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BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidates for the treatment of ischemic disease or injury and may be an alternative treatment for cell therapy. This aim of the study was to evaluate whether exosomes derived from adipose mesenchymal stem cell (ADSC) can protect the skin flap during ischemia-reperfusion (I/R) injury and induce neovascularization. METHODS: To investigate the effects of exosomes in the I/R injury of flap transplantation in vivo, flaps were subjected to 6â¯h of ischemia by ligating the left superficial inferior epigastric vessels (SIEA) followed by blood perfusion. Exosomes derived from normal ADSC (ADSC-exos) and exosomes derived from ADSC preconditioned with H2O2 (H2O2-ADSC-exos) were injected into the flaps. Then, the blood perfusion unit (BPU) of the flaps was measured by Laser Doppler Perfusion Imaging (LDPI) and microvessel density was determined by the endothelial with cell marker CD31 with Immunohistochemistry (IHC) staining. Inflammatory cell infiltration of the skin flap and apoptosis were detected by hematoxylin & eosin staining (H&E) and the TdT-mediated biotinylated dUTP nick end-labeling (TUNEL) technique. RESULTS: In vivo, exosomes significantly increased flap survival and capillary density compared to I/R on postoperative day 5, and decreased the inflammatory reaction and apoptosis in the skin flap (Pâ¯<â¯0.05). Furthermore, H2O2-ADSC-exos had better outcomes compared to normal exosomes (Pâ¯<â¯0.05). ADSC-exos could significantly increase human umbilical vein endothelial cell (HUVEC) proliferation (Pâ¯<â¯0.05), but no statistic difference was found in exosomes derived from different microenvironments (Pâ¯>â¯0.05). HUVEC co-cultured with H2O2-ADSC-exos increased the migration ratio and generated more cord-like structures compared to ADSC-exos and the control group (Pâ¯<â¯0.05). CONCLUSION: ADSC-exos can enhance skin flap survival, promote neovascularization and alleviate the inflammation reaction and apoptosis in the skin flap after I/R injury. The use of a specific microenvironment for in vitro stem cell culture, such as one containing a low concentration of H2O2, will facilitate the development of customized exosomes for cell-free therapeutic applications in skin flap transplantation.
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Tejido Adiposo/citología , Exosomas/metabolismo , Peróxido de Hidrógeno/farmacología , Células Madre Mesenquimatosas/citología , Daño por Reperfusión/patología , Colgajos Quirúrgicos/irrigación sanguínea , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Exosomas/efectos de los fármacos , Exosomas/trasplante , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , PerfusiónRESUMEN
BACKGROUND: Adipose-derived stromal cells (ADSCs)-derived exosomes (ADSC-Exos) account for the proangiogenic potential of stem cell. This study aimed to investigate the effect of ADSC-derived exosomes (ADSC-Exos) on the survival in fat grafting. METHODS: A nude mouse model of subcutaneous fat grafting was adopted. Hypoxic preconditioned ADSC-Exos and ADSC-Exos were injected around the grafted tissue. The fat graft sample was weighed and examined by hematoxylin and eosin (H&E) staining and immunohistochemistry. Laser Doppler flowmetry and CD31 immunofluorescence staining were used to analyze neovascularization. RESULTS: ADSC-Exo and hypoxic ADSC-Exo groups had a significantly higher weight of fat graft and more perilipin-positive adipocytes than the control groups from 2 to 8 weeks after grafting, and the hypoxic ADSC-Exo group had better outcomes (all Pâ¯<â¯0.05). H&E staining showed that ADSC-Exos attenuated infiltration of inflammatory cells around the fat grafts. Laser Doppler flowmetry showed that the two ADSC-Exo groups had better blood perfusion in the graft tissue than the control groups (all Pâ¯<â¯0.05). Immunofluorescence demonstrated that the hypoxic ADSC-Exo group had significantly more CD31-positive cells than the ADSC-Exo group. In vitro study showed that hypoxic ADSC-Exos treatment significantly increased the migration (at 12 and 24â¯h) and in vitro capillary network formation (at 12â¯h) in the human umbilical vein endothelial cells (HUVECs) as compared with the ADSC-Exo group and control group (all Pâ¯<â¯0.05). CONCLUSIONS: Co-transplantation of ADSC-Exos can effectively promote the survival of graft, neovascularization and attenuated inflammation in the fat grafts. Hypoxia treatment can further enhance the beneficial effect of ADSC-Exos.
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Tejido Adiposo/crecimiento & desarrollo , Tejido Adiposo/trasplante , Exosomas/trasplante , Supervivencia de Injerto/fisiología , Precondicionamiento Isquémico/métodos , Células Madre Mesenquimatosas/ultraestructura , Neovascularización Fisiológica/fisiología , Tejido Adiposo/citología , Animales , Exosomas/ultraestructura , Femenino , Trasplante de Células Madre Mesenquimatosas/métodos , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
UNLABELLED: Emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving treatment of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play important roles in HCC; however, the mechanisms by which miRNAs target the EMT and their therapeutic potential remains largely unknown. To better explore the roles of miRNAs in the EMT process, we established an EMT model in HCC cells by transforming growth factor beta 1 treatment and found that several tumor-related miRNAs were significantly decreased. Among these miRNAs, miR-125b expression was most strongly suppressed. We also found down-regulation of miR-125b in most HCC cells and clinical specimens, which correlated with cellular differentiation in HCC patients. We then demonstrated that miR-125b overexpression attenuated EMT phenotype in HCC cancer cells, whereas knockdown of miR-125b promoted the EMT phenotype in vitro and in vivo. Moreover, we found that miR-125b attenuated EMT-associated traits, including chemoresistance, migration, and stemness in HCC cells, and negatively correlated with EMT and cancer stem cell (CSC) marker expressions in HCC specimens. miR-125b overexpression could inhibit CSC generation and decrease tumor incidence in the mouse xenograft model. Mechanistically, our data revealed that miR-125b suppressed EMT and EMT-associated traits of HCC cells by targeting small mothers against decapentaplegic (SMAD)2 and 4. Most important, the therapeutic delivery of synthetic miR-125b mimics decreased the target molecule of CSC and inhibited metastasis in the mice model. These findings suggest a potential therapeutic treatment of miR-125b for liver cancer. CONCLUSION: miR-125b exerts inhibitory effects on EMT and EMT-associated traits in HCC by SMAD2 and 4. Ectopic expression of miR-125b provides a promising strategy to treat HCC.
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Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Células Madre Neoplásicas/patología , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Distribución Aleatoria , Sensibilidad y Especificidad , Transfección , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To isolate and steadily culture kidney stem cells (KSCs) from rat renal papilla, and to identify the biological characteristics of KSCs. METHODS: KSCs were isolated from the tips of renal papilla in 4 weeks-old Sprague-Dawley rats. The morphology of KSCs was observed under inversion microscope, and the phenotye characteristics of kSCs were identified through flow cytometry and immunofluorescence. The abilities of KSCs in adipogenic and osteogenic differentiation were evaluated. The differences of gene expression between KSCs and rat renal tubular epithelial cells (RTECs)were compared using quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: KSCs showed a spindle-shaped and arborization-like growth pattern. Immunofluorescence indicated that KSCs staining with alpha-sooth muscle actin (α-SMA), Vimentin, N-Cadherin, Nestin, CD133 marker, and without E-cadherin, cytokeratin-18 (CK-18), zona occludens protein-1 (ZO-1). The positive staining of CD29, CD90, CD73 were 99. 0%, 95. 8%, 99. 9% respectively, the positive staining of CD45 was 3. 4%. The positive stainings of stem cell marker CD133 and Nestin were 33. 2% and 70. 2% respectively, while the double staining rate was 31. 4%., KSCs showed positive staining by oil red 0 after adipogenic differentiation, and orange calcium deposition by alizarin red staining after osteogenic differentiation. qRT-PCR showed that the expressions of embryonic stem cell marker Nanog, Oct4/pou5f1,Sox2/sry-box-2 in KSCs were higher than those in RTECs (P< 0.01), and the expressions of mesenchymal marker c-SMA, Vimentin were also higher in KSCs (P<0. 01). Compared with RTECs, the expressions of mature epithelium marker E-Cadherin, CK18 in KSCs were lower (P< 0. 01). CONCLUSION: KSCs were isolated successfully and steadily cultured from the rat renal papilla, which were identified with featured biological characteristics.
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Riñón/citología , Células Madre/citología , Adipogénesis , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Separación Celular , Células Cultivadas , Citometría de Flujo , Osteogénesis , Ratas , Ratas Sprague-DawleyRESUMEN
UNLABELLED: Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. CONCLUSION: S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013).
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Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , Proteínas S100/metabolismo , Animales , Carcinoma Hepatocelular/patología , Proliferación Celular , Progresión de la Enfermedad , Humanos , Neoplasias Hepáticas/patología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteína de Unión al Calcio S100A4 , Factor de Transcripción STAT3/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
The enzymatic activities of Furin, Transmembrane serine proteinase 2 (TMPRSS2), Cathepsin L (CTSL), and Angiotensin-converting enzyme 2 (ACE2) receptor binding are necessary for the entry of coronaviruses into host cells. Precise inhibition of these key proteases in ACE2+ lung cells during a viral infection cycle shall prevent viral Spike (S) protein activation and its fusion with a host cell membrane, consequently averting virus entry to the cells. In this study, dual-drug-combined (TMPRSS2 inhibitor Camostat and CTSL inhibitor E-64d) nanocarriers (NCs) are constructed conjugated with an anti-human ACE2 (hACE2) antibody and employ Red Blood Cell (RBC)-hitchhiking, termed "Nanoengineered RBCs," for targeting lung cells. The significant therapeutic efficacy of the dual-drug-loaded nanoengineered RBCs in pseudovirus-infected K18-hACE2 transgenic mice is reported. Notably, the modular nanoengineered RBCs (anti-receptor antibody+NCs+RBCs) precisely target key proteases of host cells in the lungs to block the entry of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), regardless of virus variations. These findings are anticipated to benefit the development of a series of novel and safe host-cell-protecting antiviral therapies.
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COVID-19 , Catepsina L , SARS-CoV-2 , Inhibidores de Serina Proteinasa , Animales , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Catepsina L/antagonistas & inhibidores , Catepsina L/metabolismo , COVID-19/prevención & control , COVID-19/virología , Eritrocitos , Pulmón/metabolismo , Péptido Hidrolasas/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Inhibidores de Serina Proteinasa/uso terapéuticoRESUMEN
OBJECTIVE: To explore the expression of Foxa2 in different pathological types of gastric polyps and examine the correlation with cancerous risk. METHODS: According to computerize random number, a total of 2000 patients were selected to receive endoscopic biopsy during November 2011 to October 2012. Tissues were harvested from 170 with gastric polyps and suspicious cancerous lesions and their histological types detected. There were hyperplastic polyps(n = 35), adenomatous polyps(n = 31), fundic gland polyps(n = 42), advanced gastric cancer tissues (n = 32)and normal gastric mucosa tissues (n = 30). ABC immunohistochemical staining and reverse transcription(RT)-PCR were employed to detect the expression of Foxa2 in these different types of tissues. Imagepro plus was used for quantitative and statistical analyses. RESULTS: A low-level expression of Foxa2 was 3.6% ± 1.3% in normal gastric mucosa group. And its expreesion gradually higher in proliferative inflammatory polyp group(33.1% ± 8.0%), adenomatous polyp group (71.4% ± 1.7%) and gastric cancer group(96.3% ± 0.9%, all P < 0.05). Its expression was 35.6% ± 5.6% in fundic gland polyps, similar to that of proliferative inflammatory polyp group (P > 0.05), it was markedly lower than the gastric cancer group (P < 0.05) and higher than the normal gastric mucosa group (P < 0.05). Correlation analyses of clinicopathological parameters showed that no significant correlation existed between its expression and patient gender, age, predilection, Helicobacter. pylori infection or proton pump inhibitor used (all P > 0.05). However, the size of polyps was correlated with Foxa2 (rs = 0.69, P < 0.05). CONCLUSION: The expression level of Foxa2 in different types of gastric polyps may be used as a clinical predicator of polyps risk.
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Pólipos Adenomatosos/metabolismo , Pólipos Adenomatosos/patología , Factor Nuclear 3-beta del Hepatocito/metabolismo , Neoplasias Gástricas/patología , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Neoplasias Gástricas/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) play a critical role in promoting cancer progression. However, it is not clear whether MSCs are located in breast cancer tissues and correlated with tumor proliferation. The aim of this study was to investigate the presence of MSCs in breast cancer tissues and evaluate their interactions with cancer cells. We successfully isolated and identified MSCs from primary breast cancer tissues. Breast cancer-associated MSCs (BC-MSCs) showed homogenous immunophenotype, and possessed tri-lineage differentiation potential (osteoblast, adipocyte, and chondrocyte). When co-transplanted with cancer cells in a xenograft model in vivo, BC-MSCs significantly increased the volume and weight of tumors. We observed that BC-MSCs stimulated mammosphere formation in the transwell co-culture system in vitro. This effect was significantly suppressed by the EGF receptor inhibitor. We verified that BC-MSCs could secrete EGF and activate cancer cell's EGF receptors. Furthermore, our data showed that EGF derived from BC-MSCs could promote mammosphere formation via the PI3K/Akt signaling pathway. Our results confirmed the presence of MSC in primary breast cancer tissues, and they could provide a favorable microenvironment for tumor cell growth in vivo, partially enhance mammosphere formation via the EGF/EGFR/Akt pathway.
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Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Factor de Crecimiento Epidérmico/fisiología , Células Madre Mesenquimatosas/metabolismo , Células Madre Neoplásicas/metabolismo , Esferoides Celulares/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Diferenciación Celular , Proliferación Celular , Forma de la Célula , Técnicas de Cocultivo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Carga Tumoral , Células Tumorales CultivadasRESUMEN
UNLABELLED: The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin-2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM-mediated invasion and metastasis of cancer cells is found to require up-regulation of matrix metalloproteinase-9 (MMP-9) through the activation of focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) axis. CONCLUSION: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP-9 expression through the FAK-ERK pathway.
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Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundario , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Sistema de Señalización de MAP Quinasas/fisiología , Sintaxina 1/metabolismo , División Celular/fisiología , Movimiento Celular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Células Hep G2 , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Hígado/metabolismo , Hígado/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Invasividad NeoplásicaRESUMEN
Cancer stem cells (CSCs) are reported to play essential roles in chemoresistance and metastasis. Pathways regulating CSC self-renewal and proliferation, such as Hedgehog, Notch, Wnt/ß-catenin, TGF-ß, and Myc, may be potential therapeutic targets. Here, a functional screening from the focused library with 365 compounds is performed by a step-by-step strategy. Among these candidate molecules, phenyl-2-pyrimidinyl ketone 4-allyl-3-amino selenourea (CU27) is chosen for further identification because it proves to be the most effective compound over others on CSC inhibition. Through ingenuity pathway analysis, it is shown CU27 may inhibit CSC through a well-known stemness-related transcription factor c-Myc. Gene set enrichment analysis, dual-luciferase reporter assays, expression levels of typical c-Myc targets, molecular docking, surface plasmon resonance, immunoprecipitation, and chromatin immunoprecipitation are conducted. These results together suggest CU27 binds c-Myc bHLH/LZ domains, inhibits c-Myc-Max complex formation, and prevents its occupancy on target gene promoters. In mouse models, CU27 significantly sensitizes sorafenib-resistant tumor to sorafenib, reduces the primary tumor size, and inhibits CSC generation, showing a dramatic anti-metastasis potential. Taken together, CU27 exerts inhibitory effects on CSC and CSC-associated traits in hepatocellular carcinoma (HCC) via c-Myc transcription activity inhibition. CU27 may be a promising therapeutic to treat sorafenib-resistant HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Compuestos de Selenio , Selenio , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Detección Precoz del Cáncer , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Ratones , Simulación del Acoplamiento Molecular , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Selenio/metabolismo , Selenio/farmacología , Compuestos de Selenio/metabolismo , Compuestos de Selenio/farmacología , Sorafenib/metabolismo , Sorafenib/farmacologíaRESUMEN
OBJECTIVE: To explore the method of constructing tissue-engineered skin using melanocytes and bone marrow mesenchymal stem cells (BMSCs) in vivo. METHODS: Melanocytes were isolated from human foreskin. BMSCs were isolated from human bone marrow. Both of them were co-cultured at a ratio of 1:10, and then were implanted into the collagen membrane to construct the tissue-engineered skin, which was applied for wound repair in nude mice. The effectiveness of wound repair and the distribution of melanocytes were evaluated by morphological observation, in vivo 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) fluorescent staining tracing, HE staining, S-100 immunohistochemistry, and transmission electron microscopy. RESULTS: The wounds were satisfactorily repaired among the nude mice. The melanocytes were distributed in the skin with normal structure, as confirmed by DAPI fluorescent staining tracing, HE staining, S-100 immunohistochemistry, and transmission electron microscopy. CONCLUSION: Melanocytes and BMSCs, after proper in vitro culture at an appropriate ratio, can construct the tissue-engineered skin with I type collagen membrane.
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Células de la Médula Ósea/citología , Melanocitos/citología , Células Madre Mesenquimatosas/citología , Piel Artificial , Ingeniería de Tejidos , Animales , Células Cultivadas , Técnicas de Cocultivo , Colágeno Tipo I , Humanos , Ratones , Ratones Desnudos , Piel/lesionesRESUMEN
Hepatocellular carcinoma (HCC) is the most common liver cancer with high mortality. Here, we found that hnRNPU is overexpressed in HCC tissues and is correlated with the poor prognosis of HCC patients. Besides, hnRNPU is of high significance in regulating the proliferation, apoptosis, self-renewal, and tumorigenic potential of HCC cells. Mechanismly, c-Myc regulates hnRNPU expression at the transcriptional level, and meanwhile, hnRNPU stabilizes the mRNA of c-MYC. We found that the hnRNPU and c-Myc regulatory loop exerts a synergistic effect on the proliferation and self-renewal of HCC, and promotes the HCC progression. Taken together, hnRNPU functions as a novel transcriptional target of c-Myc and promotes HCC progression, which may become a promising target for the treatment of c-Myc-driven HCC.
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Apoptosis/fisiología , Carcinoma Hepatocelular/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo U/fisiología , Neoplasias Hepáticas/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Humanos , Ratones Endogámicos NOD , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays. RESULTS: The erythroid cells produced from hEBs could differentiate into enucleated cells and expressed globins in a time-dependent manner. They expressed not only embryonic globins but also the adult-globin with the maturation of the erythroid cells. In addition, our data showed that the hEBs-derived erythroid cells were able to act as oxygen carriers, indicating that hESCs could generate functional mature erythroid cells. CONCLUSION: Cell extract exposure with the addition of cytokines resulted in robust erythroid -like differentiation of hEBs and these hEBs-derived erythroid cells possessed functions similar to mature red blood cells.
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Células Madre Embrionarias/citología , Eritropoyesis/efectos de los fármacos , Extractos Hepáticos/farmacología , Feto Abortado/química , Citocinas , Eritrocitos , Expresión Génica/efectos de los fármacos , HumanosRESUMEN
Liver cancer stem cells (L-CSCs) are considered to be an important therapeutic target for hepatocellular carcinoma (HCC). This study provides a new in vitro long-term culture model for a specific subpopulation of L-CSCs enriched by cell surface markers. We combined CD13, CD133 and EpCAM to selectively enrich L-CSCs, which we then cultured in modified chemically defined medium. The enriched L-CSCs exhibited enhanced proliferation, self-renewal and long-term clonal maintenance ability as compared with non-CSCs. Compared with wild-type hepatocellular carcinoma, the expression of stemness surface markers, oncogenes, drug resistance and tumorigenicity in enriched L-CSCs was significantly increased. In summary, the subpopulation of L-CSCs still maintains cancer stem cell-related phenotypes after 14 days of culture.
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Antígeno AC133/metabolismo , Biomarcadores de Tumor/metabolismo , Antígenos CD13/metabolismo , Carcinoma Hepatocelular/patología , Molécula de Adhesión Celular Epitelial/metabolismo , Neoplasias Hepáticas/patología , Células Madre Neoplásicas/patología , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/metabolismo , Células Tumorales CultivadasRESUMEN
Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Recent evidence suggests that stem cells are localized in the microenvironment of low oxygen. We hypothesized that hypoxia could maintain the undifferentiated phenotype of embryonic stem cells. We have co-cultured a human embryonic cell line with human fetal liver stromal cells (hFLSCs) feeder cells stably expressing hypoxia-inducible factor-1 alpha (HIF-1alpha), which is known as the key transcription factor in hypoxia. The results suggested HIF-1alpha was critical for preventing differentiation of hES cells in culture. Consistent with this observation, hypoxia upregulated the expression of Nanog and Oct-4, the key factors expressed in undifferentiated stem cells. We further demonstrated that HIF-1alpha could upregulate the expression of some soluble factors including bFGF and SDF-1alpha, which are released into the microenvironment to maintain the undifferentiated status of hES cells. This suggests that the targets of HIF-1alpha are secreted soluble factors rather than a cell-cell contact mechanism, and defines an important mechanism for the inhibition of hESCs differentiation by hypoxia. Our findings developed a transgene feeder co-culture system and will provide a more reliable alternative for future therapeutic applications of hES cells.
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Células Madre Embrionarias/citología , Feto/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hígado/citología , Células Madre Pluripotentes/citología , Células del Estroma/citología , Células del Estroma/metabolismo , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Diferenciación Celular , Proliferación Celular , Forma de la Célula , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Cariotipificación , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , SolubilidadRESUMEN
OBJECTIVE: In order to explore the regulating effects of Egr-1 promoter sequences in transcriptional targeting by 5-fluorouracil (5-Fu) on the expression of hematopoietic growth factor genes. METHODS: The human GM-CSF cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES and then inserted into the expression vector pCIneo under control of the Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin(TM). The transfected cell clones (HFCL/EG) have been selected by the addition of G418. The cells are exposed to the clinically important anticancer agent 5-fluorouracil. The activity of EGFP in HFCL/EG cells was detected by flow cytometry. The post-chemotherapeutical expression of GM-CSF in HFCL/EG was confirmed with ELISA and Western blot and RT-PCR respectively. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production post-exposure to 5-Fu was examined. The HFCL/EG cells were transplanted intravenously into B16 melanoma in C. B-17 combined immunodeficient (SCID) mice. 5-Fu was given i.p. at Day 3. The white blood cell number in peripheral blood, the expression of EGFP and GM-CSF and in human stromal cell engrafted in recipient mice were detected by flow cytometry and RT-PCR respectively. Tumor volume in tumor-bearing mice was calculated. RESULTS: The results indicated that the activity of EGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to 5-Fu increased as compared to non-5-Fu group with flow cytometry, RT-PCR and ELISA respectively. N-acetylcysteine significantly decreased the concentration of GM-CSF in HFCL/EG cells treated with 5-FU. In contrast to two control groups, HFCL/EG (Egr-1 regulatory element-derived expression of GM-CSF gene therapy) resulted in a proportionally obvious increase in the number of white blood cell after chemotherapy and no significant difference was found for tumor inhibition in recipient mice. CONCLUSIONS: These in vitro data provide an experimental basis for use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from 5-Fu-injury effects.
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Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Fluorouracilo/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Hematopoyesis , Regiones Promotoras Genéticas , Animales , Células de la Médula Ósea/efectos de los fármacos , Línea Celular , Femenino , Expresión Génica , Vectores Genéticos , Humanos , Ratones , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
OBJECTIVE: To investigate the effects of different culture conditions on the isolation and expansion of stem cells from second-trimester amniotic fluids. METHODS: Amniotic fluids were obtained from 15 pregnant women undergone amniocenteses for medical indications between 16 - 24 gestation weeks by transabdominal amniocenteses from September 2007 to June 2008. Amniotic fluids (10 - 20 ml) samples were collected and each was cultured under different conditions or groups. (1) Low-glucose DMEM (LD) medium supplemented with 10% of fetal bovine serum (group of 10%FBS); (2) LD medium with 20% of FBS (group of 20%FBS); (3) LD medium with 15% of FBS and 4 ng/ml of basic fibroblast growth factor (group of bFGF); (4) LD medium with 10% of FBS as well as the culture plate coated with gelatin (group of gelatin). The effects of different conditions were evaluated by comparing the number of primary colonies, the cell morphology and the ability of expansion. The isolated stem cells were identified by flow cytometry, RT-PCR and differentiation ability to adipocyte. RESULTS: (1) The success rates of primary culture of the group of 10%FBS, 20%FBS, bFGF and gelatin were 60%, 73%, 73% and 60% respectively (P > 0.05). The numbers of colonies were 0.9 +/- 0.5, 2.6 +/- 1.5, 2.9 +/- 1.5, 1.1 +/- 0.8 (P < 0.01 when group of 10%FBS and gelatin compared with group of 20%FBS and bFGF); among the primary colonies, fibroblast-like colonies accounted for 46%, 49%, 64%, 44% respectively (P > 0.05). (2) The second passage cells obtained from all of these four groups could differentiate into adipocyte after induction. (3) In the group of bFGF, stem cells were isolated from 5 samples and expanded to nearly 10(7) cells after 5 passages (P < 0.01 compared with other groups). (4) Karyotype were normal in all samples. (5) Stem cells from bFGF group showed positive expression of SSEA-4, Oct-4 and Nanog gene detected by flow cytometry and RT-PCR. CONCLUSION: Stem cells can be isolated from second-trimester amniotic fluids; moderate serum concentration and supplementation of bFGF can improve the efficiency of isolation and expansion of amniotic fluid of stem cells.
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Líquido Amniótico/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre/citología , Separación Celular/métodos , Células Cultivadas , Medios de Cultivo/química , Femenino , Citometría de Flujo , Humanos , Embarazo , Segundo Trimestre del Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y Etiquetado , Células Madre/efectos de los fármacosRESUMEN
OBJECTIVE: To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. METHODS: hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined. RESULTS: The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. CONCLUSION: hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.
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Tejido Adiposo/citología , Diferenciación Celular/efectos de los fármacos , Hepatocitos/citología , Células Madre Mesenquimatosas/citología , Albúminas/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Separación Celular , Células Cultivadas , Medios de Cultivo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/metabolismo , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa-Fetoproteínas/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) has a high mortality rate due to the lack of effective treatments and drugs. Arsenic trioxide (ATO), which has been proved to successfully treat acute promyelocytic leukemia (APL), was recently reported to show therapeutic potential in solid tumors including HCC. However, its anticancer mechanisms in HCC still need further investigation. In this study, we demonstrated that ATO inhibits tumorigenesis and distant metastasis in mouse models, corresponding with a prolonged mice survival time. Also, ATO was found to significantly decrease the cancer stem cell (CSC)-associated traits. Minichromosome maintenance protein (MCM) 7 was further identified to be a potential target suppressed dramatically by ATO, of which protein expression is increased in patients and significantly correlated with tumor size, cellular differentiation, portal venous emboli, and poor patient survival. Moreover, MCM7 knockdown recapitulates the effects of ATO on CSCs and metastasis, while ectopic expression of MCM7 abolishes them. Mechanistically, our results suggested that ATO suppresses MCM7 transcription by targeting serum response factor (SRF)/MCM7 complex, which functions as an important transcriptional regulator modulating MCM7 expression. Taken together, our findings highlight the importance of ATO in the treatment of solid tumors. The identification of SRF/MCM7 complex as a target of ATO provides new insights into ATO's mechanism, which may benefit the appropriate use of this agent in the treatment of HCC.
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Antineoplásicos/farmacología , Trióxido de Arsénico/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Células Madre Neoplásicas/metabolismo , Factor de Respuesta Sérica/metabolismo , Animales , Antineoplásicos/uso terapéutico , Trióxido de Arsénico/uso terapéutico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/secundario , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Ontología de Genes , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/mortalidad , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Células Madre Neoplásicas/efectos de los fármacos , Pronóstico , Factor de Respuesta Sérica/antagonistas & inhibidores , Factor de Respuesta Sérica/genética , Trasplante HeterólogoRESUMEN
OBJECTIVE: To evaluate the effects of human mesenchymal stem cells (hMSCs) transplantation via portal vein to treat acute liver injury in mice induced with acetaminophen. METHODS: A model of acute liver injury was established by acetaminophen gavage with a dose of 500 mg/kg. Twenty severe combined immune deficient mice (SCID mice) were randomly divided into 2 groups; one with hMSCs transplantation via their portal veins, the other group served as controls and only saline was infused into their veins. Liver function tests, fluorescein staining and reticular fiber staining of liver histological preparations and fluorescence- and light-microscopy were applied to observe the biochemical and pathological changes in the mice before and after the transplantation of hMSCs. RESULTS: Liver function of the hMSCs group was significantly better than that of the controls (P less than 0.05). Fluorescence microscopy revealed that the hMSCs appeared in the areas of the periportal veins at first and then extended to the central vein areas; the reticular fiber staining indicated that hMSCs could repair the architecture of the hepatic acini. No prominent fibrosis and pseudolobules were found. CONCLUSIONS: hMSCs transplantation via portal vein to SCID mice with acute liver injury induced by acetaminophen can improve their liver function effectively; hMSCs growth in their livers and acinus reconstruction can be affected. We think it is a good method to treat acute liver injury.