osteoblast differentiation markers, including alkaline phosphatase activity, nodule ... osteoblast-related protein that plays an important role in the regulation ...
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E:tkaryotic Gene Expression
factors on mammalian cell viability, number, and proliferation (Alley et al., 1988). Anti-OA antibody had no significant effect on osteoblast proliferation or viability (Fig. 4). We further examined the effect of ami-OA antibody during differentiation phases: matrL"\': formation and mineralization. It has been established that osteoblast differentiation could be evaluated by different markers, such as alkaline phosphatase (ALP) (Aubin et al., 1995), osteocalcin expression (Lian et al., 1991), calcium deposition, and nodule formation (Stein et al., 1990). The effect of anti-OA antibody on ALP activity (Fig. SA) and nodule formation (Figs. 5B and 5C) at day 14, and calcium deposition (Fig. 6A) and osteocalcin production (Fig. 6B) at day 21, was examined. Anti-OA antibody markedly inhibited ALP activity (Fig. SA), nodule formation (Figs. 5B and 5C), calcium deposition (Fig. 6A) and osteocalcin production (Fig. 6B). These data suggest a role tor OA in the differentiation and function of the osteoblast.
and an average of about 80% was reproducibly achieved (Figs. 3A and 3B). To evaluate the stability of the anti-OA antibody, primary osteoblasts were transfected at day 3 with nonimmune IgY, as a control, or anti-OA antibody; then, at day 7 of the culture, cells were fL'{ed and incubated with anti-chicken-C y3 conjugated secondary antibody. No signal was detected in nonimmune IgY transfected osteoblasts (Fig. 3C), whereas signal was found in anti-OA antibody transfected cells, indicating stability and binding specificity of antiOA antibody (Fig. 3D). This delivery approach provides high transfection efficiency, stability and protection of the ami-OA antibody (Fig. 3). To select the appropriate dose that can significantly affect osteoblast differentiation without affecting its viability, primary osteoblasts were transfected with different doses (1, 5, 15, and 30 ~g/mL) of anti-OA antibody and their effect on osteoblast differentiation and viability was examined. A dose of 15 j.lg/mL significantly inhibited calcium deposition (differentiation marker) in primary osteoblast culture \vithout affecting cell viabilirv, (data not shO\vn). Since OA is expressed throughout the culture period (Fig. 1), we examined the effect of antiOA antibody on osteoblast proliferation during the 1st week ofculture. Primary osteoblast cultures were transfected with 15 j.lg/mL of nonimmune Ig'Y, as a control, or anti-OA antibody, and, at day 5, MTT assa~' (Fig. 4A) and cell number counting (Fig. -J.B) were performed. The MIT assay is routineh" used to examine the effects of different
IV. DISCUSSION
In the present study, we evaluated the OA protein expression pattern in primary osteoblast cultures and examined its functional role in osteoblast development and differentiation. For this purpose, an anti-OA antibody was used to detect OA protein in primary osteoblast culture by immunofluorescent localization and Western blot analysis.
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