OBJECTIVETo investigate the effects of resveratrol (RV) in reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) and the related mechanism.

METHODSPrimary MEFs were isolated from E13.5 embryos and used within three passages. Retroviruses expressing Sox2 and Oct4 were produced by transfecting GP2-293t cells with recombinant plasmids (MSCV)-Sox2 and MSCV-Oct4. Supernatants containing retroviruses were obtained after 48-hour transfection and MEFs were then infected. Different concentrations (0, 5, 10 and 20 μmol/L) of RV were added to embryonic stem cell (ESC) medium to culture MEFs 48 h post-infection. iPSC clones emerged and were further cultured. Expression of pluripotent markers of iPSCs was identified by cell immunofluorescence and reverse transcription-polymerase chain reaction. Both cytotoxicity and cell proliferation were assayed by Western blot analysis after RV was added into ESC medium. The ultrastructure change of mitochondria was observed by electron microscopy.

RESULTSMore than 2.9-fold and 1.3-fold increases in colony number were observed by treatment with RV at 5 and 10 μmol/L, respectively. The reprogramming efficiency was significantly decreased by treatment with 20 μmol/L RV. The proliferation effect on MEFs or MEFs infected by two factors Sox2/Oct4 (2 factors-MEFs, 2F-MEFs) was investigated after RV treatment. At 20 μmol/L RV, induced cell apoptosis and proliferation inhibition were more obvious than those of 5 and 10 μmol/L treatments. Clones were selected from the 10 μmol/L RV-treated group and cultured. Green fluorescent protein expression from one typical clone was silenced one month later which expressed ESC-associated marker genes Gdf3, Nanog, Ecat1, Fgf4 and Foxd3. Electron transmission microscope showed obvious cavitations in mitochondria. The expression of hypoxia-inducible factor-1α was up-regulated when 2F-MEFs were treated with RV compared to the control group.

CONCLUSIONRV improved the efficiency of reprogramming 2F-MEFs into iPSCs at low and moderate concentrations (5 and 10 μmol/L). The effect of 10 μmol/L RV on reprogramming was much greater than that of 5 μmol/L RV. However, high concentration of RV (20 μmol/L) led to more severe cavitations in mitochondria and caused cytotoxic effects. Taken together, these findings suggest that RV mimics hypoxia in cells and promotes reprogramming at a low concentration.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; Induced Pluripotent Stem Cells ; drug effects ; Mice ; Octamer Transcription Factor-3 ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; SOXB1 Transcription Factors ; physiology ; Stilbenes ; pharmacology

OBJECTIVETo study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.

METHODSExperiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.

RESULTSThe cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.

CONCLUSIONLIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.

Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Genes, Homeobox ; Humans ; Leukemia Inhibitory Factor ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Octamer Transcription Factor-3 ; metabolism ; Organic Chemicals ; Proliferating Cell Nuclear Antigen ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Umbilical Cord ; cytology

OBJECTIVEIn order to explore the effects of lead on the growth and development of cultured hippocampal neural cells and on the expression of Oct-2, the II subtype POU domain protein.

METHODSExperiment cell model was established using primary culture of hippocampal neural cells from SD rat embryos. Target cells were exposed to lead acetate in the different concentrations, i.e. 10(-1), 10(0), 10(1), 10(2), 10(3) micromol/L, while the control group was given the same quantity of the culture medium. The immunohistochemistry method was utilized to detect the expressions of Neurofilament (NF) and Glial Fibrillary Acidic Protein (GFAP), the markers for neuron and astrocyte, respectively, and the expression of Oct-2 as well.

RESULTSThe results showed that 10 micromol/L lead acetate treatment caused diminishing of neuronal cell body and the decreases of both axon lengths and inter-cellular connections. In addition, 1 micromol/L lead acetate significantly increased the number of GFAP-positive cells compared with the control group (P < 0.05). By image analysis system, 1 micromol/L lead acetate treatment was found to induce a statistically significant increase of the positive area rate concerning Oct-2 expression in hippocampal neurons and astrocytes, while both positive area rate and integral density of light of Oct-2 expression were found to increase markedly in the groups treated by 10 micromol/L lead acetate (P < 0.01).

CONCLUSIONSLead acetate treatment may contribute to the inhibitions of both growth and differentiation of hippocampus neurons, and to the stimulation of glial cell hyperplasia simultaneously. In addition, the CNS impairments caused by lead is partly correlated with the enhancement of Oct-2 expression.

Animals ; Astrocytes ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; biosynthesis ; genetics ; Dose-Response Relationship, Drug ; Embryo, Mammalian ; Female ; Glial Fibrillary Acidic Protein ; biosynthesis ; genetics ; Hippocampus ; cytology ; metabolism ; Lead ; toxicity ; Neurofilament Proteins ; biosynthesis ; genetics ; Neurons ; cytology ; metabolism ; Octamer Transcription Factor-2 ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; biosynthesis ; genetics

Increasing evidence suggests that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. Additionally, it is becoming apparent that cyclooxygenase (COX) signaling is associated with canine mammary tumor development. The goals of the present study were to investigate COX-2 expression patterns and their effect on CSC-mediated tumor initiation in primary canine mammary tissues and tumorsphere models using immunohistochemistry. Patterns of COX-2, CD44, octamer-binding transcription factor (Oct)-3/4, and epidermal growth factor receptor (EGFR) expression were examined in malignant mammary tumor (MMT) samples and analyzed in terms of clinicopathological characteristics. COX-2 and Oct-3/4 expression was higher in MMTs compared to other histological samples with heterogeneous patterns. In MMTs, COX-2 expression correlated with tumor malignancy features. Significant associations between COX-2, CD44, and EGFR were observed in low-differentiated MMTs. Comparative analysis showed that the levels of COX-2, CD44, and Oct-3/4 expression varied significantly among TSs of three histological grades. Enhanced COX-2 staining was consistently observed in TSs. Similar levels of staining intensity were found for CD44 and Oct-3/4, but EGFR expression was weak. Our findings indicate the potential role of COX-2 in CSC-mediated tumor initiation, and suggest that COX-2 inhibition may help treat canine mammary tumors by targeting CSCs.
Animals ; Antigens, CD44/genetics/metabolism ; Biomarkers, Tumor/genetics/metabolism ; Cell Transformation, Neoplastic/*genetics/metabolism ; Cyclooxygenase 2/*genetics/metabolism ; Dog Diseases/*genetics/metabolism ; Dogs ; Female ; Immunohistochemistry/veterinary ; Mammary Neoplasms, Animal/*genetics/metabolism ; Mammary Neoplasms, Experimental/*genetics/metabolism ; Neoplastic Stem Cells/*metabolism ; Octamer Transcription Factor-3/genetics/metabolism ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Retrospective Studies

OBJECTIVETo investigate the specificity and sensitivity of Oct2 protein expression in lymphoma cells and its significance in diagnosis and classification of lymphoma.

METHODSFormalin-fixed and paraffin-embedded materials from 129 cases of lymphoma and 10 cases of reactive lymphoid hyperplasia (RLH) were studied by EnVision immunohistochemistry for Oct2 protein.

RESULTSOct2 was mainly expressed in germinal center cells of RLH. It was diffusely expressed in B-cell lymphoma cells. 97.7% cases (85/87) of B-cell lymphoma and 3.8% cases (1/26) of T-cell lymphoma were positive for Oct2 protein. In comparison, the expression rates for CD20 and CD79alpha in B-cell lymphomas were 90.8% (79/87) and 84.7% (61/72) respectively. The difference in expression rates between Oct2 protein and CD20 was not statistically significant (P > 0.05) There was, however, significant difference in expression rates between Oct2 protein and CD79alpha (P < 0.05). The expression rates of Oct2 protein in nodular lymphocyte-predominant Hodgkin lymphoma and classic Hodgkin lymphoma were 3/3 and 46.2% (6/13) respectively. The difference in expression rates of Oct2 protein in these two groups showed no statistical significance (P > 0.05).

CONCLUSIONAs a relatively sensitive and specific marker for B cells, Oct2 can serve as a useful antibody for the diagnosis and differential diagnosis of lymphoma.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD20 ; metabolism ; CD79 Antigens ; metabolism ; Child ; Diagnosis, Differential ; Female ; Germinal Center ; metabolism ; Hodgkin Disease ; diagnosis ; metabolism ; Humans ; Lymphoma ; classification ; diagnosis ; metabolism ; Lymphoma, B-Cell ; diagnosis ; metabolism ; Lymphoma, T-Cell ; diagnosis ; metabolism ; Male ; Middle Aged ; Octamer Transcription Factor-2 ; metabolism ; Pseudolymphoma ; diagnosis ; metabolism

BACKGROUNDKeratinocyte serum-free medium (K-SFM) is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells (CSCs) using K-SFM.

METHODSA K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum (FCS) was used as a control. CSCs were identified with flow cytometry using CD44(+)/CD24(-) as molecular markers. The expression of a variety of CSC markers (Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin) was analyzed with real-time PCR.

RESULTSMuch higher percentage of CSCs was achieved with K-SFM: 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin.

CONCLUSIONK-SFM is an optimal culture medium to maintain and to enrich breast CSCs.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; Cadherins ; genetics ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; Culture Media, Serum-Free ; Female ; Homeodomain Proteins ; genetics ; Humans ; Keratinocytes ; cytology ; Nanog Homeobox Protein ; Neoplasm Proteins ; genetics ; Neoplastic Stem Cells ; cytology ; metabolism ; Octamer Transcription Factor-3 ; genetics ; Real-Time Polymerase Chain Reaction

The effects of mangiferin on uric acid excretion, kidney function and related renal transporters were investigated in hyperuricemic mice induced by potassium oxonate. Mice were divided into normal control group, and 5 hyperuricemic groups with model control, 50, 100, and 200 mg x kg(-1) mangiferin, and 5 mg x kg(-1) allopurinol. Mice were administered by gavage once daily with 250 mg x kg(-1) potassium oxonate for seven consecutive days to create the model. And 3 doses of mangiferin were orally initiated on the day 1 h after potassium oxonate was given, separately. Serum uric acid, creatinine and urea nitrogon levels, as well as urinary uric acid creatinine levels were measured. Mouse uromodulin (mUMOD) levels in serum, urine and kidney were determined by ELISA method. The mRNA and protein levels of related renal transporters were assayed by RT-PCR and Western blotting methods, respectively. Compared to model group, mangiferin significantly reduced serum uric acid, creatinine and urea nitrogon levels, increased 24 h uric acid and creatinine excretion, and fractional excretion of uric acid in hyperuricemic mice, exhibiting uric acid excretion enhancement and kidney function improvement. Mangiferin was found to down-regulate mRNA and protein levels of urate transporter 1 (mURAT1) and glucose transporter 9 (mGLUT9), as well as up-regulate organic anion transporter 1 (mOAT1) in the kidney of hyperuricemic mice. These findings suggested that mangiferin might enhance uric acid excretion and in turn reduce serum uric acid level through the decrease of uric acid reabsorption and the increase of uric acid secretion in hyperuricemic mice. Moreover, mangiferin remarkably up-regulated expression levels of renal organic cation and carnitine transporters (mOCT1, mOCT2, mOCTN1 and mOCTN2), increased urine mUMOD levels, as well as decreased serum and kidney mUMOD levels in hyperuricemic mice, which might be involved in mangiferin-mediated renal protective action.
Animals ; Blood Urea Nitrogen ; Carrier Proteins ; genetics ; metabolism ; Creatinine ; blood ; Glucose Transport Proteins, Facilitative ; genetics ; metabolism ; Hyperuricemia ; blood ; chemically induced ; physiopathology ; urine ; Kidney ; metabolism ; physiopathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Octamer Transcription Factor-1 ; genetics ; metabolism ; Organic Anion Transport Protein 1 ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Organic Cation Transporter 2 ; Oxonic Acid ; Protective Agents ; pharmacology ; RNA, Messenger ; metabolism ; Random Allocation ; Solute Carrier Family 22 Member 5 ; Uric Acid ; blood ; urine ; Uromodulin ; blood ; urine ; Xanthones ; pharmacology