Effect Of Phosvitin On Bone Development Of Chicken Embyos

Phosvitins possess the highest level of phosphorylation of all known proteins in eggs, and it contains approximately80%of an egg yolk’s phosphorous. Phosvitin serves as the main source of phosphorus for chicken embryo bone formation. The research object of this diploma paper was phosvitin. We focused on the effects of phosvitin on chicken embryonic skeletal development and carried out a series of work. The main research content and research results are summarized as follows:We studied the variation of skeletal-related indicators during incubation. Experimental results show that9-10d embryo age was a turning point:Albumen pH gradually stabilized after9d, keep at pH7.5; yolk pH had a sudden rise in10d, reached a maximum8.19, and after a slight decline it was relatively stable in7.5; Yolk phosphorus content appeared sharp decline in10d,these were associated with that embryos begined to form bone at9d. Comparative analysis of Phosphorus loss、ALP activity and body length’s growth rate Found that the three are basically the same trends, First increased and then decreased. The fastest Phosphorus loss occurred at days13-16, reaching a maximum of0.027mg/g.d, ALP activity initially increased, reaching a maximum of1.12U/g prot, the growth rate increased significantly at days17-18, at1.1cm/d The growth rate growth rate lagged behind phosphorus loss and ALP activity, these were because yolk phosphorus transferred to embryos and then use by embryonic bone development needes time.We studied the changes of phosvitin during9-20d embryo age. The results of Gel electrophoresis showed that phosvitin’s molecular weight decreased, during embryonic development; Analysis of P/N ratio and infrared spectra proved that phosvitin’s phosphate levels decreased during incubation; through analysis infrared spectroscopy date and circular dichroism date, we found thatγ-random coil and β-turn structure transformed to the a-helix structure during incubation, phosvitin tends ordering. In summary, there were correlated changes in the P/N ratio and secondary structure of phosvitin, ALP activity, and body length growth. ALP activity and body length growth slightly lagged behind changes in the P/N ratio and the secondary structure of phosvitin. Phosvitin’s phosphate content decreased, the γ-random coil and β-turn gradually transformed into a-helixes, and the protein’s secondary structure tended to become more orderly; these changes mainly occurred on days13-16. Bone formation of the chicken embryos occurred primarily on days14-18, whereas ALP activity and body length growth increased substantially. All of these findings demonstrate that the phosphorus in phosvitin is involved in chicken embryo bone formation through dephosphorylation.Cultured MC3T3-E1cell were treated with phosvitin in different concentrations(0、50、100、500、1000ug/mL) for24h、48h. Cell proliferation was assessed by CCK-8colorimetric assay. Cell cycle and apoptosis were assessed by flow cytometry analysis. The results showed that:low concentrations of protein can be found to promote proliferation and inhibit apoptosis, high concentrations of proteins inhibit cell proliferation and induce apoptosis. After the cells were treated with100μg/mL phosvitin for48h, cell proliferation activity reached108.61%, the cell proliferation rate reached23.49%, which is significantly higher then control and high concentrations. At the same time, cells undergoing apoptosis was6.42%, the apoptosis rate was2.47%, which is significantly lower than the control group and the high concentration group.Cultured MC3T3-E1cell were treated with different Mineralization inducers (control、 Pv、Pv+Vc、Pv+β-GP、Vc+β-GP), ALP activity in cells, mineralized nodules content and related gene expression levels was measured during induction of mineralization. The experiments first established optimal seeding density is1×104/cm2. The results show that phosvitin alone can not induce cell mineralization but can promote osteoblast cell activity: ALP activity reached1.0U/gprot at21d, significantly higher than the negative control group, and the expression level of Col-I mRNA, OCN mRNA increased significantly, while the expression level of Runx-2mRNA, BMP-2mRNA almost no change, and alizarin red staining was negative. Pv+β-GP groups can induce mineralization, ALP activity reached2.1U/gprot at21d, slightly lower than the positive control groups, and the expression level of Runx-2mRNA, BMP-2mRNA, Col-I mRNA, OCN mRNA all increased significantly, and alizarin red staining results were positive. Compared with the positive control group, the measurement results of Pv+β-GP groups were not significantly different. We speculate that phosvitin replaced Vc as a powerful antioxidant/reducing agent involved in the synthesis of collagen, thereby promoting osteoblast activity during the induction of mineralization. Phosvitin probably through upregulating the expression of Col-I mRNA and OCN mRNA, promote the expression levels of ALP increased, thus enhancing the mineralization activities. But the detailed cytological and molecular mechanisms of phosvitin’s impact on osteoblasts need further study.