Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Functions of key genes involved in TGF-β/Smad signaling pathway in progression of pulmonary fibrosis / 环境与职业医学

Background Although transforming growth factor-β (TGF-β)/Smad signaling pathway is important in regulating the occurrence and development of pulmonary fibrosis, the pathogenesis of pulmonary fibrosis remains elusive. Objective To explore the functions of genes associated with TGF-β/Smad signaling pathway in the progression of pulmonary fibrosis. Methods A NIH-3T3 fibroblast model induced by TGF-β1 was established. The experiment samples were divided into a control group and a TGF-β1 treatment group. The control group was exposed to normal saline, while the TGF-β1 treatment group was exposed to 10 ng·mL−1 TGF-β1 for 12 h. The RNAs of the two groups were extracted, sequenced, and analyzed by bioinformatics methods to identify seven key genes in TGF-β pathway, including Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3. The gene expression levels of five markers [Collagen1α1, Collagen1α2, α-smooth muscle actin (α-SMA), TGF-β1, and TGF-β3] and the seven key genes were detected by quantitative real-time PCR (qRT-PCR). The proteins of the two groups were extracted. The important marker protein expression levels of Smad3, the phosphorylation of Smad3 (P-Smad3), and α-SMA were detected by Western blotting. At the same time, 30 healthy SPF-grade C57BL/6 mice were randomly divided into three groups, with 10 mice in each group: a control group, a SiO2 inhalation exposure group for 28 d (10 mice), and a SiO2 inhalation exposure group for 56 d (10 mice). The mice in the two treatment groups were exposed to a natural SiO2 environment for 4 h per day with a 10-min pause for breathing fresh air at 2 h intervals. The lung tissues of the mice were taken after execution. The changes of pulmonary fibrosis were detected by Masson staining, and mRNAs and proteins were extracted to detect the expression of the above key genes and proteins. Results The expression levels of the five marker genes Collagen1α1, Collagen1α2, α-SMA, TGF-β1, and TGF-β3 were significantly increased in the TGF-β1-induced NIH-3T3 fibroblasts than those in the control group (P < 0.01); the expression levels of P-Smad3 and α-SMA proteins increased significantly (P < 0.01); the expression results of the seven key genes screened in the TGF pathway were that Dcn and Smad3 were obviously down-regulated (P < 0.01), and Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 were obviously up-regulated (P < 0.01). The changes in gene expression levels of the transcriptome sequencing showed the same trend. The results of Masson staining showed that the content of collagen fibers in the lung tissues also increased in the SiO2 inhalation exposure groups over time. In the mouse experiment, five marker genes were obviously up-regulated compared with the control group (P < 0.01); no obvious change was found in the expression of Smad3 protein, and the expression levels of P-Smad3 and α-SMA were obviously higher in the SiO2 exposure groups than those in the control group (P < 0.01); the expression levels of Dcn and Smad3 showed a down-regulated trend, while the expression levels of Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 showed an up-regulated trend with the increase of SiO2 inhalation exposure days (P < 0.01). The expression levels of the above five marker genes, three important marker proteins, and seven key genes were consistent with the expression trends of TGF-β1-induced NIH-3T3 fibroblasts. Conclusion The expression levels of pulmonary fibrosis-related marker genes and proteins change significantly in TGF-β1-induced fibroblast cells, and the lung tissues of mice under natural SiO2 inhalation exposure has obvious fibrosis characteristics. Seven genes (Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3) may be involved in the regulation of pulmonary fibrosis by the TGF-β/Smad signaling pathway.

Reactive oxygen species and senescence modulatory effects of rice bran extract on 4T1 and NIH-3T3 cells co-treatment with doxorubicin

Objective:To determine the effect of rice bran extract (RBE) in combination with doxorubicin on 4T1 triple-negative breast cancer cells and NIH-3T3 cells. Methods:RBE was obtained by maceration with n-hexane. The phytochemical profile of RBE was observed using high-performance liquid chromatography. Cytotoxic effect of RBE was evaluated through MTT assay. In addition, flow cytometry was used for cell cycle and apoptosis analysis. Cellular senescence was observed using SA-β-Gal assay and intracellular reactive oxygen species (ROS) levels were evaluated using DCFDA staining. The pro-oxidant property of RBE was also evaluated through 1-chloro-2,4-dinitrobenzene spectrophotometry and molecular docking. Results:RBE was obtained with a yield of 18.42％ w/w and contained tocotrienols as the major compound. RBE exerted no cytotoxic effect on 4T1 and NIH-3T3 cells. However, RBE in combination with doxorubicin decreased 4T1 cell viability synergistically (combination index<0.9) and induced apoptosis and senescence on 4T1 cells. RBE significantly decreased senescence in doxorubicin-treated NIH-3T3 cells. Additionally, RBE did not increase ROS levels in doxorubicin-treated 4T1 cells. Meanwhile, the combination of RBE and doxorubicin reduced ROS levels in NIH-3T3 cells. RBE significantly reduced glutathione-S-transferase activity and alpha-tocotrienol interacted with glutathione-S-transferase in the glutathione binding site. Conclusions:Rice bran may be used as a co-chemotherapeutic agent to improve the therapeutic effectiveness of doxorubicin while protecting against the cellular senescence effects of doxorubicin on healthy cells.

High-throughput screening of single Chinese plant extracts on specific anti-hepatoma drugs / 中国药理学与毒理学杂志

OBJECTIVE To evaluate the effect of single traditional Chinese medicine(TCM) herb extracts on hepatoma and normal fibroblast cells using high-throughput screening in order to obtain extracts with specific anti-hepatoma effect. METHODS 242 commonly used TCM herbs were extracted by petroleum ether,ethanol and water,respectively. The total number of TCM extracts was 554. The cyto?toxicity of samples was evaluated by MTT in human hepatoma cells Bel7402 and mice normal fibroblasts NIH3T3. RESULTS 7.4%of the total extracts had an inhibitory effect greater than 50%for Bel7402,but 14.8% for fibroblasts NIH3T3 cells. Extracts with an inhibitory effect above 50% on both Bel7402 and NIH3T3 cells accounted for 4.4%of the total extracts. Our results showed that the sample DF173 had preferable cytotoxicity effect on hepatoma carcinoma cells in a good dose-effect relationship. DF173 is an ethanol extract from Stephania tetrandra,which is a commonly used herb in TCM. The cytotoxic IC50 of DF173 against Bel7402 was 8.27 mg·L-1,and 19.48 mg·L-1 on NIH3T3. CONCLUSION The components of TCM herbs are highly complicated. The combination of tumor cells with normal fibroblast cells to evaluate the cytotoxicity effect during anti-tumor drug screening will contribute much to the discovery of TCM drugs with high anti-tumor efficiency and lower toxicity.

Differentially Expressed Proteins in Nitric Oxide-Stimulated NIH/3T3 Fibroblasts: Implications for Inhibiting Cancer Development

PURPOSE: Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. MATERIALS AND METHODS: The cells were treated with 300 microM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. RESULTS: Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (alpha1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (epsilon subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (epsilon subunit) are unknown in relation to carcinogenesis. CONCLUSION: Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Cell density and solvent are critical parameters affecting formazan evaluation in MTT assay

The aim of this study was to establish the more accurate protocol for fibroblast cell viability using MTT assay. NIH/3T3 fibroblasts were seeded at the following cell densities: 3.125x10³; 1.156x10(4); 3.125x10(4); 1.156x10(5) and 3.125x10(5) cells/cm². Following 24h of seeding, MTT was added to the wells. After 4h of the MTT addition, different solvents were added to solubilize the formazan crystals: 1) HCl/SDS group- 20% SDS and 0.01 M HCl; 2) EtOH/ HAc group-50% ethanol and 1% acetic acid; 3) DMSO group- 99.5% dimethyl sulfoxide; and 4) PropOH group- 99.5% isopropanol. The absorbance values were measured using a spectrophotometer at 570 nm. The data were analyzed by 2-way ANOVA (p<0.05) and showed that the absorbance average varied according to the number of cells and solvents: HCl/SDS (0 to 0.13), EtOH/HAc (0 to 0.22), DMSO (0.76 to 1.31) and PropOH (0.66 to 1.04). The DMSO and PropOH groups presented the most appropriate protocols for NIH/3T3 fibroblasts cell viability, especially at the density of 1.156x10(4) cells/cm².

Development of experimental in vitro burn model

PURPOSE: To propose an experimental burn model in NIH-3T3 cell line. METHODS: Induction of thermal injury in cultures of mouse fibroblast - NIH-3T3- cell line and determination of cell viability by MTT and imunofluorescence. RESULTS: The heating of the Petri dish increased proportionally to the temperature of the base and the time of exposure to microwave. In this in vitro burn model, using the cell line NIH-3T3 was observed drastic cellular injury with significant changes in cell viability and activity. It showed drastically modified cell morphology with altered membrane, cytoskeleton and nucleus, and low cellularity compared to the control group. CONCLUSION: The burn model in vitro using the cell line NIH-3T3 was reproductive and efficient. This burn model was possible to determine significant changes in cell activity and decreased viability, with drastic change in morphology, cell lysis and death. .

PTPMeg2 inhibits STAT3 transcriptional activity in NIH3 T3/STAT3 CA cell model / 中国药理学通报

Aim To investigate the NIH3T3/STAT3CA cell proliferation ability and the STAT3 transcriptional activity affected by PTPMeg2 . Methods MTT assay and xenograft nude mice model were used to investigate the NIH3 T3/STAT3 CA cell proliferation inhibited by PTPMeg2 in vitro and in vivo. Co-immunoprecipitation assay was used to measure the interaction between PT-PMeg2 and STAT3CA. STAT3 transcriptional activity was measured by dual-luciferase assay. Results The NIH3 T3/STAT3 CA cell proliferation ability was signifi-cantly inhibited by PTPMeg2 in vitro and in vivo com-pared with the control group ( P <0.05 ) . The tran-scriptional activity was increased by PTPMeg2 , but not the PTPMeg2 mutant (PTPMeg2C515S) and the ShPT-PMeg2 . Conclusion PTPMeg2 plays a role in inhibi-ting the proliferation ability of NIH3 T3/STAT3 CA cells through inhibiting the STAT3 transcriptional activity.

Paraquat Induces Apoptosis through Cytochrome C Release and ERK Activation

Paraquat has been suggested to induce apoptosis by generation of reactive oxygen species (ROS). However, little is known about the mechanism of paraquat-induced apoptosis. Here, we demonstrate that extracellular signal-regulated protein kinase (ERK) is required for paraquat-induced apoptosis in NIH3T3 cells. Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis. Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors. Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

A Study of Antidepressant and Mood Stabilizer's Effect on Phase of Peripheral Circadian Rhythm in Rat Fibroblast / 대한정신약물학회지

OBJECTIVE: Circadian rhythm is cyclic variations in biological activity, which is crucial for physiology in mammals and alterations in circadian rhythm may be related to mood disorders. However, the effect of mood disorder medications on peripheral tissue is less understood. In this study, we investigated whether treatment with antidepressants, antipsychotics, and mood stabilizers in rat fibroblast influenced circadian rhythm. METHODS: Lithium, bupropion, risperidone, lamotrigine, and paroxetine were used on cultured NIH3T3, peripheral rat fibroblast and we observed the periodic oscillation and rhythmicity of Per2, Bmal1 mRNA for 48 hours. RESULTS: We found that lithium has a dose-dependent effect on phase change of circadian rhythm and buproipion has a tendency to dampen the amplitude. Risperidone, lamotrigine, and paroxetine had no influence on circadian rhythm in NIH3T3 cell. CONCLUSION: These results suggest that lithium treatment may have an effect on circadian rhythm lengthening in peripheral fibroblast tissue.

Preparation and culture of NIH3T3 cells harboring microencapsulated VEGF gene / 第二军医大学学报

Objective: To prepare NIH3T3 cells harboring microencapsulated VEGF gene and investigate the proliferation, activity and metabolic function of the modified cells. Methods: Microencapsulated VEGF modified NIH3T3 cells were prepared through an alginate-BaCl2 process. Morphological appearance of the microencapsulation and the cell morphology were observed under inverted phase microscope; untreated NIH3T3 cells served as control. The concentrations of VEGF in the culture supernatant (collected every 48 hours) were measured by ELISA; the proliferation and vitality of the cells were examined by MTT assay and flow cytometry with PI staining. Results: The microcapsules were round in shape and the cells grew well. There was no significant difference in the concentrations of VEGF,MTT values and vitalities of cells between the 2 groups. Conclusion: The growth and metabolic functions of NIH3T3 cells are not influenced by microencapsulated NIH3T3 cells harboring VEGF gene. The bio-properties of modified cells are similar to those of the control cells,which lays a foundation for transplantation of microencapsulated VEGF modified NIH3T3 cells in vivo.

Adenovirus-mediated VEGF expression in NIH3T3 cells / 第二军医大学学报

Objective: To investigate VEGF expression in NIH3T3 cells infected by adenovirus containing hVEGF165 gene and its influence on proliferation of NIH3T3 cells, and to observe the expression of hVEGF and its angiogenic effect in vivo. Methods: Adenoviral vector containing hVEGF165 gene was constructed and was used to infect NIH3T3 cells. The infection efficiency of adenovirus vector was examined by immunofluorescence and flow cytometry. Expression of VEGF in NIH3T3 cells and its levels in the culture medium were examined by immunohistochemical (IHC) staining, RT-PCR, and ELISA. The infected NIH3T3 cells were implanted in skin defect at rat back and the acellular dermis on the wound was obtained one week later; the expression of hVEGF was detected by IHC in the dermis and the density of vessels was determined under microscope. Results: NIH3T3 cells were effectively transfected by adenovirus containing VEGF gene in vitro, the transfection efficiency was in a dose-effect manner with multiplicities of infection (MOI) of the adenovirus. When MOI was 100, the infection efficiency was more than 95%. The expression of VEGF mRNA and protein was detected by RT-PCR and IHC 24 h after transfection. ELISA result showed that the high level of VEGF on the 3rd day after transfection and the level reached its peak 7 d after infection (1 052 pg/ml); VEGF expression was detectable 13 d after transfection. MTT assay demonstrated no significant difference in cellular proliferation between the transfection and non transfection group. Expression of hVEGF was also detected in vivo in mice, and the density of vessels in the experimental group was significantly higher than that in the control group (P<0.01). Conclusion: Adenoviral vector can effectively transfect VEGF gene into NIH3T3 cells; VEGF gene can be detected in vitro and in vivo; and it can promote neovascularization in the transplanted tissues.

Papel dos corpúsculos lipídicos na progressão do ciclo celular

Corpúsculos lipídicos são organelas dinâmicas envolvidas no metabolismo de lipídeos, no tráfego de membrana e na sinalização intracelular. A lipogênese está associada com um mal prognóstico em diversas doenças neoplásicas, sugerindo um papel dessas organelas no desenvolvimento do câncer. Foi mostrado anteriormente que corpúsculos lipídicos são elementos centrais na síntese de PGE2 e na proliferação celular em linhagens celulares de câncer de cólon, e podem estar implicados na patologia do adenocarcinoma de cólon. Baseado nesses dados, a regulação dos corpúsculos lipídicos na progressão do ciclo celular foi investigada. Células NIH3T3 foram sincronizadas por privação de soro por 24 horas, e então suplementadas com meio contendo 10% de soro fetal bovino para avaliar sua entrada e progressão pelo ciclo celular. Análise por iodeto de propídeo revelou que, a partir da suplementação com soro, células entraram na fase S do ciclo celular após 24 horas e progrediram para as fases G2/M após 36 e 48 horas. Esses dados foram confirmados com análise dos níveis de fosforilação de Rb por western blot e dos níveis de expressão das ciclinas D2, E2, A2 e B2 por PCR em tempo real. Posteriormente, a regulação dos corpúsculos lipídicos durante a progressão do ciclo celular foi avaliada. Células com arresto na fase G1 mostraram um menor número de corpúsculos lipídicos, com localização perinuclear. Por outro lado, foram observados um aumento no número de corpúsculos lipídicos e uma distribuição homogênea dessas organelas pelo citoplasma durante a fase S. Além disso, células NIH3T3 mostraram aumento no número de corpúsculos lipídicos e localização dispersa dessas organelas após transformação com a oncoproteína H-rasV12. Juntos esses resultados mostram que corpúsculos lipídicos são regulados durante a progressão do ciclo celular, que esta regulação depende da entrada na fase S do ciclo celular, e que em células transformadas esta regulação está alterada. Por fim, esses dados dão evidências de que um mecanismo coordenado regula a progressão de ciclo celular e a biogênese de corpúsculos lipídicos, que pode estar desregulado durante o desenvolvimento neoplásico

Lipid bodies are dynamic organelles involved in lipid turnover, membrane traffic and intracellular signaling. Lipogenesis has been associated with poor prognosis in several neoplasic diseases, suggesting a role for these organelles in cancer development. It has been previously reported that lipid bodies are centrally involved in PGE2 synthesis and cell proliferation in colon cancer cells, and may potentially have implications to colon adenocarcinoma pathogenesis. Based on this data, the regulation of lipid bodies during cell cycle progression was investigated. NIH3T3 cells were synchronized by serum starvation for 24 hours, and then supplemented with medium containing 10% fetal bovine serum to evaluate their entering and progression through cell cycle. Propidium iodide analysis revealed that upon serum supplementation cells reached S phase after 24 hours and followed to G2/M phase after 36-48 hours. These data was confirmed by analysis of Rb phosphorylation by western blot assay and by expression levels of cyclins D2, E2, A2, and B2 assayed by quantitative real-time PCR. The quantity and subcellular localization of lipid bodies during cell cycle progression was evaluated. Cells arrested on G1 phase showed a lower number of lipid bodies with perinuclear localization. On the other hand, an increased number of lipid bodies with a homogeneous distribution through the cytoplasm were observed during S phase. Moreover, NIH3T3 cells displayed increased number and dispersed localization of lipid bodies upon transformation with H-rasV12 oncoprotein. Taken together, these results suggest that lipid bodies are highly regulated during cell cycle, and also that this regulation is altered in transformed cells. Finally, these data provide evidence for a coordinate mechanism that regulates cell cycle progression and lipid body biogenesis, which might be deregulated during cancer development

An Expedient Reliable Double Fluorescent Reporter System for φC31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for φC31 integrase was developed in NIH3T3 cells. The reporter plasmid coding green fluorescent protein (GFP) coupled with red fluorescent protein (RFP) was eo-transfected with the plasmid coding φC31 integrase, to show the activity of integrase in the cells. Fluorescence activated cell sorter (FACS) was used to measure the proportion of the cells containing red and green fluorescence. The increment of green cells was positively related to the increase in the transfection with plasmid coding φC31 integrase. Approximately 90% of green cells were observed under a ratio of [plasmid-φC31-integrase]/[reporter plasmid] at 10 : 1. This suggests that the φC31 integrase reporter system provides a probe for the function of φC31 integrase in cells.

Inhibition of IL-13-induced collagen I by sIL-13Rα2 in NIH-3T3 fibroblast cells and the profile of sIL-13Rα2/IL-13 in mice with schistosomiasis / 中国人兽共患病学报

To determine the inhibition of IL-13 by recombinant sIL-13Rα2 in NIH-3T3 fibroblast cells for its potential therapeutic value in hepatic fibrosis caused by Schistosoma japanicum in mice . IL-13 and sIL-13Rα2 from liver of BALB/c mice infected with S.japonicum at different infection time (weeks 0,6,8,10 and 12) were analyzed by ELISA and RT-PCR. The recombinant sIL-13Rα2 expression plasmidwas constructed, followed by transfection into NIH-3T3 fibroblast cells. TypeⅠcollagen produced by NIH-3T3 cells were examined by RT-PCR and Western blotting. It was demonstrated that the expression of IL-13 increased gradually after infection, reached peak density (16.1586 pg/mL)at week 8 and then reduced but was still higher than the level of control mice(3.4146 pg/mL;P =0.017 ). The secretion of sIL-13R α2 reached to its peak 10 weeks after infection(4827.426 pg/mL)and then reduced slowly but still higher than normal(4057.112 pg/mL; P=0.021). Meanwhile, the changes in mRNA level of IL-13 and sIL-13R α2 were coincided with that examined by ELISA. Both IL-13 and sIL-13Rα2 reached their peak density (P=0.033) at week 8 and 10 (P=0.025) respectively, and they were followed by a slower degree of decrease. The sIL-13Rα2 could significantly inhibit the effect of IL-13 on NIH-3T3 fibroblast cells, showing decreased mRNA level(P =0.012)and protein level of typeⅠcollagen compared with normal groups(P =0.031). It is concluded that the sIL-13Rα2 can inhibit the effect of IL-13 on NIH-3T3 fibroblast cells which leads to a reduced production of typeⅠcollagen, demonstrating its potential therapeutic value in hepatic fibrosis of schistosomiasis.

The optimal conditions to improve retrovirus-mediated transduction efficiency to NIH 3T3 cells / 소아과

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.

Effect and underlying mechanism of antioxidant quercetin in mouse and in NIH-3T3 cells / 基础医学与临床

Objective To compare the difference in quercetin against oxidative stress response in mouse and in NIH-3T3 cells before and after H2O2 treatment,to explore the underlying mechanism for the quercetin antioxidant.Methods The cultured NIH-3T3 cells were randomly divided into 4 groups: quercetin(Q) pre-protective group(Qb) firstly treated with quercetin for 24 h followed by incubation with H2O2 for 30 min;post-protective group(Qa) treated with H2O2 for 30 min followed by incubation with quercetin for 24 h;H2O2 group(H2O2) after exposure to H2O2 for 30 min,incubated with DMEM medium and the control group(C) only cultured with DMEM medium.The survival rate and apoptotic rate were detected respectively with MTT and TUNEL in NIH-3T3 cell sus-pension samples.The expression of cyclin D1,PTEN,NF-?B,HSP-70,BCl-2,BAX and caspase-3 were examined with immunocytochemistry and immunoblotting.Besides,20 Wistar rats were divided into control group and experimental group,the latter was given with quercetin in the doze of 0.13 mmol/kg.The levels of T-AOC,SOD,GSH-Px,GSH,MDA,NOS and NO2-/NO3-were detected both in the cleaved NIH-3T3 cells and in the plasma from both experimental and control animals prior to and post-1 h,2 h and after 24 h.Results When the Qb group was compared with H2O2 or Qa group,the survival rate was higher and the apoptotic rate was lower.When the H2O2 group was compared with C group,the expression of cyclin D1、PTEN or BCl-2 was down-regulated;while that of BAX、HSP-70、NF-?B or caspase-3 was up-regulated;the level of T-AOC,SOD,GSH-Px or GSH was decreased;that of NOS、NO2-/NO3-or MDA enhanced in the cleft NIH-3T3 cells.When the plasma level of the anti-oxidative enzyme system prior to-compared with post-1h and 2h-treatment with Q,the level of T-AOC,SOD,GSH-Px and GSH,especially the former two,were higher;MDA,lower;NOS or NO2-/NO3-promoted.However,the above parameters basically became normal 24 h after treatment with Q.Conclusion Quercetin down-regulates the promoted expression of HSP70,NOS,NO2-/NO3-and NF-?B etc.in H2O2-treatment NIH-3T3 cells.Qb could reverse the H2O2 damage effects more markedly.Moreover,the quercetin exerts anti-oxidant protective effect through modulating the anti-oxidative enzyme system both in vivo and in vitro.However,based on the cell heterogeneity in none-or pre/post-H2O2-treatment state,a difference in quercetin antioxidant response is noted.

Alternariol enhanced DNA polymerase ? expression in NIH3T3 cells / 基础医学与临床

Objective To study the effects of Alternariol(AOH) on DNA polymerase ?(DNA POL?)expression in NIH3T3 cells.Methods RT-PCR,Immunocytochemistry and Western blot were used to detected mRNA and the protein levels of DNA POL? in NIH3T3 cell line induced by AOH.Results The expression of DNA POL? in NIH3T3 cells contaminated by AOH was significantly higher than that in the control group(P

An Expedient Reliable Double Fluorescent Reporter System for ?C31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for ?C31 integrase was developed in NIH3T3 cells.The reporter plasmid coding green fluorescent protein(GFP) coupled with red fluorescent protein(RFP) was co-transfected with the plasmid coding ?C31 integrase, to show the activity of integrase in the cells.Fluorescence activated cell sorter(FACS) was used to measure the proportion of the cells containing red and green fluorescence.The increment of green cells was positively related to the increase in the transfection with plasmid coding ?C31 integrase.Approximately 90% of green cells were observed under a ratio of plasmid-?C31-integrase/reporter plasmid at 10∶1.This suggests that the ?C31 integrase reporter system provides a probe for the function of ?C31 integrase in cells.

Prediction Model for the Cellular Immortalization and Transformation Potentials of Cell Substrates

The establishment of DNA microarray technology has enabled high-throughput analysis and molecular profiling of various types of cancers. By using the gene expression data from microarray analysis we are able to investigate diagnostic applications at the molecular level. The most important step in the application of microarray technology to cancer diagnostics is the selection of specific markers from gene expression profiles. In order to select markers of immortalization and transformation we used c-myc and H-ras(V12) oncogene-transfected NIH3T3 cells as our model system. We have identified 8751 differentially expressed genes in the immortalization/transformation model by multivariate permutation F-test (95% confidence, FDR <0.01). Using the support vector machine algorithm, we selected 13 discriminative genes which could be used to predict immortalization and transformation with perfect accuracy. We assayed H-ras(V12)-transfected "transformed" cells to validate our immortalization/transformation classification system. The selected molecular markers generated valuable additional information for tumor diagnosis, prognosis and therapy development.