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2.
Science ; 371(6531): 839-846, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33602855

RESUMO

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.


Assuntos
Doenças dos Ductos Biliares/terapia , Ductos Biliares Intra-Hepáticos/fisiologia , Ductos Biliares/citologia , Terapia Baseada em Transplante de Células e Tecidos , Células Epiteliais/citologia , Organoides/transplante , Animais , Bile , Ductos Biliares/fisiologia , Ductos Biliares Intra-Hepáticos/citologia , Ducto Colédoco/citologia , Células Epiteliais/fisiologia , Vesícula Biliar/citologia , Regulação da Expressão Gênica , Humanos , Fígado/fisiologia , Transplante de Fígado , Transplante de Células-Tronco Mesenquimais , Camundongos , Organoides/fisiologia , RNA-Seq , Obtenção de Tecidos e Órgãos , Transcriptoma
3.
Hepatology ; 73(1): 247-267, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32222998

RESUMO

BACKGROUND AND AIMS: Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. APPROACH AND RESULTS: Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. CONCLUSIONS: Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.


Assuntos
Ductos Biliares Extra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/citologia , Células Epiteliais/citologia , Organoides/fisiologia , Animais , Bile , Ductos Biliares Extra-Hepáticos/fisiologia , Ductos Biliares Intra-Hepáticos/fisiologia , Diferenciação Celular , Ducto Colédoco/citologia , Células Epiteliais/fisiologia , Vesícula Biliar/citologia , Regulação da Expressão Gênica , Humanos , Queratina-19/análise , Fígado/fisiologia , Camundongos , RNA-Seq , Obtenção de Tecidos e Órgãos
4.
Cancer Sci ; 112(5): 1822-1838, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33068050

RESUMO

Biliary tract cancer (BTC) arises from biliary epithelial cells (BECs) and includes intrahepatic cholangiocarcinoma (IHCC), gallbladder cancer (GC), and extrahepatic cholangiocarcinoma (EHCC). Although frequent KRAS mutations and epigenetic changes at the INK4A/ARF locus have been identified, the molecular pathogenesis of BTC is unclear and the development of corresponding anticancer agents remains inadequate. We isolated epithelial cell adhesion molecule (EpCAM)-positive BECs from the mouse intrahepatic bile duct, gallbladder, and extrahepatic bile duct, and established organoids derived from these cells. Introduction of activated KRAS and homozygous deletion of Ink4a/Arf in the cells of each organoid type conferred the ability to form lethal metastatic adenocarcinoma with differentiated components and a pronounced desmoplastic reaction on cell transplantation into syngeneic mice, indicating that the manipulated cells correspond to BTC-initiating cells. The syngeneic mouse models recapitulate the pathological features of human IHCC, GC, and EHCC, and they should therefore prove useful for the investigation of BTC carcinogenesis and the development of new therapeutic strategies. Tumor cells isolated from primary tumors formed organoids in three-dimensional culture, and serial syngeneic transplantation of these cells revealed that their cancer stem cell properties were supported by organoid culture, but not by adherent culture. Adherent culture thus attenuated tumorigenic activity as well as the expression of both epithelial and stem cell markers, whereas the expression of epithelial-mesenchymal transition (EMT)-related transcription factor genes and mesenchymal cell markers was induced. Our data show that organoid culture is important for maintenance of epithelial cell characteristics, stemness, and tumorigenic activity of BTC-initiating cells.


Assuntos
Neoplasias do Sistema Biliar/genética , Colangiocarcinoma/genética , Células Epiteliais/fisiologia , Genes ras , Organoides , Células-Tronco/fisiologia , Fator 1 de Ribosilação do ADP/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Extra-Hepáticos/anatomia & histologia , Ductos Biliares Extra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/citologia , Neoplasias do Sistema Biliar/patologia , Colangiocarcinoma/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Modelos Animais de Doenças , Molécula de Adesão da Célula Epitelial , Células Epiteliais/química , Transição Epitelial-Mesenquimal , Feminino , Vesícula Biliar/anatomia & histologia , Vesícula Biliar/citologia , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/patologia , Deleção de Genes , Genes Supressores de Tumor , Fígado/anatomia & histologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias/métodos , Organoides/metabolismo , Organoides/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Análise Serial de Tecidos/métodos , Microambiente Tumoral/fisiologia
5.
Biosci Rep ; 40(9)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32869841

RESUMO

Some microRNAs (miRs or miRNAs) have been reported to function as tumor suppressors in gallbladder cancer (GBC). However, the specific effect of miR-205-5p on GBC remains unclear. The objective of the present study was to unravel the effects of miR-205-5p on the drug resistance in GBC. For this purpose, the expression of miR-205-5p and protein kinase C ϵ (PRKCE) was quantified in the peripheral blood sample harvested from GBC patients and healthy volunteers. Then the relationship between miR-205-5p and PRKCE was validated. After isolating the GBC stem cells, ectopic expression and depletion experiments were conducted to analyze the effect of miR-205-5p and PRKCE on cell proliferation, drug resistance, apoptosis, and colony formation rate as well as the expression of apoptotic factors (Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), and cleaved caspase 3). Finally, the mouse xenograft model of GBC was established to verify the function of miR-205-5p in vivo. Intriguingly, our results manifested that miR-205-5p was down-regulated, while PRKCE was up-regulated in peripheral blood samples and stem cells of patients with GBC. Moreover, miR-205-5p targeted PRKCE and negatively regulated its expression. The overexpression of miR-205-5p or silencing of PRKCE inhibited the drug resistance, proliferation, and colony formation rate while promoting apoptosis of GBC stem cells. Additionally, the overexpression of miR-205-5p attenuated drug resistance to gemcitabine but promoted the gemcitabine-induced cell apoptosis by inhibiting the PRKCE in vivo. Overall, an intimate correlation between miR-205-5p and PRKCE is a key determinant of drug resistance of GBC stem cells, thus, suggesting a novel miR-205-5p-based clinical intervention target for GBC patients.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias da Vesícula Biliar/terapia , MicroRNAs/metabolismo , Proteína Quinase C-épsilon/genética , Adulto , Idoso , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Colecistectomia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Vesícula Biliar/citologia , Vesícula Biliar/patologia , Vesícula Biliar/cirurgia , Neoplasias da Vesícula Biliar/sangue , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Voluntários Saudáveis , Humanos , Masculino , Camundongos , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , MicroRNAs/sangue , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
6.
mBio ; 11(5)2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963006

RESUMO

Carcinoma of the gallbladder (GBC) is the most frequent tumor of the biliary tract. Despite epidemiological studies showing a correlation between chronic infection with Salmonella enterica Typhi/Paratyphi A and GBC, the underlying molecular mechanisms of this fatal connection are still uncertain. The murine serovar Salmonella Typhimurium has been shown to promote transformation of genetically predisposed cells by driving mitogenic signaling. However, insights from this strain remain limited as it lacks the typhoid toxin produced by the human serovars Typhi and Paratyphi A. In particular, the CdtB subunit of the typhoid toxin directly induces DNA breaks in host cells, likely promoting transformation. To assess the underlying principles of transformation, we used gallbladder organoids as an infection model for Salmonella Paratyphi A. In this model, bacteria can invade epithelial cells, and we observed host cell DNA damage. The induction of DNA double-strand breaks after infection depended on the typhoid toxin CdtB subunit and extended to neighboring, non-infected cells. By cultivating the organoid derived cells into polarized monolayers in air-liquid interphase, we could extend the duration of the infection, and we observed an initial arrest of the cell cycle that does not depend on the typhoid toxin. Non-infected intoxicated cells instead continued to proliferate despite the DNA damage. Our study highlights the importance of the typhoid toxin in causing genomic instability and corroborates the epidemiological link between Salmonella infection and GBC.IMPORTANCE Bacterial infections are increasingly being recognized as risk factors for the development of adenocarcinomas. The strong epidemiological evidence linking Helicobacter pylori infection to stomach cancer has paved the way to the demonstration that bacterial infections cause DNA damage in the host cells, initiating transformation. In this regard, the role of bacterial genotoxins has become more relevant. Salmonella enterica serovars Typhi and Paratyphi A have been clinically associated with gallbladder cancer. By harnessing the stem cell potential of cells from healthy human gallbladder explant, we regenerated and propagated the epithelium of this organ in vitro and used these cultures to model S. Paratyphi A infection. This study demonstrates the importance of the typhoid toxin, encoded only by these specific serovars, in causing genomic instability in healthy gallbladder cells, posing intoxicated cells at risk of malignant transformation.


Assuntos
Dano ao DNA , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Vesícula Biliar/citologia , Salmonella paratyphi A/patogenicidade , Adulto , Idoso , Animais , Células Cultivadas , Feminino , Vesícula Biliar/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Sorogrupo , Virulência/genética
7.
Toxicol In Vitro ; 67: 104912, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32512147

RESUMO

Gallbladder cancer (GBC) is the commonest biliary tract cancer with an ill-defined etiology. We examined the role of Cd+2 exposures in a primary human gallbladder (GB) cell line model in this study. Cd+2 exposures induced decreased cell viability, reactive oxygen species (ROS) generation, altered Akt/ERK signaling pathway activation, PGE2 and COX-2 expression in a human primary gallbladder epithelial cell model. Pharmacological inhibitors were used to determine the key drivers of elevated COX-2 expression due to Cd+2 exposure. Our results show Cd+2 causes a dose-dependent reduction in GB cell viability (EC50 value - 18.6 µM). Dose-dependent activation of phospho-Akt and phospho-ERK signaling pathways via increased phosphoprotein expression was observed due to Cd+2. Signaling activation of Akt and ERK was prevented by 5 mM N-Acetyl Cysteine (NAC), establishing the role of ROS as a key driver in the activation process. Importantly, we observed Cd+2 also caused a dose dependent change in the COX-2 and PGE2 expression levels. PI3K-Akt and NF-kB signaling pathways play a key role in Cd+2 exposure induced COX-2 activation in the gallbladder epithelial cells. In conclusion, our study measures the toxicological effects of Cd+2 exposures on human GB epithelial cells for the first time and establishes the role of Cd+2 as a possible driver of the Akt/ERK pathway overactivity and chronic inflammation in gallbladder carcinogenesis.


Assuntos
Cádmio/toxicidade , Ciclo-Oxigenase 2/metabolismo , Células Epiteliais/efeitos dos fármacos , Vesícula Biliar/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dinoprostona/metabolismo , Células Epiteliais/metabolismo , Glutationa/metabolismo , Humanos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
8.
Pol J Pathol ; 70(3): 205-209, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31820864

RESUMO

Interstitial cells of Cajal (ICC) were first described by Santiago Ramon y Cajal over 100 years ago. They are thought to play an important role in the regulation of gastrointestinal motility. There is increasing evidence that the decline in their number in the gallbladder wall contributes to the formation of concrements. The aim of the study was to determine the exact location of interstitial cells of Cajal in the gallbladder wall in patients with calculous and non-calculous cholecystitis. Sixty-eight patients were examined, of whom 50 were cases of cholelithiasis and 18 were of non-calculous cholecystitis. The technique of immunohistochemistry with the CD117 antibody was used to determine the cells of Cajal, while to distinguish them from mast cells the technique with mast cell tryptase (MCT) was applied. Redistribution of the interstitial cells of Cajal from the muscle membrane to lamina propria of mucous tissue was observed in the cases of cholelithiasis, while in the group of non-calculous cholecystitis most of the ICC was located within the muscle tissue.


Assuntos
Colecistite/patologia , Colelitíase/patologia , Vesícula Biliar/citologia , Células Intersticiais de Cajal/citologia , Vesícula Biliar/patologia , Humanos , Imuno-Histoquímica , Proteínas Proto-Oncogênicas c-kit
9.
Stem Cell Res Ther ; 10(1): 289, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547878

RESUMO

BACKGROUND: Stem cell-derived pancreatic ß-like cells hold great promise for treating diabetes. Gallbladder belongs to the extrahepatic bile duct system and possesses stem-like cells. These stem cells could be expanded in vitro and have the potential of differentiating into hepatocytes, cholangiocytes, or pancreatic cells. As the gallbladder is highly available, gallbladder stem cells provide a new cell source of pancreatic ß-like cells. In this study, we aimed to investigate an approach for the generation of pancreatic ß-like cells from gallbladder stem cells (GSCs) without genetic modification. METHODS: A CK19CreERT;Rosa26R-GFP mouse was used to isolate CK19+ cells, which represented EpCAM+ stem cells in the gallbladder. They were cultured in the modified Kubota's medium for expansion and further analyzed. Then, we developed a strategy to screen a combination of small molecules that can generate insulin-secreting cells from gallbladder stem cells. These cells were identified with markers of pancreatic cells. Finally, they were seeded into the cellulosic sponge and transplanted to the diabetic mice for functional examination in vivo. RESULTS: Gallbladder stem cells could be expanded for more than 15 passages. They expressed typical hepatic stem cell markers including CK19, EpCAM, Sox9, and albumin. By screening method, we found that adding Noggin, FR180204, and cyclopamine could efficiently induce gallbladder stem cells differentiating into insulin-secreting cells. These cells expressed Pdx1, Nkx6.1, and insulin but were negative for Gcg. After transplantation with the cellulosic sponge, they could ameliorate hyperglycemia in the diabetic mice. CONCLUSION: This study provides a new approach which can generate insulin-secreting cells from the gallbladder without genetic modification. This offers an option for ß cell therapy in treating type 1 diabetes.


Assuntos
Células-Tronco Adultas/citologia , Técnicas de Reprogramação Celular/métodos , Vesícula Biliar/citologia , Células Secretoras de Insulina/citologia , Células-Tronco Adultas/metabolismo , Albuminas/genética , Albuminas/metabolismo , Animais , Transdiferenciação Celular , Células Cultivadas , Diabetes Mellitus Experimental/terapia , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transplante de Células-Tronco/métodos , Transativadores/genética , Transativadores/metabolismo
10.
Folia Histochem Cytobiol ; 57(2): 94-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237344

RESUMO

INTRODUCTION: A reduced number of interstitial Cajal-like cells (ICLCs) in the gallbladder have been proposed to play a role in the pathogenesis of cholelithiasis. Therefore, this prospective study was conducted to investigate the relationship between gallbladder contractility and the number of gallbladder ICLCs in patients with cholelithiasis. MATERIAL AND METHODS: Patients admitted to the Department of Hepatobiliary Surgery for cholecystectomy were divided into the cholelithiasis (n = 18) and non-cholelithiasis (n = 8) groups based on their clinical data. Patients' clinical data were collected on admission, and B-mode ultrasonography was performed to assess their gallbladder contractility. The resected gallbladder specimens were fixed, paraffin sections mounted on slides, and the immunofluorescence staining with the anti-human CD-117 and anti-human tryptase antibodies was performed to identify ICLSs and mast cells, respectively. The number of ICLCs was counted in 10 high-power fields (HPFs) randomly. RESULTS: Independent sample t-tests revealed differences between the cholelithiasis and non-cholelithiasis groups in the number of ICLCs (mean ± standard deviation: 88.61 ± 28.22 vs. 115.89 ± 27.87 per HPFs, P = 0.032) and gallbladder contractility (43.94% ± 18.50% vs. 61.00% ± 20.50%, P = 0.046). Pearson and Spearman cor-relation analyses revealed no significant correlation between the number of ICLCs and gallbladder contractility. CONCLUSION: The results suggest that the number of gallbladder ICLCs in the wall of the gallbladder of patients with or without cholelithiasis is not a decisive factor affecting gallbladder contractility.


Assuntos
Colelitíase/fisiopatologia , Esvaziamento da Vesícula Biliar/fisiologia , Vesícula Biliar/citologia , Vesícula Biliar/fisiologia , Telócitos/citologia , Adulto , Idoso , Animais , Anticorpos/imunologia , Contagem de Células , Colelitíase/patologia , Feminino , Vesícula Biliar/patologia , Cabras , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-kit/imunologia , Coelhos , Telócitos/patologia , Triptases/imunologia
11.
J Cell Mol Med ; 22(7): 3323-3329, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29700981

RESUMO

A novel type of interstitial tissue cells in the biliary tree termed telocytes (TCs), formerly known as interstitial Cajal-like cells (ICLCs), exhibits very particular features which unequivocally distinguish these cells from interstitial cells of Cajal (ICCs) and other interstitial cell types. Current research substantiates the existence of TCs and ICCs in the biliary system (gallbladder, extrahepatic bile duct, cystic duct, common bile duct and sphincter of Oddi). Here, we review the distribution, morphology and ultrastructure of TCs and ICCs in the biliary tree, with emphasis on their presumptive roles in physiological and pathophysiological processes.


Assuntos
Ductos Biliares/citologia , Doenças da Vesícula Biliar/patologia , Vesícula Biliar/citologia , Células Intersticiais de Cajal/ultraestrutura , Telócitos/ultraestrutura , Animais , Colecistite/patologia , Colelitíase/patologia , Humanos , Células Intersticiais de Cajal/patologia , Células Intersticiais de Cajal/fisiologia , Telócitos/patologia , Telócitos/fisiologia
12.
Toxicol Pathol ; 46(2): 169-183, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29334850

RESUMO

With the increasing use of animal-based biomaterials for regenerative medical applications, the need for their safety assessment is paramount. A porcine cholecyst-derived scaffold (CDS), intended as a muscle repair graft, prepared by a nondetergent/enzymatic method was engrafted in a rat abdominal wall defect model. Host tissue-scaffold interface samples were collected 2, 8, and 16 weeks postimplantation and evaluated by histopathology, immunohistochemistry, and electron microscopy. The nature of the tissue reaction was compared with those induced by a jejunum-derived scaffold (JDS) prepared by the same method and a commercial-grade small intestinal submucosa (CSIS) scaffold. A study of the immunopathological response in major lymphoid tissues and immunophenotyping for M1 and M2 macrophages was performed at the host tissue-scaffold interface. Further, "irritancy scores" for CDS and JDS were determined using CSIS as the reference material. Both CDS and JDS appeared to be potential biomaterials for muscle grafts, but the former stimulated a skeletal muscle tissue remodeling response predominated by M2 macrophages. The data support the notion that biomaterials with similar biocompatibility, based on local tissue response on implantation, may cause differential immunogenicity. Additionally, CDS compared to JDS and CSIS was found to be less immunotoxic.


Assuntos
Parede Abdominal/patologia , Vesícula Biliar , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Materiais Biocompatíveis/farmacologia , Modelos Animais de Doenças , Vesícula Biliar/citologia , Imuno-Histoquímica , Masculino , Teste de Materiais , Músculo Esquelético , Ratos , Ratos Sprague-Dawley , Medicina Regenerativa/métodos , Suínos
13.
World J Gastroenterol ; 23(31): 5787-5797, 2017 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-28883705

RESUMO

AIM: To detect the expression of threonine and tyrosine kinase (TTK) in gallbladder cancer (GBC) specimens and analyze the associations between TTK expression and clinicopathological parameters and clinical prognosis. METHODS: A total of 68 patients with GBC who underwent surgical resection were enrolled in this study. The expression of TTK in GBC tissues was detected by immunohistochemistry. The assessment of TTK expression was conducted using the H-scoring system. H-score was calculated by the multiplication of the overall staining intensity with the percentage of positive cells. The expression of TTK in the cytoplasm and nucleus was scored separately to achieve respective H-score values. The correlations between TTK expression and clinicopathological parameters and clinical prognosis were analyzed using Chi-square test, Kaplan-Meier method and Cox regression. RESULTS: In both the nucleus and cytoplasm, the expression of TTK in tumor tissues was significantly lower than that in normal tissues (P < 0.001 and P = 0.026, respectively). Using the median H-score as the cutoff value, it was discovered that, GBC patients with higher levels of TTK expression in the nucleus, but not the cytoplasm, had favorable overall survival (P < 0.001), and it was still statistically meaningful in Cox regression analysis. Further investigation indicated that there were close negative correlations between TTK expression and tumor differentiation (P = 0.041), CA 19-9 levels (P = 0.016), T stage (P < 0.001), nodal involvement (P < 0.001), distant metastasis (P = 0.024) and TNM stage (P < 0.001). CONCLUSION: The expression of TTK in GBC is lower than that in normal tissues. Higher levels of TTK expression in GBC are concomitant with longer overall survival. TTK is a favorable prognostic biomarker for patients with GBC.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias da Vesícula Biliar/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Idoso , Antígeno CA-19-9/sangue , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Seguimentos , Vesícula Biliar/citologia , Vesícula Biliar/patologia , Vesícula Biliar/cirurgia , Neoplasias da Vesícula Biliar/sangue , Neoplasias da Vesícula Biliar/mortalidade , Neoplasias da Vesícula Biliar/cirurgia , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos
14.
PLoS One ; 12(8): e0181812, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28813430

RESUMO

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to ß-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic ß-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall ß-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to ß cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs-derived from multiple unrelated donors-into pancreatic ß-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes.


Assuntos
Reprogramação Celular , Vesícula Biliar/citologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Transdiferenciação Celular , Transplante de Células , Células Cultivadas , Técnicas de Reprogramação Celular , Análise por Conglomerados , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , MicroRNAs/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , Transgenes
15.
In Vivo ; 31(2): 169-173, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28358696

RESUMO

BACKGROUND: We have previously reported a procedure for isolating and culturing biliary epithelial cells (BECs). The aim of this study was to reconsider the method for obtaining pure BECs using the mouse gallbladder. MATERIALS AND METHODS: Cells that were obtained from the gallbladder alone were sorted by fluorescence-activated cell sorting (FACS) for purifying based on the expression of the epithelial cell adhesion molecule (EpCAM). The viability rate was measured based on the negative expression of 7-aminoactinomycin D (7-AAD). RESULTS: More than 75% of cells from the gallbladder were determined to be pure BECs. An analysis of the EpCAM revealed that 73.3% of the cells were 7-AAD-negative. Finally, the 0.82×106 pure BECs that survived were obtained and seeded on a collagen gel plate. However, these pure BECs showed almost no proliferation. CONCLUSION: Pure BECs could be accumulated using FACS. However, the number of BECs was insufficient for the culturing process.


Assuntos
Separação Celular/métodos , Células Epiteliais/citologia , Citometria de Fluxo/métodos , Vesícula Biliar/citologia , Animais , Contagem de Células , Sobrevivência Celular , Células Cultivadas , Dactinomicina/análogos & derivados , Dactinomicina/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/metabolismo , Vesícula Biliar/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Contraste de Fase , Reprodutibilidade dos Testes
16.
Stem Cell Res ; 17(3): 587-596, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27833043

RESUMO

Direct reprogramming is a promising approach for the replacement of ß cells in diabetes. Reprogramming of cells originating from the endodermal lineage, such as acinar cells in the pancreas, liver cells and gallbladder cells has been of particular interest because of their developmental proximity to ß cells. Our previous work showed that mouse gallbladder epithelium can be partially reprogrammed in vitro to generate islet-like cells (rGBC1). Here, the reprogramming protocol was substantially improved, yielding cells (rGBC2) closer to functional ß cells than the 1st generation method with higher conversion efficiency and insulin expression. In addition to insulin synthesis and processing, rGBC2 presented many hallmark features of ß cells, including insulin secretion in response to high glucose stimulation. Gene expression analysis indicated that rGBC2 clustered closer with ß cells and had a metabolic gene expression profile resembling neonatal ß cells. When transplanted into immune-deficient animals, rGBC2 were stable for at least 5months and further matured in vivo. Taken together, this approach provides further understanding of endodermal lineage conversion and potential for development of cell replacement therapy for type 1 diabetes patients.


Assuntos
Reprogramação Celular/fisiologia , Vesícula Biliar/citologia , Células Secretoras de Insulina/citologia , Animais , Modelos Animais de Doenças , Feminino , Vesícula Biliar/metabolismo , Perfilação da Expressão Gênica , Humanos , Insulina/biossíntese , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD
17.
Gut Liver ; 10(5): 851-8, 2016 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-27563024

RESUMO

BACKGROUND/AIMS: Gallstone pathogenesis is linked to mucin hypersecretion and bacterial infection. Several mucin genes have been identified in gallbladder epithelial cells (GBECs). We investigated MUC expression in cholesterol-associated gallbladder disease and evaluated the relationship between mucin and bacterial infection. METHODS: The present study involved 20 patients with cholesterol stones with cholecystitis, five with cholesterol stones with cholesterolosis, six with cholesterol polyps, two with gallbladder cancer, and six controls. Canine GBECs treated with lipopolysaccharide were also studied. MUC3, MUC5AC, MUC5B, and MUC6 antibodies were used for dot/slot immunoblotting and immunohistochemical studies of the gallbladder epithelial tissues, canine GBECs, and bile. Reverse-transcription polymerase chain reaction was performed to evaluate MUC3 and MUC5B expression. RESULTS: MUC3, MUC5AC, MUC5B, and MUC6 were expressed in the normal gallbladder epithelium, and of those, MUC3 and MUC5B exhibited the highest expression levels. Greatly increased levels of MUC3 and MUC5B expression were observed in the cholesterol stone group, and slightly increased levels were observed in the cholesterol polyp group; MUC3 and MUC5B mRNA was also upregulated in those groups. Canine GBECs treated with lipopolysaccharide also showed upregulation of MUC3 and MUC5B. CONCLUSIONS: The mucin genes with the highest expression levels in gallbladder tissue in cholesterol-associated diseases were MUC3 and MUC5B. Cholesterol stones and gallbladder infections were associated with increased MUC3 and MUC5B expression.


Assuntos
Colecistite/metabolismo , Células Epiteliais/metabolismo , Doenças da Vesícula Biliar/metabolismo , Mucinas Gástricas/metabolismo , Hipercolesterolemia/metabolismo , Animais , Estudos de Casos e Controles , Colecistite/etiologia , Cães , Vesícula Biliar/citologia , Doenças da Vesícula Biliar/etiologia , Humanos , Hipercolesterolemia/complicações , Mucina-5AC/metabolismo , Mucina-3/metabolismo , Mucina-5B/metabolismo , Mucina-6/metabolismo
18.
Parasitol Res ; 115(11): 4129-4138, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27389734

RESUMO

We describe a new myxosporean species, Zschokkella trachini n. sp., infecting the gallbladder of greater weever Trachinus draco Linnaeus 1758 from Tunisia. This is the first record of Zschokkella species in T. draco. Plasmodia were polysporic producing six to eight mature spores; they were attached to the gallbladder epithelium or free floating in the bile. Mature spores were sub-ovoid in the frontal view, measured 15.2 ± 0.6 (14.4-16.0) µm in length and 9.8 ± 0.7 (9.0-10.8) µm in width. Two equal spherical polar capsules 4.0 ± 0.4 (3.6-4.5) µm in diameter, were located separately at the spore's extremities. The prevalence of infection ranged from 23.5 to 87.7 %. Morphological data and molecular analysis of the small subunit rDNA gene identified this parasite as a new species of Zschokkella. Neighbour joining clustered the parasite in a sub-clade containing other Zschokkella species parasiting the gallbladder of marine fish hosts, located within the coelozoic clade of the major freshwater clade. This is the second Zschokkella species reported from Tunisia.


Assuntos
Doenças dos Peixes/parasitologia , Myxozoa/classificação , Doenças Parasitárias em Animais/parasitologia , Perciformes/parasitologia , Animais , Doenças dos Peixes/epidemiologia , Vesícula Biliar/citologia , Vesícula Biliar/parasitologia , Myxozoa/citologia , Myxozoa/genética , Myxozoa/isolamento & purificação , Doenças Parasitárias em Animais/epidemiologia , Filogenia , Estações do Ano , Esporos , Tunísia/epidemiologia , Água/parasitologia
19.
EMBO Rep ; 17(5): 769-79, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26993089

RESUMO

Pioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of endoderm-derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R-spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5-positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non-regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R-spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder-derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride-treated mice. Human gallbladders were also a source of organoid-forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.


Assuntos
Proteínas de Transporte/metabolismo , Vesícula Biliar/citologia , Células-Tronco/metabolismo , Trombospondinas/metabolismo , Animais , Biomarcadores , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Diferenciação Celular/genética , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Fígado/citologia , Camundongos , Camundongos Transgênicos , Organoides , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Células-Tronco/efeitos dos fármacos , Trombospondinas/genética , Trombospondinas/farmacologia , Transcriptoma
20.
Dig Dis Sci ; 61(3): 900-4, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26547756

RESUMO

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) have been shown to be present in the extrahepatic biliary tract of animals and humans. However, ICC distribution in choledochal cysts (CC) has not been investigated. A study was conducted to investigate the distribution of ICC in the extrahepatic biliary tract, including CC, in pediatric human specimens. METHOD: The specimens were divided into two main groups as gallbladders and common bile ducts. Gallbladders were obtained from the cholelithiasis, CC operations and autopsies. Common bile ducts were obtained from autopsies. Tissues were stained using c-kit immunohistochemical staining. ICC were assessed semi-quantitatively by applying morphological criteria and were counted as the number of cells/0.24 mm(2) in each area under light microscopy. RESULTS: A total of 35 gallbladders and 14 CC were obtained from operations. Ten gallbladders plus common bile ducts were obtained from autopsies. The mean numbers of ICC in the gallbladders of cholelithiasis and the gallbladders of CC were 12.2 ± 4.9 and 5.3 ± 1.2, respectively (p = 0.003). The mean numbers of ICC in the common bile ducts and CC were 9.8 ± 2.9 and 3.4 ± 1.4, respectively (p = 0.001). CONCLUSION: The scarcity of ICC in the extrahepatic biliary tract may be responsible for the etiopathogenesis of the CC.


Assuntos
Cisto do Colédoco/patologia , Ducto Colédoco/citologia , Vesícula Biliar/citologia , Células Intersticiais de Cajal/citologia , Adolescente , Estudos de Casos e Controles , Contagem de Células , Criança , Pré-Escolar , Colecistectomia , Cisto do Colédoco/metabolismo , Cisto do Colédoco/cirurgia , Colelitíase/cirurgia , Ducto Colédoco/metabolismo , Feminino , Vesícula Biliar/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Células Intersticiais de Cajal/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-kit/metabolismo
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