| To study the effects of 1α,25-dihydroxyvitamin D3 injection on proliferation, differentiation, cell cycles, [Ca2+]i, and ultrastructure of osteoblasts, osteoblasts were isolated from SD rat cranial bones and cultured in vitro. These reaults provided rationale for clinical application.1 Isolation and identification of rat osteoblastsTo obtain osteoblasts, cranial bones were harvested from SD rat, digested by trypsin and collagenase. Then the cells were brachytely centrifuged for purify and seeded to culture flask. The culture medium replaced after 24 h to remove un-adherence cells. Shape and growth of OB were observed under inverted microscope. They were identified by ALP stained and mineralizated node stained. The activity of osteoblast was detected by microtitration (MTT) during a culture cycle. It was concluded that the modified twice-enzyme isolation method was an ideal technique to obtain and culture osteoblasts with typical characteristics and high purity for the experiment.2 Effects of 1α,25-dihydroxyvitamin D3 on proliferation and differentiation of osteoblasts cultured in vitroIn order to observe the proliferation and functions of 1α,25-dihydroxyvitamin D3 on the OB in vitro, OB were cultured with various concentration of 1α,25-dihydroxyvitaminD3 after 80% cell fusion. After 1, 3, 5 d, the proliferation and the activity of alkali phosphatase (ALP) of OB was observed. We found that at 1, 3, 5 d, all different concentration 1α,25-dihydroxyvitamin D3 inhibited proliferation of OB, there were significant differences in teams with high concentration (10-8,10-7 mol/L) of 1α,25-dihydroxyvitamin D3(P<0.01). At 1 d, all different concentration 1α,25-dihydroxyvitamin D3 promoted the ALP activtiy. At 3, 5 d, teams with 10-7mol/L 1α,25-dihydroxyvitamin D3 remarkably inhibited the ALP activtiy (P<0.01), the other teams had no significant differences(P>0.05). In conclusion, 1α,25-dihydroxyvitamin D3 can inhibit proliferation, low dosage of can inhibit differentiation, and vice versas.3 Effects of 1α,25-dihydroxyvitamin D3 on cell cycle and [Ca2+]i of osteoblasts cultured in vitroIn order to observe the effects of 1α,25-dihydroxyvitamin D3 on cell cycle and [Ca2+]i of osteoblasts cultured in vitro, Obwere isolated from calvaria bone, then dealt with various concentration of 1α,25-dihydroxyvitamin D3 (0, 10-9, 10-8, 10-7 mol/L). After 20 min and 24 h treated by 1α,25-dihydroxyvitamin D3, [Ca2+]i was evaluated. 48 h incubation later, the changes of cell phase was analyzed using flow cytometry. Compared with the control group, [Ca2+]i in group with 1α,25-dihydroxyvitamin D3 were all increased significantly (P<0.05) at time 20 min. 24 h incubation later, [Ca2+]i in the group with 10-9 mol/L 1α,25-dihydroxyvitamin D3 was the lowest (P<0.05). After 48 h, 10-9,10-8,10-7 mol/L 1α,25-dihydroxyvitamin D3 caused G2/M arrest. The result provids theoretical base for explaining 1α,25-dihydroxyvitamin D3 inhibits proliferation of OB.3 Effects of 1α,25-dihydroxyvitamin D3 on the morphous and ultrastructure of osteoblasts cultured in vitroTo investigate the effects on the development and ultrastructure of SD rat osteoblasts (OB) under different 1α,25-dihydroxyvitamin D3 levels in vitro. After 48 h, the morphous and ultrastructure were observed using scanning electron microscope (SEM) and transmission electron microscope (TEM) independently. The results showed that, compared with the control group, more microvillus and mitochondria were observed in the group with 10-9 mol/L 1α,25-dihydroxyvitamin D3. In the group with 10-8 mol/L 1α,25-dihydroxyvitamin D3, osteoblasts became flatter, and contained abundant slender cytoplasmic processes and endoplasmic reticula. Lots of filiform fibers forming network in the Extracellular Matrix of OB, secretory vesicles and calcium granule, less organelles were observed in the group with 10-7 mol/L 1α, 25-dihydroxyvitamin D3. In conclusion, the present study verified further morphologically that high concentration of 1α,25-dihydroxyvitamin D3 has obviously facilitative effects on differentiation and functional expression of SD rat osteoblasts cultured in vitro.
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