PURPOSE: We tried to assess the optimal conditions to improve low transduction efficiency and their effect on target cells. METHODS: Cultured NIH 3T3 cells were incubated with retroviral vectors bearing an enhanced green fluorescent protein (eGFP) gene. We varied the ratio of viral vectors to target cells (1:1-1:8) and the number of transfections (x1, x2), and compared transduction efficiencies. Also, the effects of polybrene on transduction efficiency and viability of target cells were assessed. Transduction of the eGFP gene was evaluated by observing NIH 3T3 cells under a fluorescence microscope and efficiencies were measured by the percentage of eGFP positive cells using FACscan. RESULTS: As the ratio of retroviral vectors to target cells increased, transduction efficiency was greatly improved, from 7% (1:1) to 38% (1:4). However, transduction efficiency did not increase any more when the ratio increased from 1:4 to 1:8. Cells transfected twice showed higher transduction efficiencies than cells transfected once, at a ratio of 1:8. The eGFP gene transduced to NIH 3T3 cells sustained its expression during repeated passages. However, after the third passage (day 9), the percentage of eGFP positive cells began to decline. The degree of this decline in eGFP expression was lower in cells transfected twice than in cells transfected once (P<0.05). The addition of polybrene did not have any toxic effect on NIH 3T3 cells and greatly increased transduction efficiency (P=0.007). In addition to vector component, transduction efficiency was very sensitive to culture confluence. Cells cultured and transfected in 24-well plate showed higher transduction efficiency, although cells cultured in 6- well plate proliferated more (P=0.024). CONCLUSION: Our data could be used as a basis for retrovirus-based gene therapy. Further study will follow using human cells as target cells.
Fluorescence ; Genetic Therapy ; Hexadimethrine Bromide ; Humans ; NIH 3T3 Cells* ; Retroviridae ; Transfection ; Zidovudine

OBJECTIVETo study the biological effects of nickel-refining dust.

METHODSThe cell phagocytosis, transformation activity, and cytotoxicity of the mouse NIH3T3 cells treated with nickel-refining dusts from two nickel-refining factories in China were observed, and the risk of carcinogenicity was studied.

RESULTS(1) Two samples of nickel-refining dusts could be phagocytosed by mouse NIH3T3 cells with different phagocytizing rates of 69.0% and 39.0% at 100.000 micro g/ml, and 78.0% and 47.0% at 200.000 micro g/ml respectively. The relative clone formation rates at 12.500 micro g/ml to 100.000 micro g/ml were 71.1% to 3.9% and 84.4% to 9.1%, respectively. The cytotoxicity expressed by clone formation rate was similar to that of Ni(2)O(3), but higher than that of TiO(2) and lower than the positive control of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). (2) MNNG, Ni(2)O(3) and the two samples of nickel-refining dusts could induce morphological transformation in NIH3T3 cells. The transformation rate at 12.500 micro g/ml to 50.000 micro g/ml were 1.9% to 3.6% and 0.9% to 2.5% respectively in a dose-dependent manner. (3) The NIH3T3 cells treated by MNNG and nickel-refining dusts could induce Con A agglutination, and may form as clone in soft agar. This finding proved the reliability of the transformed clone.

CONCLUSIONSThe present study for the first time demonstrate that nickel-refining dusts have cell transformation activity. The findings provide a new experimental evidence for the carcinogenic risk of nickel-refining dusts, and for the aetiology of lung cancer in nickel-refining workers.

Animals ; Cell Transformation, Neoplastic ; drug effects ; pathology ; Dust ; Mice ; NIH 3T3 Cells ; Nickel ; toxicity

Animals ; Coal Tar ; toxicity ; Comet Assay ; DNA Damage ; drug effects ; Mice ; NIH 3T3 Cells ; drug effects

AIMTo establish a HSP90 highly expressing cell line and study the effect of high level of HSP90 on cell stress response.

METHODSThe recombined plasmid pSmycHSP, which contained the full length DNA coding for human HSP90 B, was introduced into mouse fibroblast cell line NIH-3T3 by electroporation after being subcloned, purified and identified by limited enzyme digestion. After screened by G418, the positive clones were selected and identified by immunocytochemistry and Western-blotting. Contrasted with NIH-3T3 cells transfected with empty plasmid, the effect of high-level HSP90 on cell proliferation and cell cycle was analyzed by MTT method and flow cytometry.

RESULTSThe rising level of HSP90 was shown by immunocytochemistry and Western-blotting. Compared with control, the growth of HSP90 highly expressing cell line slowed down and the DNA content of S phase was lower.

CONCLUSIONThe NIH-3T3 derived cell line, which stably expressed high level of HSP90 was established. The effect of high-level HSP90 on cell proliferation was to retard cell growth by affecting cell cycle.

Animals ; Cell Cycle ; Cell Proliferation ; HSP90 Heat-Shock Proteins ; metabolism ; Mice ; NIH 3T3 Cells

OBJECTIVETo investigate the influence of fluorescent protein expression on the proliferation of murine NIH3T3 cells, so as to provide a theoretical basis for cell tracing technology.

METHODSNIH3T3 cells were cultured in vitro, and were randomly divided into control, pLEGFP-N1 (with transfection of pLEGFP-N1 retroviral vector), pEGFP-N1 (with transfection of pEGFP-N1 vector) and pDsRed2-C1 (with transfection of pDsRed2-C1 vector) groups. Then the cells were screened by G418 for 3 weeks. The changes in cell adhesive rate were observed and the population doubling times was determined by growth curve.

RESULTSThere was obvious fluorescent protein expression in the transfected NIH3T3 cells after G418 selection, and the highest percentage of labeled NIH3T3 cells was found in pLEGFP-N1 group. The population doubling time in pDsRed2-C1 (40.3+/-0.7 h) , PEGFP-N1 (39.6 +/- 0.6 h) and pLEGFP-N1 (36.5 +/- 0.7 h) groups was evidently longer than that in control (27.9 +/- 0.6 h, P < 0.01), with high adhesive rate in each group.

CONCLUSIONThe expression of fluorescent protein exhibited some inhibitory effect on the proliferation of NIH3T3 cells in vitro. Since the inhibitory effect by retroviral vector was weaker compared with eukaryotic vector, it should be the first choice for fluorescent protein labeling during cell transplantation.

Animals ; Cell Culture Techniques ; Cell Proliferation ; Green Fluorescent Proteins ; biosynthesis ; Mice ; NIH 3T3 Cells ; Transfection

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

The aim of this study was to evaluate the biological efficacy of a unique perpendicular protrusion of type-I collagen (Col-I) from TiO
Animals ; Cell Adhesion ; Collagen Type I ; Mice ; NIH 3T3 Cells ; Nanotubes ; Surface Properties ; Titanium

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

OBJECTIVETo study the effects of two kinds of nickel-refining fumes on DNA damage of NIH/3T3 cell and the difference.

METHODSNIH/3T3 cells were treated by two kinds of nickel fumes collected from smelting furnace and refining workshop of a nickel-smeltery, and PBS taken the place of nickel-smelting fumes was used as negative control. Several hours later, the cytotoxicity of on NIH/3T3 cells was detected with MTT colorimetric assay, and the DNA damage was also measured by comet assay (single cell gel electrophoresis).

RESULTWith the extension of exposure time and increasing of concentration, the living rate of NIH/3T3 cells was decreased; the tail rate, tail extent moment and tail DNA percent of NIH/3T3 cell induced by these two refining fumes were increased. After cells were treated with 100.00 microg/ml of nickel-smelting fume for 48 h, the living rate of NIH/3T3 cells was 24.5% and 26.5% respectively. The tail length of NIH/3T3 cell induced by these two refining fumes was not significant difference. Tail DNA percent of NIH/3T3 cell induced by smelting furnace fume was higher than negative control group (P < 0.05). The tail rate, and tail DNA percent (except 12.5 microg/ml and 50.0 microg/ml treated 2 h group) of NIH/3T3 cell induced by refining workshop fume was higher than negative control group (P < 0.05).

CONCLUSIONNickel-smelting fume could depress the survival rate of NIH/3T3 cells, and induce different degree DNA damage of NIH/3T3 cell.

Animals ; Cell Survival ; drug effects ; Comet Assay ; DNA Damage ; drug effects ; Metallurgy ; Mice ; NIH 3T3 Cells ; Nickel ; toxicity

OBJECTIVETo demonstrate the effectiveness of a retrovirus-based plasmid vector coupled with nanometer material-polyamidoamine (PAMAM) dendrimer in stable gene expression of FVIII in vitro and to study the cytotoxicity of PAMAM.

METHODSThe retrovirus-based plasmid vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa - 1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. The complex that contained PAMAM and pLNC-FVIII BD transfer FVIII BD cDNA into NIH3T3 cell line. In day 2, 5, 10, 15, 30 after transferring, the antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of FVIII BD mRNA. Inhibitory percentage of cell vitality was used for cytotoxicity of PAMAM.

RESULTSHuman FVIII was expressed for 30 days by transfected cells. The mean procoagulant activity of secreted FVIII in these 30 days was 0.929 U/ml, and the FVIII antigen was 0.188 micro g/ml by 10(6) cells in 24 hours, respectively. The level of FVIII didn't significantly decreased during these days. Inhibitory percent of cell vitality was only 5.32%.

CONCLUSIONPAMAM could effectively transfer pLNC-FVIII BD into NIH3T3 cells and FVIII could be stably and effectively expressed by the transfected cells. Cytotoxicity of PAMAM was low.

Animals ; Dendrimers ; Factor VIII ; genetics ; Genetic Vectors ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; Polyamines ; pharmacology ; Retroviridae ; genetics