3T3-L1 preadipocyte were differentiated to adipocytes, and then treated with 0, 10, 20, and 40 microg/mL of peanut sprout ethanol extract (PSEE). The main component of PSEE is resveratrol which contained 5.55 mg/mL of resveratrol. The MTT assay, Oil-Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, and the triglyceride concentration were determined in 3T3-L1 cells. MMP-2 and MMP-9 activities as well as mRNA expressions of C/EBP beta and C/EBP alpha were also investigated. As the concentration of PSEE in adipocytes increased, the cell proliferation was decreased in a dose-dependent manner from 4 days of incubation (P < 0.05). The GDPH activity (P < 0.05) and the triglyceride concentration (P < 0.05) were decreased as the PSEE treatment concentration increased. The mRNA expression of C/EBPbeta in 3T3-L1 cells was significantly low in groups of PSEE-treated, compared with control group (P < 0.05). The MMP-9 (P < 0.05) and MMP-2 (P < 0.05) activities were decreased in a dose-dependent manner as the PSEE concentration increased from 20 microg/mL. In conclusion, it was found that PSEE has an effect on restricting proliferation and differentiation of adipocytes.
3T3-L1 Cells ; Adipocytes ; Animals ; Cell Proliferation ; Ethanol ; Fibroblasts ; Glycerolphosphate Dehydrogenase ; Matrix Metalloproteinases ; Mice ; RNA, Messenger ; Stilbenes

BACKGROUND: Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. METHODS: In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. RESULTS: Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. CONCLUSION: We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.
Bioreactors ; Bone Substitutes ; Calibration ; Cell Proliferation ; Fibroblasts ; Glass ; Methods ; NIH 3T3 Cells ; Perfusion ; Porosity ; Tissue Engineering

OBJECTIVETo construct and identify the stable expression system of NIH3T3 fibroblast with eukaryotic expression vector of human transforming growth factor beta3 (pcDNA3.1 (-)/TGFbeta3). So as to investigate the proliferation of NIH3T3 fibroblasts transfected with hTGFbeta3 gene stably.

METHODSThe stable transfection of NIH3T3 fibroblasts with recombinant plasmid expressing hTGFbeta3 was established by using LipofectamineTM2000 and G418 selection. The mRNA and protein expression of TGFbeta3 were detected by the RT-PCR and Western blot method, respectively. Microscope and MTT were adopted to examine the proliferation of the stable expression system of fibroblasts with hTGFbeta3.

RESULTSAfter G418 selection, RT-PCR and Western blot analysis, 7 out of 10 cell lines transfected with pcDNA3.1 (-)/TGFbeta3 expressed with very high level of TGFbeta3, as compared with vector control transfectants that showed no expression, and compared with the other cell lines that expressed relatively low level. The stable transfection of NIH3T3 fibroblasts growth slowed down significantly (P < 0.05).

CONCLUSIONThe stable expression system of NIH3T3 fibroblast with hTGFbeta3 were constructed successfully. The TGFbeta3 gene could inhibit the proliferation of NIH3T3 fibroblasts in vitro.

Animals ; Cell Proliferation ; Fibroblasts ; metabolism ; Humans ; Mice ; NIH 3T3 Cells ; Plasmids ; Transfection ; Transforming Growth Factor beta3 ; genetics ; metabolism

PURPOSE: Ascorbic acid has been reported to have an adipogenic effect on 3T3-L1 preadipocytes, while evidence also suggests that ascorbic acid reduces body weight in humans. In this study, we tested the effects of ascorbic acid on adipogenesis and the balance of lipid accumulation in ovariectomized rats, in addition to long-term culture of differentiated 3T3-L1 adipocytes. MATERIALS AND METHODS: Murine 3T3-L1 fibroblasts and ovariectomized rats were treated with ascorbic acid at various time points. In vitro adipogenesis was analyzed by Oil Red O staining, and in vivo body fat was measured by a body composition analyzer using nuclear magnetic resonance. RESULTS: When ascorbic acid was applied during an early time point in 3T3-L1 preadipocyte differentiation and after bilateral ovariectomy (OVX) in rats, adipogenesis and fat mass gain significantly increased, respectively. However, lipid accumulation in well-differentiated 3T3-L1 adipocytes showed a significant reduction when ascorbic acid was applied after differentiation (10 days after induction). Also, oral ascorbic acid administration 4 weeks after OVX in rats significantly reduced both body weight and subcutaneous fat layer. In comparison to the results of ascorbic acid, which is a well-known cofactor for an enzyme of collagen synthesis, and the antioxidant ramalin, a potent antioxidant but not a cofactor, showed only a lipolytic effect in well-differentiated 3T3-L1 adipocytes, not an adipogenic effect. CONCLUSION: Taking these results into account, we concluded that ascorbic acid has both an adipogenic effect as a cofactor of an enzymatic process and a lipolytic effect as an antioxidant.
3T3-L1 Cells ; Adipocytes/drug effects ; Adipocytes/metabolism ; Adipogenesis/drug effects ; Animals ; Antioxidants/pharmacology ; Ascorbic Acid/pharmacology ; Body Composition/drug effects ; Body Weight/drug effects ; Cell Differentiation/drug effects ; Female ; Fibroblasts/drug effects ; Fibroblasts/metabolism ; Lipolysis/drug effects ; Mice ; Ovariectomy ; Rats, Sprague-Dawley

In order to develop antitumor agent which indicates weak side effects and strong antitumor activity, cytotoxicity and antitumor effects of MG was evaluated by MTT assay and SRB assay of colorimetric assay methods on the cultured NIH 3T3 fibroblasts and human oral epithelioid carcinoma cells (KB cells). KB cells were cultured in EMEM and RPMI 1640 media containing 10% fetal bovine serum, antibiotics and fungizone. After incubation for 24 hrs, the cells were treated with MG by dose dependent manner for 48 hrs under the same condition. The MTT and SRB quantity were measured by ELISA reader (Spectra Max 250, USA). The microscopic study was carried out to observed morphological change, Ag-NORs (argyrophylic nucleolar organizer region) number and PAS positive reacton of cultured KB cells. The results were as follows; 1. The MTT50 were 16677 micrometer in NIH 3T3 cell and 65.55 micrometer in KB cells treated with MG. 2. The SRB50 were 8701.23 micrometer in NIH 3T3 cell and 168.81 micrometer in KB cells treated with MG. 3. The number of Ag-NORs was not significant in cultured NIH 3T3 cells, but the number of Ag-NORs was significantly decreased in cultured KB cells treated with MG. 4. PAS reaction of cultured NIH 3T3 cells was not changed, but PAS reaction of cultured KB cells was changed to negative reaction. 5. Morphological changes of cultured NIH 3T3 cells treated with MG was weak, but KB cell treated with MG was severe. These results indicate that MG have noncytoxicity on NIH 3T3 fibroblasts and high cytotoxicity on KB cells.
Amphotericin B ; Anti-Bacterial Agents ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; Humans ; KB Cells ; NIH 3T3 Cells ; Nucleolus Organizer Region ; Periodic Acid-Schiff Reaction

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
Animals ; Arginine ; Cell Dedifferentiation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Elongation Factor 2 Kinase/*metabolism ; Fibroblast Growth Factor 2/*metabolism ; Fibroblasts/*metabolism/pathology ; Flavonoids/pharmacology ; MAP Kinase Signaling System/drug effects/genetics ; Methylation ; Mice ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Myofibroblasts/pathology ; NIH 3T3 Cells ; Protein Methyltransferases/*metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Small Interfering/genetics

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
Animals ; Arginine ; Cell Dedifferentiation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Elongation Factor 2 Kinase/*metabolism ; Fibroblast Growth Factor 2/*metabolism ; Fibroblasts/*metabolism/pathology ; Flavonoids/pharmacology ; MAP Kinase Signaling System/drug effects/genetics ; Methylation ; Mice ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Myofibroblasts/pathology ; NIH 3T3 Cells ; Protein Methyltransferases/*metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Small Interfering/genetics

The purpose of this study was to regenerate human dental pulp tissues similar to native pulp tissues. Using the mixture of type I collagen solution, primary cells collected from the different tissues (pulp, gingiva, and skin) and NIH 3T3 (1 x 10(5) cells/ml/well) were cultured at 12-well plate at 37degrees C for 14 days. Standardized photographs were taken with digital camera during 14 days and the diameter of the contracted collagen gel matrix was measured and statistically analyzed with student t-test. As one of the pulp tissue engineering, normal human dental pulp tissue and collagen gel matrix cultured with dental pulp cells for 14 days were fixed and stained with Hematoxyline & Eosin. According to this study, the results were as follows: 1. The contraction of collagen gel matrix cultured with pulp cells for 14 days was significantly higher than other fibroblasts (gingiva, skin) (p < 0.05). 2. The diameter of collagen gel matrix cultured with pulp cells was reduced to 70.4% after 7 days, and 57.1% after 14 days. 3. The collagen gel without any cells did not contract, whereas the collagen gel cultured with gingiva and skin showed mild contraction after 14 days (88.1% and 87.6% respectively). 4. The contraction of the collagen gel cultured with NIH 3T3 cells after 14 days was higher than those cultured with gingival and skin fibroblasts, but it was not statistically significant (72.1%, p > 0.05). 5. The collagen gel matrix cultured with pulp cells for 14 days showed similar shape with native pulp tissue without blood vessels. This approach may provide a means of engineering a variety of other oral tissue as well and these cell behaviors may provide information needed to establish pulp tissue engineering protocols.
Blood Vessels ; Collagen ; Collagen Type I* ; Dental Pulp* ; Eosine Yellowish-(YS) ; Fibroblasts ; Gingiva ; Hematoxylin ; Humans ; NIH 3T3 Cells ; Skin ; Tissue Engineering*

PURPOSE: We investigated the molecular mechanism by which the Raf-1 kinase pathways that are linked to protein kinase C induce differential physiological effects, depending on the stimulus, by employing the pharmacological PKC activator PMA. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems and these cells were transiently transfected with the pMTH vector that encodes dominant-negative (DN) PKC-epsilon with using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. The Raf-1 kinase activity was measured by assessing the phosphorylation of recombinant MEK with using the immunoprecipitated Raf-1 proteins. The phosphorylated MEK protein bands were quantified by using Quantity One analysis software. RESULTS: The pharmacological PKC activator phorbol-12-myristate-13-acetate (PMA) and platelet-derived growth factor (PDGF) were able to induce the activation of Raf-1 kinase in the v-H-ras-transformed NIH3T3 fibroblasts. However, PMA was found to be much less sensitive PI3 kinase inhibitor or the chemical antioxidant than is PDGF. Especially, PMA mediated growth arrest while PDGF induced mitogenic signaling through the PKC-epsilon activation. Thus, the regulation of the Raf-1 cascade by both PDGF and PMA is likely to be intimately linked and they converge at the PKC level through different upstream pathways, as was shown by the inhibition of PDGF-induced Raf-1 kinase activation by the transient transfection with a dominant-negative mutant of PKC-epsilon. CONCLUSIONS: Taken together, these results imply that, depending on the stimulus, Raf-1 kinase leads to different physiological effects.
Cell Proliferation ; Fibroblasts ; Humans ; Lipids ; NIH 3T3 Cells ; Parents ; Phosphorylation ; Phosphotransferases ; Platelet-Derived Growth Factor ; Protein Kinase C ; Protein Kinases ; Proteins ; Proto-Oncogene Proteins c-raf ; Transfection

OBJECTIVETo investigate the ultrastructure and the alkaline phosphatase (ALP) activity changes of NIH3T3 cells incubated with secretive human bone morphogenetic protein-2 (hBMP-2) that is induced by gene transfection through transwell system.

METHODSEukaryotic expression vector (pcDNA3.1-B2) was transduced into NIH3T3 cells by Sofast, a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expression of BMP-2 in the NIH3T3 cells were determined by immunohistochemical and enzyme-linked immunosorbent assay (ELISA). NIH3T3 cells were co-cultured with hBMP-2 gene transfecting cells through transwell system, and the ultrastructure and ALP activity (the markers of osteogenetic differentiation) changes were observed.

RESULTSThere were cytoplasmic and extracellular expression of BMP-2 in transfecting NIH3T3 cells. The ultrastructure changes and the high expression of ALP suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells.

CONCLUSIONSSecretive BMP-2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; Coculture Techniques ; Fibroblasts ; cytology ; ultrastructure ; Humans ; Mice ; NIH 3T3 Cells ; Osteogenesis ; Transfection