be of congenital origin. The dilated bile ducts may, in turn, give rise to bacterial and fungal cholangitis and formation of biliary sludges and intrahepatic calcium bilirubinate stones.     

Biliary intraepithelial neoplasia: an international interobserver agreement study and proposal for diagnostic criteria.

Cholangiocarcinoma of the intrahepatic and extrahepatic bile ducts develops through a multistep histopathologic sequence. Premalignant or non-invasive neoplastic lesions of bile ducts have been historically called biliary dysplasia or atypical biliary epithelium. To this date, no standard terminology or classification system has been offered for these lesions. In 2005, a conceptual framework and diagnostic criteria for biliary intraepithelial neoplasia (BilIN) were proposed using the livers of patients with hepatolithiasis. We report herein an international interobserver agreement study on the diagnosis of biliary non-invasive neoplastic lesions with the goal to obtain a consensus on the terminology and grading. Seventeen pathologists from the United States, Europe and Asia participated in this study. They shared a digital file containing histological pictures of 30 foci of non-invasive neoplastic lesions selected from the biliary system of patients suffering from primary sclerosing cholangitis, choledochal cyst or hepatolithiasis. In the criteria, we proposed in 2005, BilIN was classified into three categories based on the degree of atypia: BilIN-1, BilIN-2 and BilIN-3. In this study, consensus was reached for the terminology of BilIN and the three-grade classification system. Interobserver agreement on the diagnosis was moderate (kappa-value=0.45). On the basis of the suggestions and opinions obtained from the 17 participants, the original criteria for BilIN were revised. We now propose a new consensus classification of BilIN that may assist in allowing a more uniform terminology for the diagnosis of biliary non-invasive neoplastic lesions. This classification should help to advance clinical and research applications.     

利用Tet-On系统构建稳定表达Pdx1的小鼠ESC细胞株

目的:利用Tet-On系统构建稳定表达胰十二指肠同源异形框1(Pdx1)的小鼠胚胎干细胞(ESC)株,为进一步研究Pdx1+定型内胚层细胞向胰腺细胞分化奠定了基础.方法:采用Tet-On系统构建具有绿色荧光蛋白标记及嘌呤霉素抗性的Pdx1过表达慢病毒载体并感染胚胎干细胞.实验分为空白对照组(ESC组)、空载慢病毒对照组...     

Aberrant expression of stem cell factor on biliary epithelial cells and peribiliary infiltration of c-kit-expressing mast cells in hepatolithiasis and primary sclerosing cholangitis: a possible contribution to bile duct fibrosis

Hepatolithiasis and primary sclerosing cholangitis (PSC) are intractable chronic biliary diseases. In hepatolithiasis, bilirubin-calcium stones are packed in multiple irregularly dilated intrahepatic bile ducts. In PSC, small bilirubin-calcium stones develop terminally. The progressive periductal fibrosis with dilated and stenotic bile ducts in these two diseases may play a role in their incurability. This immunohistochemical study has investigated the expression of some factors that might be involved in fibrogenesis in hepatolithiasis and PSC. Many mast cells positive for c-kit were found in the periductal and ductal fibrosis around the intrahepatic large bile ducts and also around the proliferative peribiliary glands. These mast cells also expressed basic fibroblast growth factor and/or tumour necrosis factor-alpha, which are known as fibrogenetic factors. It was of interest that the aberrant expression of stem cell factor (SCF), a ligand of c-kit, was demonstrated on biliary epithelia of the dilated and stenotic bile ducts showing periductal fibrosis and inflammation and also of the proliferated peribiliary glands in hepatolithiasis and PSC, while no such expression was seen in non-affected bile ducts in hepatolithiasis or in the bile ducts in normal livers. Some of the infiltrating mononuclear cells around the SCF-expressing bile ducts were also positive for SCF. It seems likely that aberrantly expressed SCF on biliary epithelial cells accumulates and stimulates mast cells via the c-kit receptor and that these up-regulated mast cells induce progressive periductal and portal fibrosis by displaying fibrogenetic factors in hepatolithiasis and PSC.     

胎儿胰胆管解剖研究

目的：探讨胎儿胰胆管合流的类型及十二指肠乳头的形状和位置。方法：选取经水囊引产死后6h内的新鲜胎儿36例,胎龄4月～9月。切取包括胆囊、胆总管、十二指肠、胰腺的标本,观察十二指肠乳头的位置、形状。再置于福尔马林中24h,脱水、透明、浸腊、包埋。组织块以乳头为中心,横行连续切片,厚6μm,每隔5张选1张,作HE染色。显微镜下观察胰胆管合流的类型。结果：(1)十二指肠大乳头半球形58.1% (21例),圆柱形25% (9例),扁平形16.9%(6例)3种形态。乳头位于十二指肠降部上1/3部8.3% (3例),中1/3部69.4% (25例),下1/3部19.4% (7例),远部2.9% (1例)。(2)存在U、V、Y和异常合流4种胰胆管合流方式,其中以Y型66.7% (24例),V型19.4%（7例）,U形11.1%（4例）,APBDU2.8%（1例）。结论：乳头形态位置及胰胆管合流的类型变化较多,了解这一解剖在临床有重要的意义。