The Study of the Mechanism and the Influence of Osteogenesis Activity by Hepcidin on Osteoblast
|Keywords||osteoblasts iron metabolism bone metabolism cell proliferation Hepcidin Osteoblasts Proliferation Apoptosis Mineralization Calcium ion L-type Ca2+ channels|
The main reason of osteoporosis is bone metabolic abnormalities, The prevention of osteoporosis is a issues with both traditional and continuous development of multiple disciplines and factors. Involves prevention, treatment method is mainly calcium based intervention, osteoblasts intervention and osteoclasts intervention, etc. Within the many influencing factors of bone metabolism disorders, The research of iron metabolism and bone metabolism are very encouraging and rejoicing in recent years. The research report between "iron ion" metabolism and osteoporosis relations is increasing, many results have clinical significance,.At present, the mainly view of this field is （1） Most older women with iron overload; （2） In-vitro, in-vivo and clinical research data show iron overload and osteoporosis index is closely related; （3） Using the method of reduce iron overload can be significantly improved the indicators of osteoporosis;（4） The method of download the iron overload is hepcidin which is found in 2001 and have good clinical application prospect.Our purpose is to study the influence of Hepcidin and iron ion concentrations change out of hFOB1.19 cells. We use iron ion and calcium ion as vehicles to study the the relationship among Hepcidin ,ferric metabolism and bone metabolism at cellular and molecular 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, then observe the cell proliferation, apoptosis, calcification activity, the change of calcium ion concentrations and the change of calcium ion channels inside hFOB 1.19. To illustrate the signifiance of iron steady for bone metabolism disorders such as osteoporosis disease to provide the experience basis.This study is divided into three parts as below: Objective To observe effect of Hepcidin, DFO, FAC on proliferation of osteoblast （hFOB1.19）, to To study the effect iron in osteoporosis. Methods Osteoblast was cultured in medium with different concentration of Hepcidin（0,100,200,400, 800, 1200 nmol/L）,DFO（0,2,10,100,200μmol/L）,FAC（0,2,10,100,200μmol/L）.Cell proliferation was detected by MTT method. Results Hepcidin inhibited proliferation of osteoblast at concentation of 1200 nmol/L, had no effect on proliferation of osteoblast at concentation of concentration of 100 nmol/L and 800 nmol/L, DFO and FAC inhibited proliferation of osteoblast at concentation of 10μmol/L and 200μmol/L. Conclusion The DFO and FAC can inhibit the proliferation of human osteoblast cells in vitro depending on its concentration, and Hepcidin had no effect on proliferation of osteoblast at a low concentration,it can inhibit the proliferation of human osteoblast cell at a high concentration.Objective To observe Effect of hepcidin on proliferation, apoptosis and mineraliation of osteoblast（hFOB1.19）. Methods The osteoblasts were divided into 3 groups:blank control group, 100 nmol/L hepcidin, 200 nmol/L hepcidin. With hepcidin for 48 hours, the ability of celI proliferation was evaluated by MTT, the rate of apoptosis was detected by Annexin V/PI staining and flow cytometry, and with hepcidin for 15 days the mineralization of hFOB1.19 was evaluated calcified nodules Von Kossa staining. Results The ability of celI proliferation treated with hepcidin has no significant difference compare with blank control group（P>0.05）. The rate of apoptosis decreased obviously and showed a dose-dependent manner after treated with 100 nmol/L, 200 nmol/L hepcidin, it has a significant difference compare with a blank control group （P<0.01）. The amounts of calcified nodes increased after treated with 100 nmol/L, 200 nmol/L hepcidin has a dose-dependent manner compare with blank control group （P<0.01）. Conclusion Hepcidin can decrease rate of apoptosis in hFOB1.19, improve the mineralization of hFOB1.19,but it has no effect on proliferation of hFOB1.19.Objective To study the role of L-type Ca2+ channels in calcium influx stimulated by hepcidin in osteoblast hFOB1.19. Methods Experiments were divided into three departments: 1. hFOB1.19 were treated with. hepcidin （100 nmol/L）and double-distilled water for 1 hour respectively; 2. hFOB1.19 were treated with Nimodipine （2×10-5 mol/L） for 10 minutes, followed by 60 minutes treatment by hepcidin （100 nmol/L） and double-distilled water ; 3. hFOB were treated with EDTA （2×10-3 mol/L） for 10 minutes, after which they were treated with Hepcidin （100 nmol/L） and double-distilled water for 60minutes. After cell collection and dyed with fluo-3/AM for 40 minutes, the fluorescence of intracellular calcium was observed with flow cytometry （FCM）. Results Compared with control group, hepcidin can increase the fluorescence of intracellular calcium obviously （P<0.01）; while no significant increase in fluorescence of intracellular calcium were seen in the two groups pretreated with Nimodipine and EDTA when they were compared with the group treated with double-distilled water （P>0.05）. Conclusion Hepcidn-stimulated elevation of intracellular calcium is due in mainly part to activation of L-type Ca2+ channels.