| The abnormality of bone metabolism is the cause of osteoporosis. Recently, the study of the relationship between ferric and bone metabolism is enheartening and joyful. Hepcidin is a type of antimicrobial polypeptide which is rich in aminothiopropionic acid. In 2001, Park separated this polypeptide from urina hominis, and named it Hepcidin. Many researches show that Hepcidin can regulate ferric metabolism as one type of hormone-like substance.Our purpose is to study the influence of Hepcidin and iron ion concentrations change out of hFOB1.19 cells on Ca2+ transportation. We use iron ion and calcium ion as vehicles to study the relationship among Hepcidin, ferric metabolism and bone metabolism at cellular level. The result of this study could be the base of the instructions for curing osteoporosis by Hepcidin. The hFOB 1.19 was cultured in the nutrient medium with Hepcidin in different concentrations, and the change of iron and calcium ion concentrations inside hFOB 1.19 was tested in different experimental session to study the influence of Hepcidin out of osteoblasts on iron and calcium transportation. This study is divided into three parts as below:Part one: The Study on the Influence of Hepcidin out of Osteoblasts on iron Transportation0bjective To study the influence of Hepcidin of different concentration out of hFOB1.19 cells on iron transportation in the different time. Methods Use Hepcidin to impact the hFOB1.19 cells, and the fluorescence intensity of iron was observed by cofocal laser scanning microscope (CLSM). Results (1) The transient group: both of the fluorescence intensity of iron decreasedslowly in the control group and the experiment group, there was not significant different between them. (2) The long-time group: At the range of 0-100 nmol/L, the fluorescence intensity of iron ion in hFOB 1.19 was increased with the increase of Hepcidin concentration out of cells. When the Hepcidin concentration exceed 100 nmol/L, the fluorescence intensity of iron was not increased anymore. Conclusion (1) The instantaneous influence of Hepcidin on iron transportation is not significant. (2) The iron concentration was increased significantly by adding Hepcidin out of hFOB 1.19 cells 20 hours later, and there was a dose dependent relationship between them when the Hepcidin concentration is in the range of 0-100 nmol/L.Part two: The instantaneous influence of Hepcidin on iron ion and calcium ion in human osteoblasts0bjective To study the instantaneous influence of Hepcidin concentration change out of hFOB1.19 cells on calcium and iron transportation, and the relationship between calcium and iron transportation. Methods hFOB1.19 cells were inoculated to the 12-well culture plates with coverslip and grown at 34℃in a humidified atmosphere containing 5%CO2. the cells were stained respectively with calcium and iron fluorescein stain when the cell density was 60%-70%, and observed the fluorescence intensity with confocal laser scanning microscope (CLSM). Added D2H2O into the control group and Hepcidin (the final concentration is 100nM/L) into the experiment group, then recorded the fluorescence intensity change of the calcium and iron. Results (1) The fluorescence intensity change of calcium: the fluorescence intensity of calcium increased and formed a calcium wave in the experiment group, while the fluorescence intensity decreased slowly in the control group, the change tendency was different between them. (2) The fluorescence intensity change of iron: both of the fluorescence intensity of iron decreased slowly, and there was not significant different between them. Conclusion (1) The calcium instantaneous transportation was increased with the increase of Hepcidin concentration out of hFOB 1.19 cells. (2) The instantaneous influence of Hepcidin on iron ion is not significant. (3) The instantaneous influence of Hepcidin on calcium transportation was not iron-dependent. Part three: The expression of ferroportin1 in human osteoblasts(hFOB1.19)and its significance0bjective To study the expression of ferroportin1 and the influence of Hepcidin on iron transportation in human osteoblasts(hFOB1.19). Methods RT-PCR was used to examine the expression of ferroportin1 and observed the fluorescence intensity of iron ion with confocal laser scanning microscope (CLSM). Results Ferroportin1 mRNA was expressed in hFOB1.19, and the fluorescence intensity of iron ion of the control group is7.21±2.78 (A0 group), while it is 20.85±5.40 (A1 group), and35.54±9.23 (A2 group) in the experiment group respectively. Conclusion The ferroportin1 protein may express in hFOB1.19 and that might have a role in iron export from hFOB1.19 cells. |
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