lipofectamine and thiazolyl-blue

The purpose of this study was to investigate the influence of a disintegrin and metalloproteinase 28 (ADAM28) on the proliferation, differentiation, and apoptosis of human dental pulp stem cells (HDPSCs) and possible mechanism.. After ADAM28 eukaryotic plasmid and antisense oligodeoxynucleotides (AS-ODNs) were constructed and respectively transfected into HDPSCs by Lipofectamine 2000, the ADAM28 expression levels among diverse groups were estimated by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. Methabenzthiazuron (MTT) and cell cycle assays were used to test the HDPSCs proliferation activity. Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide and alkaline phosphatase analysis were performed respectively to measure apoptosis and the cytodifferentiation level. Immunocytochemistry and western blotting were performed to determine the effects of ADAM28 eukaryotic plasmid on HDPSCs expressing dentin sialophosphoprotein (DSPP), dentin matrix protein 1, and bone sialoprotein.. ADAM28 could be correctly transcribed, translated, and expressed in HDPSCs. The ADAM28 AS-ODN group displayed the highest optical density value, whereas the eukaryotic plasmid group showed the lowest, which suggested that ADAM28 had a negative regulatory effect on the proliferation of HDPSCs. ADAM28 eukaryotic plasmid could significantly inhibit the HDPSC proliferation, promote specific differentiation of HDPSCs, induce apoptosis, and enhance the DSPP expression, whereas ADAM28 AS-ODN produced the opposite effects.. Our results proved that ADAM28 might actively participate in manipulating the proliferation, differentiation, and apoptosis of HDPSCs.

Topics: ADAM Proteins; Adolescent; Adult; Alkaline Phosphatase; Annexin A5; Apoptosis; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coloring Agents; Dental Pulp; Disintegrins; Extracellular Matrix Proteins; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Genetic Vectors; Humans; Integrin-Binding Sialoprotein; Lipids; Male; Membrane Glycoproteins; Oligodeoxyribonucleotides, Antisense; Phosphoproteins; Plasmids; Propidium; Sialoglycoproteins; Stem Cells; Tetrazolium Salts; Thiazoles; Transfection; Young Adult

The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid). The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit RS and RL) nanoparticles. These nanoparticles were prepared by two methods: nanoprecipitation and double emulsion. The nanoparticles were characterized by their size, zeta potential and amount of DNA adsorption. Cytotoxicity tests based on mitochondrial activity (MTT test) revealed that the nanoparticles had limited cytotoxicity and that this depended on both the cell type and the nanoparticle concentration. Transgene expression was observed using the Green Fluorescence Protein gene as reporter gene, and was evaluated by flow cytometry in FaDu, MDA-MB 231 and MCF-7 cell lines. The results showed that transfection rates ranging between 4 and 7% were achieved in FaDu and MDA-MB 231 cells with nanoparticles prepared by the nanoprecipitation method. In MCF-7 cells transfected with nanoparticles prepared by either the double emulsion or the nanoprecipitation method, the transfection efficiency was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the lipofectamine control.

Topics: Adsorption; Cell Line, Tumor; DNA; Drug Delivery Systems; Genetic Vectors; Humans; Lipids; Methacrylates; Mitochondria; Nanoparticles; Nanotechnology; Neoplasms; Plasmids; Polymers; Polymethacrylic Acids; Recombination, Genetic; Tetrazolium Salts; Thiazoles; Transfection

This study used RNA interference (RNAi) to explore the effect of NO and inducible nitric oxide synthase (iNOS) on apoptosis and proliferation in the tongue squamous carcinoma cell line Tca8113. Tca8113 cells were transfected with the plasmid pGenesil-1, which expresses iNOS short hairpin RNA (shRNA), or the negative control plasmid pSilencer-HK, and the transfected cells were compared with untransfected cells. The expression of iNOS was detected by histochemistry, and apoptosis was detected by flow cytometry. The expression of iNOS was significantly lower in the pSilencer-iNOS group than in the pSilencer-HK and empty control groups. The apoptosis rate was significantly higher in the pSilencer-iNOS group than in the pSilencer-HK and empty control groups. Growth monitoring showed that proliferation was also inhibited in cells transfected with pSilencer-iNOS. RNAi gene silencing decreased iNOS gene expression, induced apoptosis, and suppressed proliferation in Tca8113 cells.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Coloring Agents; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Histocytochemistry; Humans; Indicators and Reagents; Lipids; Nitric Oxide; Nitric Oxide Synthase Type II; Plasmids; RNA Interference; RNA, Small Interfering; Tetrazolium Salts; Thiazoles; Tongue Neoplasms; Transfection