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1.
Curr Issues Mol Biol ; 46(6): 5682-5700, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38921011

RESUMO

It is known that sialyllactose (SL) in mammalians is a major source of sialic acid (Sia), which can further form cytidine monophosphate sialic acid (CMP-Sia), and the final product is polysialic acid (polySia) using polysialyltransferases (polySTs) on the neural cell adhesion molecule (NCAM). This process is called NCAM polysialylation. The overexpression of polysialylation is strongly related to cancer cell migration, invasion, and metastasis. In order to inhibit the overexpression of polysialylation, in this study, SL was selected as an inhibitor to test whether polysialylation could be inhibited. Our results suggest that the interactions between the polysialyltransferase domain (PSTD) in polyST and CMP-Siaand the PSTD and polySia could be inhibited when the 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL) concentration is about 0.5 mM or 6'-SL and 3 mM, respectively. The results also show that SLs (particularly for 3'-SL) are the ideal inhibitors compared with another two inhibitors, low-molecular-weight heparin (LMWH) and cytidine monophosphate (CMP), because 3'-SL can not only be used to inhibit NCAM polysialylation, but is also one of the best supplements for infant formula and the gut health system.

2.
Int J Mol Sci ; 25(9)2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38731861

RESUMO

The expression of polysialic acid (polySia) on the neuronal cell adhesion molecule (NCAM) is called NCAM-polysialylation, which is strongly related to the migration and invasion of tumor cells and aggressive clinical status. Thus, it is important to select a proper drug to block tumor cell migration during clinical treatment. In this study, we proposed that lactoferrin (LFcinB11) may be a better candidate for inhibiting NCAM polysialylation when compared with CMP and low-molecular-weight heparin (LMWH), which were determined based on our NMR studies. Furthermore, neutrophil extracellular traps (NETs) represent the most dramatic stage in the cell death process, and the release of NETs is related to the pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis, and vascular disorders. In this study, the molecular mechanisms involved in the inhibition of NET release using LFcinB11 as an inhibitor were also determined. Based on these results, LFcinB11 is proposed as being a bifunctional inhibitor for inhibiting both NCAM polysialylation and the release of NETs.


Assuntos
Armadilhas Extracelulares , Lactoferrina , Moléculas de Adesão de Célula Nervosa , Ácidos Siálicos , Lactoferrina/farmacologia , Lactoferrina/metabolismo , Humanos , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Heparina de Baixo Peso Molecular/farmacologia
3.
Cancer Manag Res ; 13: 8575-8583, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34815712

RESUMO

PURPOSE: Long non-coding RNA (lncRNA) DNAJC3 antisense RNA 1 (head to head) (DNAJC3-AS1) plays a key role in the progression of several cancers. However, its biological role in hepatocellular carcinoma (HCC) is still unclear. We aimed to investigate the role of DNAJC3-AS1 in the development of HCC and reveal the potential mechanisms. MATERIALS AND METHODS: Expression analysis of DNAJC3-AS1 and microRNA-27b (miR-27b) at both mature and premature levels was determined by RT-qPCR. HCC patients were followed up for 5 years to analyze the prognostic value of DNAJC3-AS1 for HCC. The direct interaction between DNAJC3-AS1 and premature miR-27b was analyzed with RNA pull-down assay. Subcellular analysis of DNAJC3-AS1 was explored by subcellular fractionation assay. DNAJC3-AS1 overexpression and knockdown were carried out to analyze the role of DNAJC3-AS1 in miR-27b maturation. Cell proliferation was analyzed by BrdU assay. RESULTS: DNAJC3-AS1 was overexpressed in HCC and predicts the poor survival. MiR-27b was downregulated at mature miRNA level, but upregulated at premature level. DNAJC3-AS1 directly interacted with premature miR-27b and was localized to both nuclear and cytoplasm. DNAJC3-AS1 overexpression upregulated premature miR-27b and downregulated mature miR-27b, while DNAJC3-AS1 knockdown led to the opposite results. DNAJC3-AS1 suppressed the role of miR-27b in inhibiting cell proliferation. CONCLUSION: DNAJC3-AS1 promotes HCC by sponging premature miR-27b and might be a biomarker and therapeutic target for HCC.

4.
Front Bioeng Biotechnol ; 9: 792023, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35145961

RESUMO

2,3,5,6-Tetramethylpyrazine (TMP) is an active pharmaceutical ingredient originally isolated from Ligusticum wallichii for curing cardiovascular and cerebrovascular diseases and is widely used as a popular flavoring additive in the food industry. Hence, there is a great interest in developing new strategies to produce this high-value compound in an ecological and economical way. Herein, a cost-competitive combinational approach was proposed to accomplish green and high-efficiency production of TMP. First, microbial cell factories were constructed to produce acetoin (3-hydroxy-2-butanone, AC), an endogenous precursor of TMP, by introducing a biosynthesis pathway coupled with an intracellular NAD+ regeneration system to the wild-type Escherichia coli. To further improve the production of (R)-AC, the metabolic pathways of by-products were impaired or blocked stepwise by gene manipulation, resulting in 40.84 g/L (R)-AC with a high optical purity of 99.42% in shake flasks. Thereafter, an optimal strain designated GXASR11 was used to convert the hydrolysates of inexpensive feedstocks into (R)-AC and achieved a titer of 86.04 g/L within 48 h in a 5-L fermenter under optimized fermentation conditions. To the best of our knowledge, this is the highest (R)-AC production with high optical purity (≥98%) produced from non-food raw materials using recombinant E. coli. The supernatant of fermentation broth was mixed with diammonium phosphate (DAP) to make a total volume of 20 ml and transferred to a high-pressure microreactor. Finally, 56.72 g/L TMP was obtained in 3 h via the condensation reaction with a high conversion rate (85.30%) under optimal reaction conditions. These results demonstrated a green and sustainable approach to efficiently produce high-valued TMP, which realized value addition of low-cost renewables.

5.
Cancer Manag Res ; 10: 873-885, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29731667

RESUMO

PURPOSE: Liver cancer stem cells (CSCs) are known to be associated with the development, survival, proliferation, metastasis, and recurrence of liver tumors. The aim of this study was to investigate the association of liver-enriched activator protein 1 (LAP1) with hepatocellular carcinoma (HCC) and liver CSCs (LCSCs) and explore the impact of LAP1 on LCSCs. MATERIALS AND METHODS: Differences in LAP1 expression in liver cancer tissues versus matched para-tumoral liver tissues and LCSCs versus non-CSCs were analyzed by Western blotting, real-time polymerase chain reaction, immunohistochemistry, and flow cytometry. The effect of LAP1 on liver cancer cells was evaluated by the expression of CSC markers, oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle distribution and the number of apoptotic cells were analyzed to assess cell cycle and cell apoptosis. Furthermore, a mouse subcutaneous tumor implant model was established to explore the role of LAP1 in the development of HCC in vivo. Finally, the expression of CSC markers in paraffin-embedded sections was evaluated by immunofluorescence. RESULTS: LAP1 was weakly expressed in HCC tumors and cell lines and even weaker in LCSCs. LAP1 inhibited the expression of stem cell-associated genes and reduced the abilities of oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle assay revealed that LAP1 induced G1/G0 arrest. Furthermore, LAP1 decreased subcutaneous tumor-formation ability and the expression of CSC markers and Ki67 in vivo. CONCLUSION: LAP1 suppressed the stem cell features of HCC, indicating that it possessed an antitumor effect in liver cancer, both in vitro and in vivo; therefore, LAP1 may prove to be a potential target in liver CSC-targeted therapy.

6.
RSC Adv ; 8(53): 30512-30519, 2018 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-35546830

RESUMO

Acetoin is an important platform chemical with a variety of applications in foods, cosmetics, chemical synthesis, and especially in the asymmetric synthesis of optically active pharmaceuticals. It is also a useful breath biomarker for early lung cancer diagnosis. In order to enhance production of optical (S)-acetoin and facilitate this building block for a series of chiral pharmaceuticals derivatives, we have developed a systematic approach using in situ-NADH regeneration systems and promising diacetyl reductase. Under optimal conditions, we have obtained 52.9 g L-1 of (S)-acetoin with an enantiomeric purity of 99.5% and a productivity of 6.2 g (L h)-1. The results reported in this study demonstrated that the production of (S)-acetoin could be effectively improved through the engineering of cofactor regeneration with promising diacetyl reductase. The systematic approach developed in this study could also be applied to synthesize other optically active α-hydroxy ketones, which may provide valuable benefits for the study of drug development.

7.
Int J Biochem Cell Biol ; 80: 154-162, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27697592

RESUMO

Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their abilities for self-renewal and bi-potential differentiation. However, the molecular mechanism regulating proliferation and differentiation of iHepSCs is poorly understood. In this study, we provide evidence that the homeodomain transcription factor, Pitx2, is essential to maintain iHepSCs stem cell characteristics. Suppressing Pitx2 expression in iHepSCs by lentivirus mediated specific shRNA markedly reduced the expression of the hepatic stem cell-associated genes (Lgr5, EpCAM, and Sox9) with concomitant inhibition of proliferation by blocking the G1/S phase transition, and these phenotypic changes were reversed upon re-expression of Pitx2. Pitx2 knockdown also resulted in up-regulation of the p53-induced Cdk inhibitor p21, and down-regulation of its downstream effector CDK2-Cyclin E kinase complex. Furthermore, we observed that iHepSCs were more efficiently induced to differentiate into both hepatocytes and cholangiocytes when Pitx2 expression was suppressed, as compared to unmanipulated iHepSCs. These findings reveal that Pitx2 expression may be leveraged to control the status of iHepSCs during expansion in vitro to provide a strategy for further application of iHepSCs in liver cell therapy.


Assuntos
Diferenciação Celular/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Fígado/citologia , Células-Tronco/citologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo/genética , Células HEK293 , Humanos , Camundongos , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Homeobox PITX2
8.
J Mater Chem B ; 4(17): 2963-2971, 2016 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32262973

RESUMO

In this paper, amidogen graphene (NH2-Gr) and Au nanoparticle (NP) functionalized cerium oxide (CeO2) NPs (NH2-Gr/Au@CeO2) have been synthesized. The prepared NH2-Gr/Au@CeO2 exhibits strong electrochemiluminescence (ECL) activity which could be quenched efficiently by bismuth sulfide (Bi2S3). Based on this principle, a novel sandwich ECL immunosensor was constructed for the detection of prostate specific antigen (PSA). NH2-Gr was used as a loading matrix for CeO2 NPs because of its superior conductivity and a large surface area. Au NPs were used to combine anti-PSA 1 (Ab1) to CeO2via the Au-NH2 covalent bond and meanwhile to enhance the sensitivity of the immunosensor. NH2-Gr/Au@CeO2 was used as the ECL response layer. Ag NP functionalized Bi2S3 was used to label the secondary antibody (Ab2). After a sandwich-type immunoreaction, a remarkable decrease of the ECL signal was observed. The signal-off sensor showed a wide linear range response from 1 pg mL-1 to 10 ng mL-1 with a low detection limit (LOD) of 0.3 pg mL-1. The applicability of the proposed ECL immunosensor was evaluated by detecting PSA in human serum samples.

9.
ACS Appl Mater Interfaces ; 7(34): 19260-7, 2015 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-26271682

RESUMO

A high-sensitivity electrochemiluminescence (ECL) sensor was conducted to detect carcinoembryonic antigen (CEA). Nanocomposites of graphene oxide/carboxylated multiwall carbon nanotubes/gold/cerium oxide nanoparticles (GO/MWCNTs-COOH/Au@CeO2) were used as antibody carriers and sensing platforms to modify on glassy carbon electrodes (GCE). CeO2 nanoparticles were first exploited as an ECL luminescent material and the possible ECL mechanism was proposed in this work. GO/MWCNTs-COOH was used as a loading matrix for CeO2 nanoparticles because of the superior conductivity and large specific surface area. Au nanoparticles were further deposited on this matrix to attach anti-CEA and enhance the sensitivity of immunosensor. The proposed sensing platform showed excellent cathodic ECL performance and sensitive response to CEA. The effects of experimental conditions on the ECL performance were investigated. The proposed immunosensor showed the broad linear range (0.05-100 ng/mL) and the low detection limit (LOD, 0.02 ng/mL, signal-to-noise ratio = 3) according to the selected experimental conditions. The excellent analysis performance for determination of CEA in the human serum samples simplied this immunosensor displayed high sensitivity and excellent repeatability. More importantly, this conducted immunosensor broadens the use scope of CeO2 nanoparticles.


Assuntos
Técnicas Biossensoriais/métodos , Antígeno Carcinoembrionário/sangue , Cério/química , Técnicas Eletroquímicas/métodos , Ouro/química , Grafite/química , Medições Luminescentes/métodos , Nanotubos de Carbono/química , Humanos , Concentração de Íons de Hidrogênio , Nanocompostos/química , Nanotubos de Carbono/ultraestrutura , Reprodutibilidade dos Testes
10.
Biosens Bioelectron ; 74: 104-12, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26120817

RESUMO

A high sensitive label-free electrochemiluminescence (ECL) immunosensor was fabricated for the detection of prostate specific antigen (PSA) based on potassium niobate-Au nanoparticles@bismuth sulfide (KNbO3-Au NPs@Bi2S3) modified glassy carbon electrode (GCE). The prepared Bi2S3 nanosheets exhibited strong and stable cathodic ECL activity. The synthesized KNbO3-Au NPs was firstly used to fabricate ECL modified electrodes and Bi2S3 nanosheets worked as luminophores for the first time in ECL sensors. Au NPs were used to combine with Bi2S3 and anti-PSA via the Au-S covalent bond and Au-NH2 covalent bond without the usage of crosslinking agents respectively, further enhancing the sensitivity and stability of immunosensor. Under the optimum experimental conditions, the ECL signal of KNbO3-Au NPs@Bi2S3 linearly decreased with the increase of PSA concentration in the range of 0.005-5 ng/mL with a detection limit of 3 pg/mL. The preparated label-free ECL immunosensor exhibited high sensitivity and selectivity, good repeatability and long-term stability. The applicability of the proposed ECL immunosensor was also evaluated by detecting PSA in real samples.


Assuntos
Bismuto/química , Ouro/química , Medições Luminescentes/instrumentação , Nanoestruturas/química , Nióbio/química , Óxidos/química , Potássio/química , Antígeno Prostático Específico/sangue , Sulfetos/química , Animais , Técnicas Eletroquímicas/instrumentação , Eletrodos , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Nanopartículas/química , Nanopartículas/ultraestrutura , Nanoestruturas/ultraestrutura , Antígeno Prostático Específico/análise
11.
Hepatology ; 60(1): 349-61, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24711261

RESUMO

UNLABELLED: A better understanding of hepatocyte senescence could be used to treat age-dependent disease processes of the liver. Whether continuously proliferating hepatocytes could avoid or reverse senescence has not yet been fully elucidated. We confirmed that the livers of aged mice accumulated senescent and polyploid hepatocytes, which is associated with accumulation of DNA damage and activation of p53-p21 and p16(ink4a)-pRB pathways. Induction of multiple rounds continuous cell division is hard to apply in any animal model. Taking advantage of serial hepatocyte transplantation assays in the fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mouse, we studied the senescence of hepatocytes that had undergone continuous cell proliferation over a long time period, up to 12 rounds of serial transplantations. We demonstrated that the continuously proliferating hepatocytes avoided senescence and always maintained a youthful state. The reactivation of telomerase in hepatocytes after serial transplantation correlated with reversal of senescence. Moreover, senescent hepatocytes harvested from aged mice became rejuvenated upon serial transplantation, with full restoration of proliferative capacity. The same findings were also true for human hepatocytes. After serial transplantation, the high initial proportion of octoploid hepatocytes decreased to match the low level of youthful liver. CONCLUSION: These findings suggest that the hepatocyte "ploidy conveyer" is regulated differently during aging and regeneration. The findings of reversal of hepatocyte senescence could enable future studies on liver aging and cell therapy.


Assuntos
Proliferação de Células , Senescência Celular/fisiologia , Hepatócitos/citologia , Hepatócitos/transplante , Regeneração Hepática/fisiologia , Animais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citometria de Fluxo , Hepatócitos/fisiologia , Hidrolases/genética , Óperon Lac , Fígado/citologia , Fígado/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Poliploidia , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo
12.
Cell Stem Cell ; 13(3): 328-40, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-23871605

RESUMO

Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1ß and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.


Assuntos
Células-Tronco Adultas/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/terapia , Fibroblastos/fisiologia , Regeneração Tecidual Guiada , Hepatócitos/fisiologia , Hidrolases/metabolismo , Fígado/citologia , Animais , Ductos Biliares Intra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/embriologia , Linhagem Celular , Linhagem da Célula , Transdiferenciação Celular , Fator 1-beta Nuclear de Hepatócito/metabolismo , Fator 3-gama Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/metabolismo , Hidrolases/genética , Fígado/embriologia , Fígado/lesões , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Organogênese , Piridinas/administração & dosagem , Transplante de Células-Tronco
13.
Biosens Bioelectron ; 47: 68-74, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23557979

RESUMO

In this paper, a carbon coated magnetic nanoparticle (Fe3O4-C) was first synthesized via solvothermal reaction and carbonization of glucose under hydrothermal condition. The electrochemiluminescence (ECL) property of Fe3O4-C was studied, and exhibited a peak at 1.21V. In the goal to amplify the ECL intensity for sensitive detection, a novel coaxial carbon coated magnetic nanomaterial (MWNTs-Fe3O4-C) was synthesized. Fourier transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), powder X-ray diffraction (XRD) and powder X-ray photoelectron spectrometry (XPS) were applied as powerful tools to characterize and to demonstrate the named nanomaterial. MWNTs-Fe3O4-C showed better ECL property than Fe3O4-C. Furthermore, an ultrasensitive ECL immunosensor based on MWNTs-Fe3O4-C was developed for the determination of carcinoembryonic antigen (CEA). The prepared ECL immunosensor exhibited high sensitivity, good reproducibility, long-term stability, and acceptable precision on the detection of CEA in clinical human serum samples.


Assuntos
Técnicas Biossensoriais , Antígeno Carcinoembrionário/isolamento & purificação , Nanopartículas de Magnetita/química , Nanoestruturas/química , Carbono/química , Antígeno Carcinoembrionário/sangue , Compostos Férricos/síntese química , Compostos Férricos/química , Humanos , Imunoensaio , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
14.
Int J Nanomedicine ; 7: 2641-52, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22679374

RESUMO

In the rapid development of nanoscience and nanotechnology, many researchers have discovered that metal oxide nanoparticles have very useful pharmacological effects. Cuprous oxide nanoparticles (CONPs) can selectively induce apoptosis and suppress the proliferation of tumor cells, showing great potential as a clinical cancer therapy. Treatment with CONPs caused a G1/G0 cell cycle arrest in tumor cells. Furthermore, CONPs enclosed in vesicles entered, or were taken up by mitochondria, which damaged their membranes, thereby inducing apoptosis. CONPs can also produce reactive oxygen species (ROS) and initiate lipid peroxidation of the liposomal membrane, thereby regulating many signaling pathways and influencing the vital movements of cells. Our results demonstrate that CONPs have selective cytotoxicity towards tumor cells, and indicate that CONPs might be a potential nanomedicine for cancer therapy.


Assuntos
Apoptose/efeitos dos fármacos , Cobre/farmacologia , Nanopartículas Metálicas/administração & dosagem , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Cobre/toxicidade , Ditiotreitol/farmacologia , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Células HeLa , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Testes de Toxicidade
15.
J Cell Biochem ; 108(3): 693-704, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19693776

RESUMO

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte-like phenotypes. When transplanted intrasplentically into carbon tetrachloride-injured livers of SCID mice, EGFP-tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three-dimensional architecture, and differentiate into hepatocyte-like cells expressing human albumin and alpha-1-anti-trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Hepatócitos/citologia , Fígado/patologia , Células-Tronco Mesenquimais/citologia , Animais , Biomarcadores/metabolismo , Tetracloreto de Carbono , Fusão Celular , Linhagem da Célula , Proliferação de Células , Separação Celular , Células Cultivadas , Células Clonais , DNA/análise , Hepatócitos/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , Ploidias
16.
J Cell Biochem ; 106(1): 16-24, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19021148

RESUMO

BMI-1 (B-cell-specific Moloney murine leukemia virus integration site 1), a novel oncogene, has attracted much attention in recent years for its involvement in the initiation of a variety of tumors. Recent evidence showed that BMI-1 was highly expressed in neoplastic skin lesions. However, whether dysregulated BMI-1 expression is causal for the transformation of skin cells remains unknown. In this study, we stably expressed BMI-1 in a human keratinocyte cell line, HaCaT. The expression of wild-type BMI-1 induced the malignant transformation of HaCaT cells in vitro. More importantly, we found that expression of BMI-1 promoted formation of squamous cell carcinomas in vivo. Furthermore, we showed that BMI-1 expression led to the downregulation of tumor suppressors, such as p16INK4a and p14ARF, cell adhesion molecules, such as E-Cadherin, and differentiation related factor, such as KRT6. Therefore, our findings demonstrated that dysregulated BMI-1 could indeed lead to keratinocytes transformation and tumorigenesis, potentially through promoting cell cycle progression and increasing cell mobility.


Assuntos
Transformação Celular Neoplásica/metabolismo , Queratinócitos/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Ciclo Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos SCID , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
17.
Stem Cells ; 24(2): 322-32, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16109753

RESUMO

Liver progenitor cells have drawn a great deal of attention both for their therapeutic potential and for their usefulness in exploring the molecular events surrounding liver development and regeneration. Despite the intensive studies on liver progenitors from rats, equivalent progenitor cells derived from mice are relatively rare. We used retrosine treatment followed by partial hepatectomy to elicit liver progenitors in mice. From these animals showing prominent ductular reactions, mouse-derived liver progenitor cell lines (LEPCs) were isolated by single-cell cloning. Phenotypic and lineage profiling of the LEPC clones were performed using immunochemistry, reverse transcription-polymerase chain reaction, and a dual-color system comprising the reporter EGFP under the control of the cytokeratin 19 promoter and the DsRed reporter under the control of the albumin promoter. LEPCs expressed liver progenitor cell markers. LEPCs also expressed some markers shared by bone marrow-derived hematopoietic stem cells c-Kit and Thy-1 but not CD34 and CD45. When cultured as aggregates in Matrigel, LEPCs differentiated into hepatocyte upon treatment with 50 ng/ml epithelial growth factor or differentiated into biliary lineage cells upon treatment with 20 ng/ml hepatocyte growth factor. In the presence of 2% dimethyl sulfoxide and 2% Matrigel, LEPCs acquired predominantly bile lineage phenotypes, with occasional patches of cells exhibiting hepatocyte phenotypes. Upon transplantation into CCl4-injured-liver, LEPCs engrafted into liver parenchyma and differentiated into hepatocytes. Considering the amenability of the mouse to genetic manipulation, these mouse-derived LEPCs may be useful tools as in vitro models to study molecular events in liver development and regeneration and can shed light in studying the therapy potential of liver stem cells.


Assuntos
Transplante de Células , Hepatócitos/metabolismo , Regeneração Hepática , Fígado/fisiologia , Células-Tronco/metabolismo , Animais , Ductos Biliares/citologia , Biomarcadores/análise , Diferenciação Celular , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Células HeLa , Hepatectomia , Humanos , Fígado/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Alcaloides de Pirrolizidina/farmacologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Transfecção
18.
World J Gastroenterol ; 9(1): 112-6, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12508363

RESUMO

AIM: To establish a mice model harboring hepatitis B virus x gene (adr subtype) for studying the function of hepatitis B virus X protein, a transactivator of viral and cellular promoter/enhancer elements. METHODS: Expression vector pcDNA3-HBx, containing CMV promoter and hepatitis B virus x gene open reading fragment, was constructed by recombination DNA technique. Hela cells were cultured in DMEM and transfected with pcDNA3-HBx or control pcDNA3 plasmids using FuGENE6 Transfection Reagent. Expression of pcDNA3-HBx vectors in the transfected Hela cells was confirmed by Western blotting. After restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes. The pups were evaluated by multiplex polymerase chain reaction (PCR) at genomic DNA level. The x gene transgenic mice founders were confirmed at protein level by Western blotting, immunohistochemistry and immunogold transmission electron microscopy. RESULTS: Expression vector pcDNA3-HBx was constructed by recombination DNA technique and identified right by restriction endonuclease digestion and DNA direct sequencing. With Western blotting, hepatitis X protein was detected in Hela cells transfected with pcDNA3-HBx plasmids, suggesting pcDNA3-HBx plasmids could express in eukaryotic cells. Following microinjection of coding sequence of pcDNA3-HBx, the embryos were transferred to oviducts of pseudopregnant females. Four pups were born and survived. Two of them were verified to have the HBx gene integrated in their genomic DNA by multiplex PCR assay, and named C57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3 respectively. They expressed 17KD X protein in liver tissue by Western blotting assay. With the immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of transgenic mice, which was furthermore confirmed by immunogold transmission electon microscopy. CONCLUSION: We have constructed the expression vector pcDNA3-HBx that can be used to study the function of HBx gene in eukaryotic cells in vitro. We also established HBx gene (adr subtype) transgenic mice named C57-TgN (HBx)SMMU harboring HBx gene in their genome and express X protein in hepatocytes, Which might be a valuable animal system for studying the roles of HBx gene in hepatitis B virus life cycle and development of hepatocellular carcinoma in vivo.


Assuntos
Antígenos da Hepatite B/metabolismo , Transativadores/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Regulação Viral da Expressão Gênica , Células HeLa , Hepatite B/genética , Antígenos da Hepatite B/genética , Humanos , Fígado/ultraestrutura , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias
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