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1.
Mater Today Bio ; 21: 100689, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37448665

RESUMO

Herein, an activable fluorescent probe for peroxynitrite (ONOO-), named NOP, was constructed for the accurate imaging in the processes of formation and inhibition of drug-induced liver injury induced by Acetaminophen (APAP). During the in-solution tests on the general optical properties, the probe showed advantages including good stability, wide pH adaption, high specificity and sensitivity in the monitoring of ONOO-. Subsequently, the probe was further applied in the model mice which used APAP to induce the injury and used inhibiting agents (GSH, Glu, NAC) to treat the induced injury. The construction of the liver injury model was confirmed by the pathological staining and the serum indexes including ALT, AST, ALP, TBIL as well as LDH. During the formation of the drug-induced liver injury, the fluorescence in the red channel enhanced in both time-dependent and dose-dependent manners. In inhibition tests, the inhibition of the liver injury exhibited the reduction of the fluorescence intensity. Therefore, NOP could achieve the accurate imaging in the processes of formation and inhibition of drug-induced liver injury. The information here might be helpful for the early diagnosis and the screening of potent treating candidates in liver injury cases.

2.
Strahlenther Onkol ; 197(5): 438-448, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33320287

RESUMO

PURPOSE: To explore whether miR-573 can suppress pancreatic cancer cell proliferation, migration, and invasion by targeting TSPAN1. METHODS: The expression of miR-573 and TSPAN1 in pancreatic cancer tissues and cells lines was analyzed using RT-qPCR. The human pancreatic cancer cell line PANC­1 was transfected with miR-573 mimic, pcDNA3.1-TSPAN1, or genOFFTM st-h-TSPAN1. The effects of miR-573 and TSPAN1 on cell proliferation, colony formation, migration, and invasion were analyzed by CCK­8, colony formation, transwell migration, and invasion assay, respectively. Target genes of miR-573 were screened using bioinformatics tools and confirmed by dual-luciferase reporter assay and real-time PCR. The effects of miR-573 in vivo were observed using tumor xenografts. RESULTS: We found that miR-573 is downregulated and TSPAN1 is upregulated in pancreatic cancer tissues and cells lines. Function assays demonstrated that overexpression of miR-573 inhibited cell proliferation, colony formation, migration, and invasion of pancreatic cancer cells, as well as suppressing tumor growth in vivo. Target genes of miR-573 were predicted using bioinformatics tools and confirmed by dual-luciferase reporter assay and RT-qPCR or western blotting. Downregulation of TSPAN1 also inhibited cell proliferation, colony formation, migration, and invasion of pancreatic cancer cells. Furthermore, overexpression of TSPAN1 attenuated miR-573-induced inhibition of pancreatic cancer cell proliferation and migration. CONCLUSION: Our findings indicated that miR-573 suppresses pancreatic cancer cell proliferation, migration, and invasion through targeting TSPAN1. TSPAN1 targeted by miR-573 might be a potential therapeutic target for clinical treatment of pancreatic cancer.


Assuntos
MicroRNAs/fisiologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Pancreáticas/patologia , RNA Neoplásico/fisiologia , Tetraspaninas/antagonistas & inibidores , Animais , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/genética , Genes Reporter , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/biossíntese , MicroRNAs/genética , MicroRNAs/uso terapêutico , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Tetraspaninas/biossíntese , Tetraspaninas/genética , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 245: 118879, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32920440

RESUMO

A novel curcumin-analogous fluorescent sensor, DNP, was developed for cysteine detection with a bilateral-response click-like mechanism. DNP indicated high selectivity and practical sensitivity. It could recognize Cys from other biologically relevant molecules, especially, from GSH and Hcy. The most interesting point was that, with typical azide groups for sensing, DNP indicated a covalent binding procedure with Cys instead of a presupposed simple reduction for reductive sulfide. Moreover, the recognition occurred at both sides of the sensor. DNP could be utilized into the detection of endogenous and exogenous Cys in living cells. Though the specific optical performances of DNP still need optimization, this work supplied novel information for broadening the vision on fluorophores and mechanisms, for the monitoring of Cys and even other sulfur-containing species.


Assuntos
Curcumina , Cisteína , Corantes Fluorescentes , Glutationa , Células HeLa , Homocisteína , Humanos
4.
Oncol Rep ; 36(6): 3559-3567, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27748934

RESUMO

The role of salidroside in colon cancer remains unknown. Here we show that salidroside, a phenylpropanoid glycoside extracted from Rhodiola rosea, exhibited potent anti-proliferative properties in human colorectal cancer cells via inducing apoptosis and autophagy. We ascertained that salidroside exerts an inhibitory effect on the proliferation of human colorectal cancer cells in a dose-dependent manner. In addition, salidroside induced cell apoptosis, accompanied by an increase of chromatin condensation and nuclear fragmentation, and a decrease of Bcl-2/Bax protein expression ratio. We also found that salidroside induced autophagy, evidenced by increased LC3+ autophagic vacuoles, positive acridine orange-stained cells, enhanced conversion of LC3-I to LC3-II, and elevation of Beclin-1. Treatment with autophagy-specific inhibitors [3-methyladenine (3-MA) and bafilomycin A1 (BA)] enhanced salidroside-induced apoptosis, indicating that salidroside-mediated autophagy may protect HT29 cells from undergoing apoptotic cell death. Additionally, salidroside decreased the phosphorylation of PI3K, Akt and mTOR. Treatment with PI3K inhibitor LY294002 augmented the effects of salidroside on the expression of Akt and mTOR. These findings indicate that salidroside could suppress the PI3K/Akt/mTOR signaling pathways. This study may provide a rationale for future clinical application using salidroside as a chemotherapeutic agent for human colorectal cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Glucosídeos/farmacologia , Fenóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo
5.
Cell Tissue Res ; 365(1): 157-71, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26796204

RESUMO

Diabetes mellitus is a worldwide metabolic disease which constitutes a major threat to human health. Stem cells with the ability to differentiate into insulin-producing cells (IPCs) could provide unlimited sources of transplanted cells and solve allogeneic rejection problems. The decellularized scaffolds could provide IPCs with tissue microarchitecture and intact vascular systems. The goal of this study was to engineer intact whole rat liver scaffolds and repopulate the stem cell-derived IPCs into the scaffolds to discover whether the decellularized scaffolds could facilitate the growth and development of IPCs. Decellularized liver scaffolds were obtained using 1 % Triton X-100 with 0.1 % ammonium hydroxide. Architecture and composition of the original extracellular matrix were confirmed by morphologic, histological and immunolabeling examinations. Islet-like clusters were differentiated from Wharton's jelly mesenchymal stem cells (WJMSCs) by a three-step induction procedure. The differentiation was evaluated by morphology, RT-PCR, immunofluorescence and glucose stimulation experiments. The islet-like clusters were recellularized into the decellularized scaffolds by the portal-vein infusion method and cultured by the dynamic circulation perfusion device. After cultivation, hematoxylin-eosin staining, immunofluorescence and RT-PCR were conducted. Our results demonstrated that the decellularized rat liver scaffolds have favorable biochemical properties and could support the survival of WJMSC-derived IPCs. In addition, the three-dimensional decellularized scaffolds could enhance the expression of the insulin gene compared with two-dimensional plate culture. In conclusion, these findings suggested that the decellularized scaffolds could provide a suitable platform for cellular activities of IPCs such as survival, differentiation, proliferation and insulin secretion. This study provides fundamental support for regenerating insulin-secreting organs from the decellularized scaffolds combined with stem cell-derived IPCs as a potential clinical application.


Assuntos
Ilhotas Pancreáticas/citologia , Fígado/citologia , Técnicas de Cultura de Tecidos/métodos , Alicerces Teciduais/química , Animais , Agregação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Forma Celular , DNA/metabolismo , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Humanos , Imageamento Tridimensional , Insulina/metabolismo , Secreção de Insulina , Fígado/ultraestrutura , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Perfusão , Fenótipo , Ratos Sprague-Dawley , Geleia de Wharton/citologia
6.
Artif Organs ; 40(3): E25-38, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26637111

RESUMO

Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim of the present study was to engineer intact whole rat liver scaffolds and repopulate them with hepatocytes and endothelial progenitor cells (EPCs) in a bioreactor. Decellularized liver scaffolds were obtained by perfusing Triton X-100 with ammonium hydroxide. The architecture and composition of the original extracellular matrix were preserved, as confirmed by morphologic, histological, and immunolabeling methods. To determine biocompatibility, the scaffold was embedded in the subcutaneous adipose layer of the back of a heterologous animal to observe the infiltration of inflammatory cells. Hepatocytes were reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using a portal vein infusion method. Morphologic and functional examination showed that the hepatocytes and EPCs grew well in the scaffold. The present study describes an effective method of decellularization and recellularization of rat livers, providing the foundation for liver engineering and the development of bioartificial livers.


Assuntos
Células Progenitoras Endoteliais/transplante , Matriz Extracelular/química , Hepatócitos/transplante , Fígado/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Reatores Biológicos , Proliferação de Células , Células Cultivadas , Células Progenitoras Endoteliais/citologia , Hepatócitos/citologia , Fígado/química , Fígado/ultraestrutura , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley
7.
Biomed Res Int ; 2015: 432645, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26688810

RESUMO

Type 1 diabetes is an autoimmune disease which is due to the lack of ß cells. The ideal therapy to cure the disease is pancreas transplantation, but its application is confined to a limited number of people due to the shortage of organ and the need for life-long immunosuppression. Regenerative medicine methods such as a tissue engineered pancreas seem to provide a useful method. In order to construct a microenvironment similar to the native pancreas that is suitable for not only cell growth but also cellular function exertion, a decellularized mouse pancreas was used as a natural 3D scaffold in this experiment. MIN-6 ß cells were planted in the bioscaffold. The cell engraftment was verified by HE staining and SEM. Immunostaining procedures were performed to confirm the normal function of the engrafted cells. qRT-PCR demonstrated that insulin gene expression of the recellularized pancreas was upregulated compared with conventional plate-cultured cells. In vivo experiment was also accomplished to further evaluate the function of the recellularized bioscaffold and the result was inspiring. And beyond doubt this will bring new hope for type 1 diabetic patients.


Assuntos
Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Linhagem Celular , Humanos , Células Secretoras de Insulina/citologia , Masculino , Camundongos
8.
Sci Rep ; 5: 12991, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26259918

RESUMO

RTK-like orphan receptor 2 (ROR2) is overexpressed in several cancers and has tumorigenic activity. However, the expression of ROR2 and its functional and prognostic significance have yet to be evaluated in pancreatic ductal adenocarcinoma (PDAC). Quantitative real-time polymerase chain reaction was used to characterize the expression of ROR2 mRNA in PDAC, corresponding peritumoral tissues, and PDAC cell lines. Immunohistochemical analysis with tissue microarrays was used to evaluate ROR2 expression in PDAC and to investigate the relationship of this expression to clinicopathological factors and prognosis. The expression of ROR2 mRNA and protein was significantly higher in PDAC than in normal pancreatic tissues. High cytoplasmic ROR2 expression in cancer cells was significantly associated with a primary tumor, distant metastasis, and TNM stage, and high stromal ROR2 expression was significantly associated with regional lymph node metastasis and TNM stage. The Kaplan-Meier method and Cox regression analyses showed that high ROR2 expression in tumor cytoplasm or stromal cells was significantly associated with malignant attributes and reduced survival in PDAC. We present strong evidence that ROR2 could be used as an indicator of poor prognosis and could represent a novel therapeutic target for PDAC.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/biossíntese , Carcinoma Ductal Pancreático/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/biossíntese , Adenocarcinoma/patologia , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Células Estromais/metabolismo , Células Estromais/patologia
9.
Tumour Biol ; 36(11): 8895-902, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26070867

RESUMO

Kinesin superfamily protein 2A (KIF2A), an M type nonmotile microtubule depolymerase, has received attention for its role in carcinogenesis and prognostic value in several types of cancer. In this study, we evaluated the expression of KIF2A and its potential and robustness to predict clinical outcomes in colorectal cancer (CRC) patients. The messenger RNA (mRNA) expression of KIF2A was determined in 20 pairs of cancerous and adjacent nontumor tissues by real-time polymerase chain reaction. KIF2A immunohistochemistry was performed on tissue microarray (TMA), composed of 182 CRC and 179 matched adjacent nontumor tissues from surgery, 23 chronic colitis, 43 low-grade, and 18 high-grade intraepithelial neoplasias acquired through intestinal endoscopic biopsy. Univariate and multivariate Cox regression models were used to perform survival analyses. Both KIF2A mRNA and protein product exhibited CRC tissue-preferred expression, when compared with benign tissues. The high KIF2A expression was significantly correlated to TNM stage (P = 0.046) and tumor status (T) (P = 0.007). In univariate and multivariate analyses, high KIF2A expression showed a major prognostic value regarding 5-year survival. The influences of KIF2A expression on the survival were further proven by Kaplan-Meier survival analysis. This study demonstrated CRC tissue-preferred expression pattern of the KIF2A and suggested that high KIF2A expression might serve as an independent maker for poor prognosis in CRC patients.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/genética , Cinesinas/biossíntese , Prognóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Cinesinas/genética , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , RNA Mensageiro/biossíntese
10.
J Biomater Appl ; 30(4): 379-87, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26006767

RESUMO

The cutting-edge technology of three-dimensional liver decellularized bioscaffold has a potential to provide a microenvironment that is suitable for the resident cells and even develop a new functional organ. Liver decellularized bioscaffold preserved the native extracellular matrix and three-dimensional architecture in support of the cell culture. The goal of this study was to discover if three-dimensional extracellular matrix derived from mouse liver could facilitate the growth and maintenance of physiological functions of mouse isolated islets. We generated a whole organ liver decellularized bioscaffold which could successfully preserve extracellular matrix proteins and the native vascular channels using 1% Triton X-100/0.1% ammonium protocol. To evaluate the potential of decellularized liver as a scaffold for islets transplantation, the liver decellularized bioscaffold was infused with mouse primary pancreatic islets which were obtained through Collagenase P digestion protocol. Its yield, morphology, and quality were estimated by microscopic analysis, dithizone staining, insulin immunofluorescence and glucose stimulation experiments. Comparing the three-dimensional culture in liver decellularized bioscaffold with the orthodoxy two-dimensional plate culture, hematoxylin-eosin staining, immunohistochemistry, and insulin gene expression were tested. Our results demonstrated that the liver decellularized bioscaffold could support cellular culture and maintenance of cell functions. In contrast with the conventional two-dimensional culture, three-dimensional culture system could give rise to an up-regulated insulin gene expression. These findings demonstrated that the liver bioscaffold by a perfusion-decellularized technique could serve as a platform to support the survival and function of the pancreatic islets in vitro. Meanwhile three-dimensional culture system had a superior role in contrast with the two-dimensional culture. This study advanced the field of regenerative medicine towards the development of a liver decellularized bioscaffold capable of forming a neo-organ and could be used as potential clinical application.


Assuntos
Matriz Extracelular/química , Ilhotas Pancreáticas/citologia , Fígado/química , Cultura Primária de Células/métodos , Alicerces Teciduais/química , Animais , Células Cultivadas , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Engenharia Tecidual/métodos
11.
Pediatr Transplant ; 19(4): 371-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25677260

RESUMO

T1D is an autoimmune disease, which may be caused by lack of insulin-secreting ß cells due to damage of autoimmune system. Living with T1D is a challenge for the child and the family; cell transplantation is a treatment option for diabetes in children. To establish a microenvironment suitable for cell growth and proliferation as well as for sustained cellular function, we used MIN-6 ß cells as seed cells and SF-IV collagen as a 3D composite scaffold to construct artificial pancreas in this experiment. The cell viabilities were determined by MTT assay, and the response of cells to different glucose concentrations was observed by glucose stimulation test. Artificial pancreas was transplanted into the abdominal cavity of T1D mice, and the changes of blood glucose were monitored. After 10 days, insulin expression was detected by immunohistochemical method, and the claybank stained area showed effectiveness of insulin secretion. A series of experiments showed that implantation of 3D cell scaffold into the abdominal cavity can effectively control the blood glucose level of T1D mice. It also had longer-lasting hypoglycemic effects than simple cell transplantation, which was expected to become a new method for the treatment of T1D.


Assuntos
Técnicas de Cultura de Células , Técnicas de Cocultura , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 1/sangue , Células Secretoras de Insulina/citologia , Animais , Glicemia/análise , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Imuno-Histoquímica , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL
12.
Diabetes Res Clin Pract ; 104(3): 383-92, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24794627

RESUMO

AIMS: The aim of this study was to evaluate the effect of PDX-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) in the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells and to explore this new approach of cell transplantation therapy for type 1 diabetes in mice. METHODS: iPSCs were infected with adenovirus (Ad-Mouse PDX-1-IRES-GFP, Ad-Mouse NeuroD1-IRES-GFP and Ad-Mouse Mafa-IRES-GFP) and then differentiated into insulin-producing cells in vitro. RT-PCR was applied to detect insulin gene expression, immunofluorescence to identify insulin protein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) was used to evaluate the amount of insulin at different concentration of glucose. Insulin-producing cells were transplanted into the liver parenchyma of diabetic mice. Immunohistochemistry, intraperitoneal glucose tolerance test (IPGTT) and fasting blood glucose (FBG) were performed to assess the function of insulin-producing cells. RESULTS: Insulin biosynthesis and secretion were induced in iPSCs and insulin-producing cells were responsive to glucose in a dose-dependent manner. Gene expression of the three-gene-modified embryoid bodies (EBs) was similar to the mouse pancreatic ß cell line MIN6. Transplantation of insulin-producing cells into type I diabetic mice resulted in hyperglycemia reversal. CONCLUSIONS: The insulin-producing cells we obtained from three-gene-modified EBs may be used as seed cells for tissue engineering and may represent a cell replacement strategy for the production of ß cells for the treatment of type 1 diabetes.


Assuntos
Adenoviridae/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células Secretoras de Insulina/citologia , Fatores de Transcrição Maf Maior/genética , Transativadores/genética , Transfecção , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Glicemia/metabolismo , Western Blotting , Diferenciação Celular/fisiologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/metabolismo
13.
BMC Cancer ; 14: 85, 2014 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-24521357

RESUMO

BACKGROUND: Pancreatic cancer is one of the most aggressive cancers, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells. And miR-200 has been identified as a powerful regulator of EMT. METHODS: Cancer Stem Cells (CSCs) of human pancreatic cancer cell line PANC-1 were processed for CD24, CD44 and ESA multi-colorstaining, and sorted out on a BD FACS Aria II machine. RT-qPCR was performed using the miScript PCR Kit to assay the expression of miR-200 family. In order to find the role of miR-200a in the process of EMT, miR-200a mimic was transfected to CSCs. RESULTS: Pancreatic cancer cells with EMT phenotype displayed stem-like cell features characterized by the expression of cell surface markers CD24, CD44 and epithelial-specific antigen (ESA), which was associated with decreased expression of miR-200a. Moreover, overexpression of miR-200a was resulted in down-regulation of N-cadherin, ZEB1 and vimentin, but up-regulation of E-cadherin. In addition, miR-200a overexpression inhibited cell migration and invasion in CSCs. CONCLUSION: In our study, we found that miR-200a played an important role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in pancreatic cancer. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to mesenchymal-to-epithelial transition (MET) phenotype using novel agents would be useful for prevention and/or treatment of pancreatic cancer.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , MicroRNAs/biossíntese , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Citometria de Fluxo/métodos , Humanos , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia
14.
Cancer Lett ; 340(1): 113-23, 2013 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-23872274

RESUMO

Pancreatic cancer is notorious for its difficult diagnosis at early stage and poor recurrence-free prognosis. This study aimed to investigate the possible involvement of Oct4 and Nanog in pancreatic cancer. The high expressions of Oct4 and Nanog in human pancreatic cancer tissues were found to indicate a worse prognostic value of patients. The pancreatic cancer stem cells (PCSCs) that isolated from PANC-1 cell line by flow cytometry exhibited high expressions of Oct4 and Nanog. To investigate whether Oct4 and Nanog play crucial role in maintaining the stemness of PCSCs, double knockdown of Oct4 and Nanog demonstrated that Oct4 and Nanog significantly reduced proliferation, migration, invasion, chemoresistance, and tumorigenesis of PCSCs in vitro and in vivo. The altered expression of the genes related to pancreatic carcinogenesis, metastasis, drug resistance and epithelial-mesenchymal transdifferentiation (EMT) might affect the biological characteristics of PCSCs. Our results suggest that Oct4 and Nanog may serve as a potential marker of prognosis and a novel target of therapy for pancreatic cancer.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Neoplásicas/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Pancreáticas/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Homeobox Nanog , Invasividade Neoplásica , Transplante de Neoplasias , Fator 3 de Transcrição de Octâmero/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Interferência de RNA , Carga Tumoral , Gencitabina
15.
Exp Diabetes Res ; 2012: 672013, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22761608

RESUMO

AIMS: The goal of cell transcription for treatment of diabetes is to generate surrogate ß-cells from an appropriate cell line. However, the induced replacement cells have showed less physiological function in producing insulin compared with normal ß-cells. METHODS: Here, we report a procedure for induction of insulin-producing cells (IPCs) from bone marrow murine mesenchymal stem cells (BM-mMSCs). These BM-mMSCs have the potential to differentiate into insulin-producing cells when a combination of PDX-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1), and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) genes are transfected into them and expressed in these cells. RESULTS: Insulin biosynthesis and secretion were induced in mMSCs into which these three genes have been transfected and expressed. The amount of induced insulin in the mMSCs which have been transfected with the three genes together is significantly higher than in those mMSCs that were only transfected with one or two of these three genes. Transplantation of the transfected cells into mice with streptozotocin-induced diabetes results in insulin expression and the reversal of the glucose challenge. CONCLUSIONS: These findings suggest major implications for cell replacement strategies in generation of surrogate ß-cells for the treatment of diabetes.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células da Medula Óssea/citologia , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas do Tecido Nervoso/metabolismo , Transativadores/metabolismo , Adenoviridae/metabolismo , Animais , Diferenciação Celular , Transplante de Células , Diabetes Mellitus/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica/métodos , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C57BL , Transfecção
16.
In Vitro Cell Dev Biol Anim ; 48(4): 236-43, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22457082

RESUMO

Induced pluripotent stem cells have been derived from various cell types via the ectopic expression of a cocktail of transcription factors. Previous studies have reported that induced pluripotent stem cells can be differentiated into multiple somatic cells, providing an invaluable resource in regenerative medicine. In this study, we compared the reprogramming efficiency of mouse embryonic fibroblasts, mouse bone marrow mesenchymal stem cells, and mouse bone marrow mononuclear cells by counting the number of alkaline phosphatase staining positive clones on day 15 after induced pluripotent stem cells induction. We found that a very low number of alkaline phosphatase-staining positive clones were derived from mouse bone marrow mesenchymal stem cells. We then evaluated the pluripotency of the clones by detecting the expression of embryonic stem cells markers and assessing their ability to form embryoid bodies and teratomas. Mouse bone marrow mesenchymal stem cells population is more homogeneous than mouse bone marrow mononuclear cells, which includes a variety of cell types. Our study indicated that the extremely low efficiency of mouse bone marrow mesenchymal stem cells induction implies that mouse bone marrow mesenchymal stem cells may not be a suitable cell type for the induction of induced pluripotent stem cells unless the efficiency of induction can be improved.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Antígenos CD/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismo
17.
Zhonghua Yi Xue Za Zhi ; 91(30): 2148-52, 2011 Aug 16.
Artigo em Chinês | MEDLINE | ID: mdl-22093995

RESUMO

OBJECTIVE: To evaluate the effects of insulin gene transcription regulators PDX-1, NeuroD1 and MafA on the differentiation of bone marrow mesenchymal stem cells (mMSCs) into insulin-producing cells. METHODS: Murine mMSCs were isolated, cultured and expanded. The base sequences of transcription factors PDX-1, NeuroD1 and MafA were obtained by total gene synthesis and the recombinant adenovirus vectors harboring target genes constructed and transfected into packaging cell line 293A. mMSCs were infected with adenovirus separately or together, and then differentiated in vitro into insulin-producing cells. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized to detect insulin gene expression, immunofluorescence for identifying the presence of insulin protein and insulin enzyme-linked immunosorbent assay (ELISA) for evaluating the secretory volume of insulin. RESULTS: The differentiation extent of mMSCs into ß-cell was analyzed. The ß-cell-specific transcriptional regulators and insulin gene were expressed in mMSCs after transfection. Immunofluorescent analyses revealed the activated expression of insulin in the cytoplasm of differentiated cells. A significant content of insulin was released in these cells in response to a certain concentrations of glucose stimulation. The insulin content of mMSCs infected with a combination of three transcription factors was significantly higher than that of the control group [(112.84 ± 9.67) mU/L vs (1.60 ± 0.22) mU/L, P < 0.05]. CONCLUSION: After modification by transcriptional factors PDX-1, NeuroD1 and MafA, mMSCs can secrete insulin through starting endogenous insulin gene transcription.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Células Secretoras de Insulina/citologia , Células-Tronco Mesenquimais/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Expressão Gênica , Vetores Genéticos , Proteínas de Homeodomínio/genética , Fatores de Transcrição Maf Maior/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Organismos Geneticamente Modificados , Transativadores/genética
18.
J Surg Res ; 168(2): 213-23, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20097376

RESUMO

BACKGROUND: To assess the effects of intrahepatic autotransplantation of bone-derived Beagle canine mesenchymal stem cells (BcMSCs) containing human insulin and EGFP in diabetic Beagle dogs. MATERIALS AND METHODS: BcMSCs were isolated from Beagle canine bone marrow, expanded, and transfected with a recombinant retrovirus MSCV carrying human insulin and EGFP. Animals were made diabetic by an intravenous administration of streptozotocin (STZ, 30 mg/kg) and alloxan (50 mg/kg), followed by intrahepatic autotransplantation of transfected BcMSCs. The variations of body weight, blood glucose, serum insulin levels, and plasma C-peptide were determined after autotransplantation. BcMSCs' survival and human insulin expression in liver and serum were examined by fluorescent microscopy, radioimmunoassay (RIA), and immunohistochemistry (IHC). RESULTS: The body weight of diabetic Beagle dogs received BcMSCs transplantation increased by 11.09% within 16 wk after treatment, and the average blood glucose levels were 19.80±3.13 mmol/L (d 7) and 9.78±3.11 mmol/L (d 112), while in untreated animals, the average values were 21.20±3.26 mmol/L (d 7) and 22.5±3.22 mmol/L (d 112), showing a significant difference (P<0.05). The detection of C-peptide excluded the possible function of regenerative ß cells. However, glucose tolerance test revealed BcMSCs group response was not as efficient as that of normal islets, although they could respond to the glucose challenge. CONCLUSION: Experimental diabetes could be relieved effectively for up to 16 wk by intrahepatic autotransplantation of BcMSCs expressing human insulin, which implies a novel approach of gene therapy for type I diabetes.


Assuntos
Diabetes Mellitus Experimental/terapia , Terapia Genética , Insulina/metabolismo , Transplante de Células-Tronco Mesenquimais , Animais , Glicemia , Peso Corporal , Peptídeo C/metabolismo , Diferenciação Celular , Cães , Imunofluorescência , Vetores Genéticos , Teste de Tolerância a Glucose , Humanos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Insulina/genética , Secreção de Insulina , Fígado/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Proteínas Recombinantes/metabolismo , Retroviridae , Transplante Autólogo
19.
Zhonghua Yi Xue Za Zhi ; 91(44): 3107-10, 2011 Nov 29.
Artigo em Chinês | MEDLINE | ID: mdl-22340650

RESUMO

OBJECTIVE: To explore the altered expressions of embryonic stem-related genes Oct4 and Nanog in pancreatic cancer stem cells (CSCs). METHODS: The uni-cell suspension of human pancreatic cancer cell line PANC-1 was prepared and incubated with CD24 and CD44 antibodies. Flow cytometer was used to separate CD24(+)CD44(+) pancreatic cancer stem cells. Tumor cell spheres were observed under light microscope. Then CSCs were induced to differentiate with 10% fetal bovine serum and the expressions of CD24 and CD44 re-evaluated by flow cytometer. Finally the cells were divided into 2 groups, group 1: CD24(+)CD44(+) and group 2: non-separated group. RT-PCR (reverse transcription-polymerase chain reaction) and Q-PCR (quantitative-polymerase chain reaction) were used to examine the transcriptions of Oct4 and Nanog in CSCs. The immunofluorescence was employed to examine the expressions of Oct4 and Nanog. Chemo-sensitivity to gemcitabine was determined by CCK8 assay in each group. RESULTS: About 1%-3% CD24(+)CD44(+) CSCs were separated from cell line PANC-1. The sorted cells were cultured in a stem cell culture medium to observe the spheroid-forming capacity. And they showed a higher colony-forming efficiency than the unsorted cells [(122 ± 6)‰, P < 0.05]. When cultured in medium with serum, these cells gradually returned to the status of parental cells with a low expression of CD24 and CD44. Both Oct4 and Nanog were highly expressed in CD24(+)CD44(+) stem cells. And the CD24(+)CD44(+) subgroup demonstrated a higher resistance to gemcitabine. CONCLUSION: Subpopulation cells CD44(+)CD24(+) have the properties of tumor stem cells. The up-regulated levels of Oct4 and Nanog may be highly correlated with the multi-potency and a higher drug-resistance of pancreatic CSCs.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Pancreáticas/metabolismo , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Proteína Homeobox Nanog
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