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In order to clarify the effect of intestinal flora on the absorption and metabolism of paeoniflorin in vivo, the metabolism of paeoniflorin by rat intestinal flora was studied under the in vitro anaerobic condition. Paeoniflorin was incubated with rat anaerobic intestinal flora for 48 h, and UPLC was used to detect the changes of paeoniflorin at different incubation time points under the following chromatographic conditionsï¼WelchromTM C18 chromatographic column (4.6 mm×100 mm, 5 µm), with 0.1% formic acid(A)-acetonitrile(B) as the mobile phase for gradient elution. The flow rate was 0.4 mLâ¢min⻹, and column temperature was 30 â. UPLC-Q-TOF-MS with positive ion mode(ESI ion source) was applied to investigate the structural characterization of metabolic products. The structures of the metabolites were identified by accurate molecular weight, TOF-MS/MS fragmentation information, combined with retention time and literature data review, and the intestinal metabolic rules were then analyzed. After incubation for 24 h, the paeoniflorin was metabolized completely, and the resulting metabolites(albiflorin, albiflorinaglycone, deacylate albiflorin, deacylate albiflorin aglycone and paeonilactone-B) were detected in rat intestinal flora. The metabolic pathway analysis showed that the isolated rat intestinal flora first transformed peoniflorin into albiflorin, and then further metabolized by glucose removal, phenyl group removal, or four-membered ring pyrolysis and rearrangement. Paeoniflorin was gradually transformed into more hydrophobic metabolites with smaller molecular mass, which were better absorbed by the intestinal tract.
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Medicamentos Herbarios Chinos/metabolismo , Microbioma Gastrointestinal , Glucósidos/metabolismo , Monoterpenos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Ratas , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: During the process of tissue remodeling in human tumor transplantation models, the roles of the inoculated tumor cells and host tissue in tumor progression is still largely unknown. The aim of this study was to investigate the relationships and interactions between these two sides using GFP-RFP double fluorescence tracing technique. METHODS: Red fluorescence protein (RFP) gene was stably transfected into glioma stem cell line SU3, then SU3-RFP cells were transplanted into the brain of athymic nude mice with green fluorescence protein (GFP) expression. After the intracerebral tumors were formed, the relationship and interaction between GFP cells and RFP cells were analyzed. Highly proliferative GFP cells were screened out, and monocloned with micro-pipetting. DNA content assay, chromosome banding and carcinogenicity test of the GFP cells were performed to observe the GFP cells' cancerous phenotype in nude mice. RESULTS: In the transplantable tumor tissue, besides a great quantity of RFP cells, there were still a proportion of GFP cells and GFP/RFP fusion cells. The proportion of RFP cells, GFP cells and GFP/RFP cells were (88.99 ± 1.46)%, (5.59 ± 1.00)%, and (4.11 ± 1.020)%, respectively. Two monoclonal host GFP cells (H1 and H9) were cloned, which demonstrated the properties of immortality, loss of contact inhibition, and ultra-tetraploid when cultured in vitro. Both H1 and H9 cells expressed CNP, a specific marker of oligodendrocytes. The GFP cells also demonstrated 100% tumorigenic rate and high invasive properties in vivo. CONCLUSIONS: In this glioma transplantation model, the transplanted tumor tissues contained not only transplanted glioma stem cells but also cancerous host GFP cells. Our findings offer important clues to further research on the relationships among different members in the tumor microenvironment.
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Encéfalo/citología , Transformación Celular Neoplásica , Glioma/patología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Células Madre Neoplásicas/citología , Neuroglía/citología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Animales , Encéfalo/metabolismo , Comunicación Celular , Línea Celular Tumoral , Glioma/metabolismo , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuroglía/metabolismo , Transfección , Microambiente Tumoral , Proteína Fluorescente RojaRESUMEN
OBJECTIVE: To explore the influences of total intravenous anesthesia (TIVA) on the feasibility and success rate of monitoring the evoked potentials (EP) and examine the correlations between EP changes and clinical outcomes in intracranial aneurysm surgery. METHODS: Thirty-one patients undergoing intracranial aneurysm surgery received TIVA. TIVA was maintained with a target controlled infusion (TCI) of propofol and a continuous infusion of remifentanyl. The bilateral SEP (somatosensory evoked potential) and MEP (motor evoked potentials) were monitored intra-operatively. And the changes of evoked potential and success rate were recorded. The preoperative and postoperative neurological outcomes and radiological manifestations were compared. RESULTS: Bilateral SEP was detected in all cases. And the control side MEP was unsuccessfully monitored. Three patients had postoperative neurological deficits. The amplitudes of MEP and SEP declined simultaneously and failed to revert back to the baseline levels in 1 case. There was postoperative hemiplegia. And computed tomography showed multiple sites of ischemic brain infarction. CONCLUSION: The administration of TIVA with propofol and remifentanyl enables successful SEP and MEP monitoring during intracranial aneurysm surgery. This protocol may detect early cerebral ischemia and reduce the incidence of ischemic stroke.
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Anestesia Intravenosa , Potenciales Evocados Motores , Potenciales Evocados Somatosensoriales , Aneurisma Intracraneal/fisiopatología , Monitoreo Fisiológico , Adulto , Femenino , Humanos , Aneurisma Intracraneal/cirugía , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE: The finding of vasculogenic mimicry (VM) in many solid tumors indicates that tumor cells themselves could participate in the construction of tumor vessels. However the origin of these cells is still not fully elucidated, and whether these vessels have the ability of blood-supply is still unclear. Preliminary studies were performed to investigate whether part of tumor neovascularity is derived from tumor stem cells (TSCs) and whether TSCs-derived vessels are functional. METHODS: Transplanted glioma tissues obtained from subcutaneous and orthotopic transplantation nude mouse models were processed into paraffin sections. In order to identify the cell origin and types of tumor vessels, sections were stained with CD31, CD34, CD133, GFAP, Ki67 and HLA, respectively. CD34-PAS staining was performed as well. A part of tumor-bearing mice were perfused with activated carbon through the systemic circulation and the distribution of activated carbon was observed. RESULTS: CD34-PAS staining showed that endothelium-dependent vessels (CD34(+), PAS(+)), VM vessels (CD34(-), PAS(+)), and the MVs (CD34(+), PAS(-)) could be seen in the transplantated tumors. Activated carbon particles were observed in all three types of vessels. CD31(+) cells adherent to the luminal surface of vessel wall. CD34(+) cells distributed along the vessels as well, but morphologically were more like a transition type between tumor cells and endothelial cells. Human specific Ki67 and HLA positive cells could be seen in the tumor vessels indicating that these vessels were derived from human tumor cells. Moreover, cells of tumor vessels were proved to be constructed by human tumor cells mainly and fusion cells of host cells and tumor cells under confocal microscope. CONCLUSIONS: Three types of blood supply sources including endothelium-dependent vessels, vasculogenic mimicry (VMs) and mosaic vessels (MVs) exist in transplantation tumors of human glioma. Glioma stem and progenitor cells (GSCPs) have the potential to differentiate and transdifferentiate into VMs and MVs.
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Neoplasias Encefálicas/irrigación sanguínea , Glioma/irrigación sanguínea , Células Madre Neoplásicas/patología , Neovascularización Patológica/patología , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Encéfalo/irrigación sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Carbono/metabolismo , Carbono/farmacocinética , Línea Celular Tumoral , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/metabolismo , Glioma/patología , Glicoproteínas/metabolismo , Antígenos HLA/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Ratones , Ratones Desnudos , Microcirculación , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Neovascularización Patológica/metabolismo , Péptidos/metabolismo , Reacción del Ácido Peryódico de Schiff , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismoRESUMEN
The centrosymmetric dinuclear Cu(II) complex, [Cu(2)Cl(4)(C(24)H(21)N(5)O(4))(2)], was synthesized by the reaction of CuCl(2)·2H(2)O with the tripodal ligand 2,2'-[3-(1H-imid-azol-4-ylmeth-yl)-3-aza-pentane-1,5-di-yl]diphthalimide (L). Each of the Cu(II) ions is coordinated by two N atoms from the ligand, two bridging Cl atoms and one terminal Cl atom. The Cu(II) coordination can be best be described as a transition state between four- and five-coordination, since one of the bridging Cl atoms has a much longer Cu-Cl bond distance [2.7069â (13)â Å] than the other [2.2630â (12)â Å]. In addition, the Cuâ¯Cu distance is 3.622â (1)â Å. The three-dimensional structrure is generated by N-Hâ¯O, C-Hâ¯O and C-Hâ¯Cl hydrogen bonds and π-π inter-actions [centroid-centroid distances = 3.658â (4) and 4.020â (4)â Å].
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BACKGROUND: Gliomas are the most common and aggressive primary brain tumors for which unfortunately no effective treatment modalities exist despite advances in molecular biology as the knowledge base to unravel the extremely complex molecular mechanisms of tumorigenesis is limited. In this study an attempt has been made to understand the molecular pathological basis of tumorigenesis which led to an identification of an oncogene, CDC2, and an epigenetic strategy has been evaluated to control the tumorigensis by downregulating this oncogene. METHODS: Tissue microarrays were utilized to investigate the expression of genes in a large number of tumor samples and to identify overexpressed genes which could be potentially causing tumorigenesis. Retroviral vectors expressing short hairpin RNAs (shRNAs) targeted against CDC2 were designed and transducted into human glioma cell line ex vivo in order to downregulate the expression of CDC2. Real-Time PCR was used to determine the level of CDC2 mRNA. Western Blotting was used to determine the level of expression of CDC2 protein as measure to quantify down regulation of CDC2 expression along with use of flow cytometry to investigate effect of shRNAs on cell cycles and detection of apoptosis. Following ex vivo study, viral particles containing small interfering RNA for CDC2 were subsequently injected into xenogeneic graft tumor of nude mice and the weight of human glioma xenografts, survival and resulting phenotypic changes of target gene were investigated. RESULTS: Human glioma tissue microarrays indicated the positive expression rates of CDC2/CyclinB1 with a positive correlation with pathologic grades (r = 0.982, r = 0.959, respectively). Retroviral vectors expressing short hairpin RNAs (shRNAs) against CDC2 caused efficient deletion of CDC2, cellular G2/M arrest concluding in apoptosis and inhibition of proliferation in human glioma cells U251 and SHG-44 cell lines ex vivo. And the viral particles containing small interfering RNA for CDC2 were subsequently injected into subcutaneous and intracranial xenogeneic graft tuomrs of nude mice. For subcutaneous tumors, injection of CDC2-shRNA retroviruses significantly decreased tumor weight and volume compared with control. Immunohistochemistry indicated that CDC2 are negative and TUNEL are positive in tumors treated with recombinant retrovirus. For mice implanted with intracranial gliomas, treatment of CDC2-shRNA retroviruses increased survival times compared with control. CONCLUSION: CDC2 gene plays an important role in the proliferation of human gliomas. Downregulation of CDC2 could potentialy inhibit human gliomas cells growth ex vivo and in vivo. From these results, it was suggested that CDC2 might be a potential target on gene therapy of human gliomas.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Ciclina B/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Adulto , Animales , Apoptosis , Proteína Quinasa CDC2 , Ciclo Celular/genética , División Celular/genética , Línea Celular Tumoral , Ciclina B1 , Quinasas Ciclina-Dependientes , Regulación hacia Abajo , Femenino , Terapia Genética , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
BACKGROUND: Despite the advances made during decades of research, the mechanisms by which glioma is initiated and established remain elusive. The discovery of glioma stem cells (GSCs) may help to elucidate the processes of gliomagenesis with respect to their phenotype, differentiation and tumorigenic capacity during initiation and progression. Research on GSCs is still in its infancy, so no definitive conclusions about their role can yet be drawn. To understand the biology of GSCs fully, it is highly desirable to establish permanent and biologically stable GSC lines. METHODS: In the current study, GSCs were isolated from surgical specimens of primary and recurrent glioma in a patient whose malignancy had progressed during the previous six months. The GSCs were cryopreserved and resuscitated periodically during long-term maintenance to establish glioma stem/progenitor cell (GSPC) lines, which were characterized by immunofluorescence, flow cytometry and transmission electronic microscopy. The primary and recurrent GSPC lines were also compared in terms of in vivo tumorigenicity and invasiveness. Molecular genetic differences between the two lines were identified by array-based comparative genomic hybridization and further validated by real-time PCR. RESULTS: Two GSPC lines, SU-1 (primary) and SU-2 (recurrent), were maintained in vitro for more than 44 months and 38 months respectively. Generally, the potentials for proliferation, self-renewal and multi-differentiation remained relatively stable even after a prolonged series of alternating episodes of cryopreservation and resuscitation. Intracranial transplantation of SU-1 cells produced relatively less invasive tumor mass in athymic nude mice, while SU-2 cells led to much more diffuse and aggressive lesions strikingly recapitulated their original tumors. Neither SU-1 nor SU-2 cells reached the terminal differentiation stage under conditions that would induce terminal differentiation in neural stem cells. The differentiation of most of the tumor cells seemed to be blocked at the progenitor cell phase: most of them expressed nestin but only a few co-expressed differentiation markers. Transmission electron microscopy showed that GSCs were at a primitive stage of differentiation with low autophagic activity. Array-based comparative genomic hybridization revealed genetic alterations common to both SU-1 and SU-2, including amplification of the oncogene EGFR and deletion of the tumor suppressor PTEN, while some genetic alterations such as amplification of MTA1 (metastasis associated gene 1) only occurred in SU-2. CONCLUSION: The GSPC lines SU-1 and SU-2 faithfully retained the characteristics of their original tumors and provide a reliable resource for investigating the mechanisms of formation and recurrence of human gliomas with progressive malignancy. Such investigations may eventually have major impacts on the understanding and treatment of gliomas.
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Neoplasias Encefálicas/patología , Glioma/patología , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/patología , Antígeno AC133 , Animales , Antígenos CD/biosíntesis , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Hibridación Genómica Comparativa , Progresión de la Enfermedad , Ependimoma/genética , Ependimoma/metabolismo , Ependimoma/patología , Femenino , Citometría de Flujo , Dosificación de Gen , Glioma/genética , Glioma/metabolismo , Glicoproteínas/biosíntesis , Humanos , Ratones , Ratones Desnudos , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Trasplante de Neoplasias , Péptidos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
OBJECTIVE: It is well known that glioma stem cells-progenitors (GSCP) proliferate indefinitely and hardly differentiate in vitro, however, the reasons remain unknown. The aim of this study was to explore the ultrastructural basis of GSCP. METHODS: GSCP, kept by our laboratory, were collected, embedded, and cut into ultrathin sections and observed under the transmission electron microscope. RESULTS: A single GSCP usually had relatively well developed mitochondria, Golgi apparatuses, ribosomes, and undeveloped rough endoplasmic reticulum, but seldom lysosomes and no typical autophagosomes were found, and the nuclear-cytoplasmic ratio was high. The nuclei frequently contained huge amounts of euchromatin and a small quantity of heterochromatin, and in most nuclei there were only one nucleolus, however, two or more nucleoli were also common. Typical apoptotic cells could hardly be found in tumor-spheres, and between neighboring cells in tumor-spheres there were incompletely developed desmosomes or intermediate junction. CONCLUSION: The ultrastructural features of glioma stem cells-progenitors showed that BTSCP were very primitive and the lack of autophagy and the underdevelopment of some other cellular organelles are probably the reasons for the differential inhibition of GSCPs.
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Neoplasias Encefálicas/ultraestructura , Glioma/ultraestructura , Células Madre Neoplásicas/ultraestructura , Línea Celular Tumoral , Membrana Celular/ultraestructura , Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Citoplasma/ultraestructura , Humanos , Uniones Intercelulares/ultraestructura , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructuraRESUMEN
OBJECTIVE: To determine the expression of SV40Tag, Rb and IRS-1 in gliomas and to identify their function in gliomagenesis and progression. METHODS: Tissue microarrays were constructed containing 118 samples including human glioma and meningioma, experimental glioma, and normal human brain tissue. The expression of SV40Tag, Rb, IRS-1, SV40Tag combined with Rb, and SV40Tag combined with IRS-1 were assayed by immunofluorescence or immunohistochemical techniques. The expression ratio and level were analyzed. RESULTS: The expressions of SV40Tag, Rb and IRS-1 were detected in gliomas and benign brain tumors. Their positive expression rate in glioma was 65.9%, 64.6% and 48.8%, respectively, with a statistically non-significant difference between the malignant and benign brain tumors. The malignant degree was positively correlated with SV40Tag and IRS-1, but negatively correlated with Rb expression. The combined expression rate of SV40Tag and Rb was 51.2%, and the combined expression rate of SV40Tag and IRS-1 was 40.2%. In the normal human brain tissue only the expression of Rb (77.8%, 7/9) and IRS-1 (22.2%, 2/9) were detected, but expression of SV40Tag could not be observed. CONCLUSION: Our findings that no expression of SV40Tag was observed in normal human brain tissue indicates that expression of SV40Tag may play an important role in the pathogenesis of glioma. It may be assumed that after SV40 virus invading human body, Rb disfunction and IRS-1 activation promote the malignant transformation of cells, which could be one of important factors in pathogenesis and procession of glioms.
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Antígenos Transformadores de Poliomavirus/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteína de Retinoblastoma/metabolismo , Adolescente , Adulto , Anciano , Animales , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Niño , Preescolar , Femenino , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Humanos , Masculino , Meningioma/metabolismo , Meningioma/patología , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Ratas , Ratas Sprague-Dawley , Análisis de Matrices Tisulares , Adulto JovenRESUMEN
In the centrosymmetric title compound, C(30)H(28)N(6)O(2), the dihedral angles between the anti-pyrine ring and the terminal phenyl and central benzene rings are 50.55â (10) and 14.62â (9)°, respectively. Some short inter-molecular C-Hâ¯O inter-actions may help to establish the packing. An intramolecular C-Hâ¯O hydrogen bond is also present.
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OBJECTIVES: To investigate the possibility of transplantation of human glioma stem cells (HGSCs) in nude mice stably expressing green fluorescent protein (GFP) so as to clearly identify the incubated HGSCs from the host tissues. METHODS: Transgenic C57BL/6J mice expressing GFP was crossed with nude mice of the line NC, then hairless male nude mice expressing GFP were crossed with hairy female pubescent mice to obtain nude mice with GFP expression the expression of GFP in the skin and organs of these nude mice were evaluated by naked eyes, and immunohistochemical and immunofluorescence assays. HGSCs were transplanted orthotopically into the caudate nuclei of nude mice expressing GFP. Immunohistochemistry was used to observe the transplanted tumor. RESULTS: The structures rich in adipose tissue of the 8th generation nude mice were dark green and the other organs were light green. However, green fluorescence was emitted from all tissues under fluorescence microscopy. Confocal fluorescence microscopy showed that the tumor cells were stained red, distinguished from the host cells distinctly in the brains bearing tumor transplanted orthotopically. CONCLUSION: Nude mice expressing GFP can be obtained by crossing the transgenic mice bearing naive immunity with nude mice. Orthotopic transplantation of HGSCs may be used in the investigation of tumor tissue reconstitution because of the easy identification between the transplantation tumor and host tissue.
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Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Células Madre Neoplásicas/trasplante , Animales , Neoplasias Encefálicas/patología , Femenino , Glioma/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: Our previous cDNA array data have shown that expression level of CDK1 increased along with the malignant progression of ganglioglioma, and decreased with the differentiation process of neural stem cells. The purpose of this study was to investigate the CDK1 expression levels in gliomas and the effects of CDK1 knockdown on phenotype of glioma cells. METHODS: Glioma tissue array was constructed, which was composed of surgical specimens of gliomas with different malignancy grades, glioma xenografts in nude mice, cellular spheroids of brain tumor stem cells, normal neural stem cells and glioma cell line. CDK1 expression was detected in glioma tissue array with immunohistochemical techniques. CDK1 expression in human brain glioma cell line and relevant xenogeneic graft tumor was inhibited by retroviral vectors expressing short hairpin RNAs (shRNAs). Both in vitro and in vivo changes of biological characteristics were further observed. RESULTS: The expression level of CDK1 increased along with the malignancy progression of glioma in clinical specimens. The positive expression rates of CDK1 in human brain glioma tissues were 22.2% (grade I), 40.0% (grade II), 69.6% (grade III) and 78.6% (grade IV), P = 0.01, respectively. The positive expression rate of CDK1 in glioma cell line and implanted xenografts was similar as the clinical tumors with high malignancy, and higher than those in neural stem cells and brain tumor stem cells (P = 0.0014). Expression of CDK1 was high in human fetal brain tissues and bone marrows of nude mice, but low in normal adult human brain tissues. Downregulation of CDK1 inhibited the proliferation activities notably both in SHG-44 cells in vitro and relevant xenogeneic graft tumors, and induced apoptosis of tumor cells prominantly as well. CONCLUSION: Overexpression of CDK1 may promote oncogenesis and progression of human gliomas. Downregulation of CDK1 expression can inhibit the proliferation activities of human malignant gliomas.
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Neoplasias Encefálicas/metabolismo , Proteína Quinasa CDC2/metabolismo , Diferenciación Celular/efectos de los fármacos , Silenciador del Gen , Glioma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias del Tronco Encefálico/metabolismo , Proteína Quinasa CDC2/genética , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ganglioglioma/genética , Ganglioglioma/metabolismo , Ganglioglioma/patología , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Humanos , Ratones , Ratones Desnudos , Estadificación de Neoplasias , Trasplante de Neoplasias , ARN Mensajero/metabolismoRESUMEN
OBJECTIVE: To investigate the target cells and molecules with sodium valproate induced differentiation of human glioma cells. METHODS: Nude mice bearing human glioma xenogenic graft subcutaneously were treated with sodium valproate. The expressions of HDAC1 and Tob genes of xenografts were analyzed with semiquantitative RT-PCR. The CD133+ cells (BTSCs) were isolated from glioma specimens by immunomagnetic sorting, and cultured in the medium containing FCS or in the serum-free medium supplemented with growth factors, respectively, followed by treatment with sodium valproate in vitro for 21 days. The cell surface markers were detected with flow cytometry and confocal microscopy. RESULTS: Sodium valproate inhibited the growth of subcutaneous xenografs bearing on nude mice (P<0.05), and up-regulated the HDAC1 expression (P<0.01), down-regulated the Tob expression (P<0.05). The cell surface markers of BTSCs were detected by flow cytometry after sodium valproate treatment for 21 days. In the FCS group, the GFAP or beta-tubulin III positive cells increased significantly (P<0.01), but in the growth factor group, no statistical differences were observed in the GFAP or beta-tubulin III expression (P>0.05). The results of confocal microscopy indicated that the GFAP+ or beta-tubulin III+ cells coexpressed with Nestin. CONCLUSIONS: HDAC1 and Tob genes were the potential target molecules in reversion of the differential inhibition of human glioma cells with sodium valproate. The BTSCs undergoing the processes of differentiation were the target cells for sodium valproate.
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Neoplasias Encefálicas/prevención & control , Diferenciación Celular/efectos de los fármacos , Glioma/prevención & control , Ácido Valproico/farmacología , Antígeno AC133 , Actinas/análisis , Animales , Antígenos CD/análisis , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/análisis , Glioma/metabolismo , Glioma/patología , Glicoproteínas/análisis , Histona Desacetilasas/genética , Humanos , Proteínas de Filamentos Intermediarios/análisis , Ratones , Ratones Desnudos , Microscopía Confocal , Proteínas del Tejido Nervioso/análisis , Nestina , Péptidos/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Research on human glioma stem cells began early in the 21st century and since then has become a rapidly growing research field with the number of publications increasing year by year. The research conducted by our diverse group of investigators focused primarily on cell culture techniques, molecular regulation, signaling pathways, cancer treatment, the stem cell microenvironment and the cellular origin and function of glioma stem cells. In particular, we put forward our view that there are inverse or forward transformations among neural stem cells, glial cells and glioma stem cells in glioma tissues under certain conditions. Based on the background of the progress of international research on human glioma stem cells, we aim to share our progress and current findings of human glioma stem cell research in China with colleagues around the world.
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OBJECTIVE: To isolate and culture tumor stem cells from glioma tissues obtained at surgical operation and to study their biological characteristics. METHODS: Glioma tissues obtained from surgically resected specimens of 8 patients were fully chopped, trypsinized, and filtered to prepare single cell suspensions. The cells were cultured in serum-free medium with EGF, LIF and bFGF. CD133(+) cells were purified by magnetic cell sorting, and cultured continuously in vitro to obtain tumor cell spheres. Tumor stem cells of the 5th passage were induced to differentiate with 10% FBS, and expression of cell differentiation markers such as Nestin, MAP2, GFAP was evaluated with immunocytochemistry techniques. RESULTS: CD133(+) cells were successfully separated and cultured from one anasplastic mixed astrocyte-ependymocyte type glioma specimen. These cells maintained a sphere-like growth status in vitro (3 months, 14 passages), and can self-renew, proliferate and conditionally differentiate into MAP2(+) and GFAP(+) cells. However, CD133(-) cells did not possess these properties. CONCLUSION: Glioma tissue contains tumor stem cells. Those cells can be cultured and passaged in vitro for a long term, and therefore to offer new approaches for studying cellular and molecular biology of glioma.
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Neoplasias Encefálicas/patología , Glioma/patología , Células Madre Neoplásicas/citología , Antígeno AC133 , Antígenos CD/metabolismo , Diferenciación Celular , Proliferación Celular , Separación Celular , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/metabolismo , Glicoproteínas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Células Madre Neoplásicas/metabolismo , Péptidos/metabolismoRESUMEN
OBJECTIVE: To pursue the changes of cell morphology, expression of differentiation related markers, and proliferation cycles of brain tumor stem cells (BTSCs) after differentiation in vitro. METHODS: Tumor stem cells of the line CD133(+) were obtained from two specimens from one clinical case with anaplasia ependymocytoma during operation, one specimen being obtained during the first operation and then second specimen being obtained during the second operation 6 months later on the recurrent tumor. CD133(+) cells were acquired by using magnetic sorting and then cultured to differentiate in medium containing 10% fetal bovine serum. The morphology of the cells was observed under phase contrast microscope. Cells were collected respectively before differentiation and 3, 7, 10, and 21 days after the differentiation. The cell surface markers such as CD133, nestin, glial fibrillary acidic protein (GFAP), and beta-tubulin III were detected with flow cytometry. Proliferation cycles were examined before differentiation and in the 7th day after differentiation. Normal neural stem cells (NSCs) obtained from fetal brain tissues were used as controls. RESULTS: (1) The BTSCs were round shape at the beginning, then changed to short fusiform, polygon and long fusiform. Seven days later the cells reversed to short fusiform and round shape. The cells accumulated into cell spheres and floated in the culture medium again. While the NSCs differentiated along their routine rules. (2) Both the undifferentiated BTSCs and NSCs showed high level expression of CD133 and nestin. After differentiation the BTSCs expressed CD133 and nestin, the expression levels decreased first and then increased. The expression rates of CD133 and nestin were (3.65 +/- 0.17)% and (28.99 +/- 1.26)% in the 7th day, (14.63 +/- 1.16)% and (45.46 +/- 1.27)% in the 21st day. While the positive expression rate of GFAP was higher than that of beta-tubulin III. In the 10th day the NSCs under differentiation lost the expression of CD133 and nestin. The percentage of GFAP positive cells and beta-tubulin III positive cells were (88.94 +/- 1.23)% and (11.94 +/- 0.36)% respectively. (3) All undifferentiated BTSCs were hypodiploid. After differentiation majority of the BTSCs were hypodiploid or hyperdiploid, The percentages of S phase and G(2)-M phase cells in the BTSCs were higher than that in the NSCs. The cell composition of recrudescent BTSCs was more complex than that of the primary BTSCs. All NSCs were diploid whether differentiated or not. Most of the NSCs were G(0)-G(1) phase cells. CONCLUSION: The differentiation direction of BTSCs is quietly different from that of the NSCs. There is an obvious dysdifferentiation in BTSCs.
Asunto(s)
Neoplasias Encefálicas/patología , Células Madre Neoplásicas/patología , Animales , Diferenciación Celular , Proliferación Celular , Cabras , Humanos , Ratones , Neuronas/citologíaRESUMEN
OBJECTIVE: To establish malignant progression associated gene expression profiles in human brain glioma. METHODS: The primary (WHO grade II), recurrent (WHO grade III) and re-recurrent (WHO grade IV) glioma specimens were sequentially collected from one single patient. Gene expression of different tumor specimens and normal brain tissue of the same patient was compared by microarrary techniques. RESULTS: 197 differentially expressed genes with differential ratio > or = 3 were observed when compared with normal brain tissue. When the specimens (3 tumor, 1 normal brain) were paired with each other, 7 groups containing 489 genes (upregulated 193, downregulated 296) were observed. According to the descending frequency of the 109 genes with known function, they were the genes associated with development, metabolism, differentiation, signal transduction, DNA binding transcription, cellular receptor, immunity, ion-channel transportation, protein translation, cell backbone motion, stress, protooncogene and anti-oncogene and cell apoptosis, respectively. CONCLUSION: From the 197 differentially expressed genes found in one glioma patient experiencing tumor malignant progression, 17 genes screened out by bioinformatics assay, may offer valuable information on molecular mechanisms on genesis and malignant progression of glioma.
Asunto(s)
Neoplasias Encefálicas/patología , Perfilación de la Expresión Génica , Glioma/patología , Neoplasias Encefálicas/genética , Glioma/genética , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The expression of RNA polymerase II subunit 3 (Rpb3) was found frequent up-regulation in Hepatocellular carcinoma (HCC) tumors. Significant associations could also be drawn between increased expressions of Rpb3 and advance HCC staging and shorter disease-free survival of patients. Overexpression of Rpb3 increased HCC cell proliferation, migratory rate and tumor growth in nude mice, whereas suppression of Rpb3 using shRNA inhibited these effects. For mechanism study, we found that Rpb3 bound directly to Snail, downregulated E-cadherin, induced HCC cells epithelial-mesenchymal transition (EMT). In particular, N-terminus of Rpb3 blocked Rpb3 binding to Snail, inhibited Rpb3-high-expression HCC cells proliferation, migration, tumor growth in nude mice, and also inhibited DEN-induced liver tumorigenesis. Furthermore, N-terminus of Rpb3 did not inhibit normal liver cells or Rpb3-low-expression HCC cells proliferation. These findings suggest that N-terminus of Rpb3 selectively inhibits Rpb3-high-expression HCC cells proliferation. N-terminus of Rpb3 may be useful in treating patients diagnosed with Rpb3-high-expression HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fragmentos de Péptidos/farmacología , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Animales , Cadherinas/biosíntesis , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Fragmentos de Péptidos/genética , Unión Proteica/genética , Interferencia de ARN , ARN Polimerasa II/biosíntesis , ARN Polimerasa II/genética , ARN Interferente Pequeño , Factores de Transcripción de la Familia SnailRESUMEN
Due to progress in the research of glioma stem cells and the glioma niche, development of an animal model that facilitates the elucidation of the roles of the host tissue and cells is necessary. The aim of the present study was to develop a subcutaneous xenograft green fluorescent protein nude mouse model and use this model to analyze the roles of host cells in tumor necrosis repair. Tumors derived from the human glioma stem/progenitor cell line SU3 were subcutaneously implanted in green fluorescent protein nude mice. The implanted tumors were then passed from animal to animal for 10 generations. Finally, subcutaneous xenografts were assayed with traditional pathology, immunopathological techniques and fluorescence photography. For each generation, the tumorigenicity rate was 100%. Subcutaneous xenografts were rich in blood vessels, and necrotic and hemorrhagic foci, which highly expressed hypoxia-inducible factor-1α, tumor necrosis factor, Ki-67, CD68 and CD11b. In the interstitial tissue, particularly in old hemorrhagic foci, there were numerous cells expressing green fluorescent protein, CD68 and CD11b. Green fluorescent protein nude mouse subcutaneous xenografts not only consistently maintained the high invasiveness and tumorigenicity of glioma stem/progenitor cells, but also consisted of a high concentration of tumor blood vessels and necrotic and hemorrhagic foci. Subcutaneous xenografts also expressed high levels of tumor microenvironment-related proteins and host-derived tumor interstitial molecules. The model has significant potential for further research on tumor tissue remodeling and the tumor microenvironment.