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Effects of Hypoxia in Rat Osteoblasts in Vitro

Author: GuJiuJun
Tutor: LiuXingYan
School: Lanzhou University
Course: Surgery
Keywords: Rat osteoblast Enzyme digestion. Appraisal Hypoxia Osteoblasts Proliferation Differentiation Mineralization Apoptosis Gene Expression
CLC: R363
Type: Master's thesis
Year: 2007
Downloads: 128
Quote: 0
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Abstract


Hypoxia on the mechanism of action of rat osteoblasts gradually increased with the aging of the human society, the incidence of osteoporosis, the high incidence of the disease has become an aging society. There are many influencing factors of osteoporosis, With the rapid increase of the population of the plateau, the development of High Altitude Medicine plateau hypoxia on the pathogenesis of osteoporosis, more and more attention. Studies show that the plateau hypoxia can promote the occurrence of osteoporosis, but the specific mechanism of action is not clear. In this paper, through the method of in vitro cell culture studies hypoxia rat osteoblast function and possible mechanisms. The first part of the rat osteoblast culture and identification purposes: the rats skull isolated osteoblasts from newborn 24hWistar enzyme digestion, and cultured in vitro and functional identification to prepare for further study. Method: osteoblasts isolated from the newborn within 24h Wistar rat skull - collagenase digestion with trypsin, azo coupling method for the expression of alkaline phosphatase in osteoblasts, calcified nodules with alizarin red tetracycline notation, Von Kossa improved staining. Results: newborn within 24h Wistar rat osteoblast culture, convenient and easy detachable train a large number of high-purity rat osteoblasts and osteoblasts in vitro passage, retained their table by enzymatic digestion type characteristics for further research. The second part of the impact of hypoxia on rat osteoblast proliferation, differentiation and mineralization of Objective: To investigate the impact of hypoxia on cultured osteoblast proliferation, differentiation and mineralization. Methods: MTT assay of the nitrophenylphosphate law and alizarin red staining method to detect the proliferation rate of osteoblasts, AKP expression and number of mineralized nodules. Results: Hypoxia osteoblast proliferation, AKP activity and mineralized nodule formation significantly lower number, and more significantly reduced with prolonged hypoxia time. CONCLUSION: Hypoxia inhibits osteoblast proliferation and differentiation and maturation in vitro culture, to extend the mineralization time, reduced bone formation, bone formation and resorption imbalance, which may be an important hypoxia formed to promote osteoporosis pathogenesis. The third part of hypoxia on rat osteoblast apoptosis Objective: To investigate the impact of hypoxia on cultured osteoblast apoptosis. Methods: TEM observations, flow cytometry (FCM) and acridine orange staining microscopic changes and apoptosis rate of osteoblasts. Results: (1) hypoxia promotes osteoblast apoptosis generation; (2) hypoxia increased osteoblast apoptosis rate was positively correlated with hypoxia time. CONCLUSION: Hypoxia promotes cultured in vitro osteoblast apoptosis significantly reduced, so that the number of osteoblasts, the bone forming ability. This may be an important mechanism for hypoxia promote osteoporosis. Part IV of hypoxia on gene expression in rat osteoblasts Objective: To observe the to express Cbfa1, CoL1α 1 , BGP, IGF-1 the impact of hypoxia on rat osteoblasts (ROB) explore an important role in regulating bone metabolism. Method: using RT-PCR assay of Cbfa1, CoL1α 1 , BGP, IGF-1 gene expression. Results: (1) hypoxia can down rat bone cells Cbfa1, CoL1α 1 , BGP gene expression, expression of IGF-1, but does not affect rat osteoblasts. (2) hypoxia down rat bone cells of Cbfa1 was CoL1α 1 BGP gene expression in a time-dependent manner, with prolonged hypoxia time gene expression decreased. Conclusion: (1) hypoxic rat osteoblasts of Cbfa1 CoL1α 1 BGP gene expression is reduced, so that the bone-forming ability decreased, osteoblast differentiation and maturation delay. This may be an important mechanism for hypoxia promote osteoporosis. (2) hypoxia to promote osteoporosis occurs role of IGF-1 gene expression has nothing to do.

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