| Objective: To observe the effect of different concentration osteogenic growth peptide (OGP) on the proliferation and differentiation of osteoblast obtained from the rib of newborn cow in vitro.Methods: To obtain osteoblasts from neonatal cow's rib adopting the method of tissue culture, conduct primary culture and sub-culturing, take the second generation cells as experimental model after having been identified by means of morphological observation and drum dyeing of ALK etc, and then co-culture these cells with different concentrations of the OGP. The effect of OGP on cell proliferation was measured by MTT method (OD value at wave-length 490nm), on cell differentiation was measured by Golden's method (ALP activity), and on cell secretion bone gla protein was observed by means of radicalization immunity (RI method, expressed by ng/ml). The effect of OGP on the expressions of osteocalcin mRNA and type I collogen were dectected by semin quantitative reverse transcription polymerase chain reaction (RT-PCR.).Results: The cells obtained from the culture in vitro have have typical osteoblasts morphological character, the masccline rate of ALP can reach 94%, and accord with the biological characteristic, suggesting that the cell obtained were high-purity osteoblasts. The sequent of cells multiplication after having been added the OGP showed that: different concentrations of OGP can promote osteoblast's growth, however, the concentrations from 10-12mol/L to 10-8 mol/L promote cells growth function present the dosage - effect relations. Compared with the control group, even the OGP of 10-12mol/L concentration can promote the osteoblast growth, and the test groups at the concentration ranging from 10-8mol/L to 10-11mol/L have the significant difference (P< 0.05) when compared with the control group, especially at 10-10mol/L concentration (P< 0.01), whereas, the effect showed the drop tendency at the concentration of 10-9mol/L, and with the OGP's concentrations increasing, the stimulation at the osteoblasts growth hasn't been strengthened any longer. Besides this, the effect of OGP at the concentration of 10-12mol/L to 10-8mol/L on promoting cells growth aslo present the time - effect relations, the absorbance value increase along with extension of response time, and the value can reach the highest when the osteoblasts were cultivate for 5 days, but the effect began to drop gradually at 7d. After having been added OGP to raise for 5 days, the alkaline phosphatase activities in the cell nutrient fluid was measured and the result showed that, compared with the control group, the effect of different concentration OGP on the promotion in osteoblast differentiation is significant (P< 0.05), the difference is OGP displays the inhibitory action on the osteoblast differentiation moreover, the inhibitory action presents the converse normal shape, takes on the dosage bi-directional capability in the suppression, the inhibitory action is remarkable at the concentration of 10-10 mol/L . After having been added OGP to raise for 5 days, the bone calcium element in the cell nutrient fluid conten was determine, the result indicated that: the effect of different concentration of OGP is not significant when compared with the control group (P> 0.05). The BGP in the cell supernate also has inhibitory action, and the action presents the dosage bi-directional capability, it is significant at the concentration of 10-10 mol/L (P< 0.05). After having been affected by OGP for 15 days, the cells were used for RNA extraction, then the RNA was carried on RT-PCR to test the mRNA of the bone calcium element mRNA and type I Collagen, all the concentration of 10-8 mol/L, 10-10 mol/L and 10-12 mol/L test group can promote the two to express.Conclusion: Although the effect of OGP on the osteoblast differentiation is not obvious, it can promote cow osteoblast bone calcium element mRNA, I Collagen mRNA to express and promote osteoblast growth. Therefore, OGP has the ruction of adjusting cow osteoblast, promotes the bone, the experiment also indicated that the cow experimentation may be used as the model of the rodentia animal. |
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