Osteoclast differentiation of Raw264.7 cells expressing enhanced green fluorescent protein in vitro / 中华口腔医学杂志

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>