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BACKGROUND: Previous studies demonstrated that eucommia bark can promote bone marrow stern cells (BMSCs) differentiated into osteoblasts, but relative mechanism is poorly understood. OBJECTIVE: To investigate the effects of eucommia bark water/methanol extracts on expressions of osteopontin (OPN) and osteoprotegedn (OPG) in rat BMSCs. METHODS: Totally 2 g eucommia bark powder were added into water or methanol to 16 mL and oscillated for 1 hour at room temperature. After soaked overnight, both extracts were centrifuged at 15 000 r/min for 10 minutes. Water extract was obtained from supernatant in water soaked powder. In methanol soaked powder, methanol extracts was obtained by concentrated supernatant in vacuo and resolved using 16 mL water. Water and methanol extracts were then filtered by 0.22 μm membrane, and conserved at -20℃. Six SD rats, aged 2 months, were selected, and the 3~(rd)passage of BMSCs were induced by water or methanol extracts with dilution of 1 × 10~(-2), 1 × 10~(-3), 1 × 10~(-4) and 1 × 10~(-5), respectively. PBS was added in the negative control group. All cells were cultured for 6 days. Expressions of OPN and OPG was measured by immunocytochemistry at 6 days with induction. The expression of OPN and OPG induced by water and methanol at 1 ×10~(-3) and 1×10~(-4) dilution was detected by RT-PCR. RESULTS AND CONCLUSION: Immunocytochemistrical results indicated that both water and methanol extracts of eucommia bark simulated OPN and OPG expression, in particular with dilution of 1×10~(-4). The methanol extracts had a stronger effect than water extract, but the expression of OPG did not change obviously. RT-PCR demonstrated that at the 3rd day of inducement, the level of OPN expression induced by water extract was higher than that of methanol extract, and no OPG expression was detected. Osteogenic differentiation of rat MSCs induced by eucommia bark water/methanol extracts relates to stimulating expression of OPN, which has no correlation to OPG. OPN expression induced by water extract is early than that of methanol extract.
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Objective: To study the effect of dexamethasone in differentiating human glioma cell line .Methods: The human glioma cell line was incubated with l mg/L dexamethasone in 1640 culture medium with 10% vitulary serum for 48 hours. The cell form was observed by contrapositive microscope,and PHA was used to induce the agglutination of these cells.Mitotic index and AgNOR amount was counted. The expression of GFAP was detected by immunocytochemisty.Rusults: SHG 44 cells incubated with dexamethasone adhered to plate firmly and its shape became astroid .The agglutination degree and mitotic index decreased significantly .The Ag NOR was atrophied and its amount decreased.The Immunocytochemistry showed the content of GFAP increased sign ficantly.Conclusion: Dexamethasone plays a role in differentiating human glioma cell.
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Objective To observe expression of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) in nerve and dominated muscle of rat after injury of sciatic nerve.Methods 12 female SD rats were divided into 4 groups, right sciatic nerve was squeezed by using forceps for 0.5hr, 1hr, 2hr and 5hr respectively. Then right sciatic nerve and gastrocnemius was isolated and RNA was extracted by using Trizol reagent and meanwhile, the left sciatic nerve and gastrocnemius was used as a normal control. NOS expression was detected by using RT PCR and RNAase protection assay (RPA), and GAPDH was used as an internal standard. The density of PCR and RPA bands was determined by using NIH image software.Results 2 NOSs did not vary in nerve tissue in 4 groups, but in muscle, nNOS increased in 1h group, decreased in 2h group and increased in 5h group; iNOS decreased in 1h and 2h groups but increased in 5h group when compared to normal control.Conclusion Injuring of nerve does not effect NOS expression itself within short period, but effects the dominated muscle via the transmission of non NOS nerve signal.