ABSTRACT
<p><b>OBJECTIVE</b>To observe the osteoclast differentiation of Raw264.7 strain stably expressing enhanced green fluorescent protein (EGFP).</p><p><b>METHODS</b>Raw264.7 cells were transfected with EGFP-Lifeact gene via retrovirus. The G3 cell clone was obtained by limited dilution technique which stably expressed EGFP under the fluorescence microscope. The morphology of G3 cells were observed. The effects of transfection on receptor activator of nuclear factor kappaB ligand (RANKL)--induced osteoclastogenesis and bone resorbing function of G3 cells were examined by tartrate resistant acid phosphatase (TRAP) staining, immunoblotting detection of cathepsin K and bone pit resorption assay. The real-time images of podosome dynamics were taken by laser confocal microscopy during osteoclast differentiation of G3 cells.</p><p><b>RESULTS</b>The Raw264.7 cells were successfully transfected with EGFP-Lifeact gene. The G3-EGFP cloned strain which could stably express EGFP even after 20 passages was constructed. There was no significant difference in morphology between G3-EGFP and wild Raw264.7 cells. The fusion rates of the transfection group and of the wild control group were (35±5)% and (39±5)%, respectively, which were not significantly different (P>0.05). The semi-quantitative ratio of cathepsin K/β-actin in the wild control group and in the transfection group was 0.83±0.07 and 1.02±0.08 (P>0.05), respectively. Bone pit results showed that the total area of the bone resorption was respectively 272,252±36,193 and 262,408±23,243 (P>0.05) and the number of the bone pits was respectively 320±51 and 339±55 (P>0.05). The photos of laser confocal microscopy showed the constant cell-cell fusion during osteoclast differentiation of G3-EGFP cells. In addition, the dynamic self-organized podosome initially assembled podosome clusts, then dynamic rings, finally formed the characteristic podosome belt pattern in mature osteoclasts.</p><p><b>CONCLUSIONS</b>Enhanced green fluorescent protein high effectively expressed in Raw264.7. Biological character does not change after transfection.</p>
Subject(s)
Humans , Acid Phosphatase , Actins , Metabolism , Bone Resorption , Cathepsin K , Metabolism , Cell Differentiation , Cell Line , Gene Expression , Green Fluorescent Proteins , Metabolism , In Vitro Techniques , Isoenzymes , Osteoclasts , Cell Biology , Metabolism , RANK Ligand , Metabolism , Receptor Activator of Nuclear Factor-kappa B , Tartrate-Resistant Acid Phosphatase , Transfection , MethodsABSTRACT
BACKGROUND:Stem cell factor(SCF)serves as a factor having a promotion effect on proliferation and differentiation of stemcells.Whether SCF affects biological features of dental pulp stem cells(DPSCs),and plays an important role in tooth regeneration remain unclear.OBJECTIVE:To observe SCF effects on the proliferation and osteogenic difierentiation of human DPSCs.METHODS:Pulp tissues were removed from healthy human teeth extracted for orthodontic purposes.The pulp tissues were digested by collagenase and dispase.The sample was cultivated in culture flasks with medium contained 1,1 0 μmol/L SCF;the normal culture medium served as control.The cell proliferation ability was detected by MTT.The osteogenic gene bone sialoprotein and osteocalcin mRNA expression were examined by real-time PCR.Alkaline phosphatase a ctivity was detectedr by alkaline phosphatase kit.RESULTS AND CONCLUSION:SCF could enhance the proliferation ability,upregulate bone sialoprotein and osteocalcin mRNA expression,and enhance the alkaline phosphatase activity,with the tendency of increased promotion with Increased concentration.Resuits indicated that SCF could enhance the proliferation and osteogenic difierentiation ability of human DPSCs.
ABSTRACT
Bone marrow mesenchymal stem cells(BMSCs) are a kind of non-hematopoietic cells living among bone marrow stroma,and are able to differentiate into mesoderm-type cells and non-mesoderm type cells.At present,there are three main methods for isolating and purifying BMSCs:bone marrow adherent culture,density gradient centrifugation and immune selection.This review listed many kinds of cells that BMSCs can differentiate into under certain conditions,discussed the related different directing conditions reported by researchers,analyzed the principle behind the artificial controlling conditions,and tried to find relatively uniform and effective directing conditions.The cells that BMSCs can differentiate into various types of cells including mesoderm-type cells,and non-mesoderm type cells:osteocytes,chondrocytes,adiocytes.As seed cells and a source of cell therapy in tissue engineering,BMSCs have been widely used in studying stem cells.