Effects Of Hypoxia In Rat Osteoblasts In Vitro

Osteoporosis is a global problem which is increase significantly in the whole world especially in aged population. Several factors are considered to relate with this disease. Among them, effects of hypoxia gain much attention recently with the increasing of population in high altitude area and medicine. The studies induced that hypoxia could promote osteoporosis. However, it is still uncertain whether hypoxia exert protective effects on bone in rat. In this study, cell culture was used to observe the effects of hypoxia on the biological it functions of rat osteoblasts and investigate the possible mechanism of such effects.Part One Identification of rat osteoblasts cultured in vitroObjective: To culture and identify the identification in vitro function of the rat osteoblasts(ROB)which were harvested from the trabecular bone of normal subjects.Method: The ROBs were isolated from newborn Wistar rat calvarias by trypin and collagenase digestion. Mineralized nodes and the expression of alkaline phosphatase were studied with alizarin red-S staining and modified Gomori's assays, respectively.Result: The ROB cells purified by enzyme digestion functioned normally in vitro as observed in vitro. They showed the osteoblastic phenotypes such as their synthesis of alkaline phosphatase and osteocalcin and formation of mineralized nodes.Conclusion: The study indicates it is easy to purify plentiful ROBs with normal functions by enzyme digestions. Which can be used in the next research of bone metabolism. Part Two The effects of hypoxia on proliferation,differentiation, content of mineralization in ROBs in vitroObjective: To study the effects of Hypoxia in cultured osteoblasts in vitro.Method: MTT, PNPP and ARS were used to observe the proliferation, activity of ALP and the number of mineral nodes of cultured in vitro.Result: It was found that hypoxia had the effects on restraining the proliferation, ALP activity, decreasing the number of mineral nodes of cultured osteoblasts.Conclusion: Hypoxia has the effects on restraining the proliferation, differentiation and mineralization of cultured osteoblasts in vitro. This may be an important mechanism that hypoxia promotes osteoporosis occur.Part Three The effects of hypoxia on apoptosis in ROBsObjective: To study the effects of Hypoxia in osteoblasts apoptosis.Methods: the nucleic acid-bind dye acridine orange were used to observe the proliferation and apoptosis of cultured in vitro.Results: It was found that Hypoxia had the effects on restraining the proliferation, decreasing the apoptosis of cultured osteoblasts.Conclusion: Hypoxia can inhibit the proliferation of osteoblasts and it can also facilitate the apoptosis of osteoblasts. This may be an important mechanism that hypoxia promotes osteoporosis occur.Part Four The effects of hypoxia on the gene expression in ROBsin vitroObjective: To observe the regulation effects of hypoxia on the expression of Cbfa1,COL1a₁ BGP and IGF-1 in ROBs and to investigate the important role of hypoxia played in the process of bone tureover. Methods: The expression of Cbfa1,COL1a₁,BGP and IGF-1 was measured by RT-PCR in ROB cells treated with hypoxia at different time-course of culture.Results: (1) Hypoxia upregulated Cbfa1,BGP,COL1a₁, expression in ROBs ; but had no effects on its expression of IGF-1 in ROB cells. (2) In ROB cells the expression of Cbfa₁ mRNA,BGP mRNA and COL1a₁ mRNA decreased in a time-dependent fashion, and with the time extending in hypoxia the expression descend obviously.Conclusion: (1) Hypoxia can decrease the ability of bone formation and delay the differentiation of ROB cells though decreasing the expression of Cbfa₁ mRNA,BGP mRNA and COL1a₁ mRNA in ROB cells. This may be an important mechanism that hypoxia promotes osteoporosis occur. (2) The mechanism had no effects on its expression of IGF-1 in ROB cells.