缺氧对大鼠成骨细胞作用机制的研究

发布时间：2019-05-26 18:41

【摘要】： 缺氧对大鼠成骨细胞作用机制的研究随着人类社会的老龄化，骨质疏松症的发病率逐渐增高，已经成为老龄化社会的高发病率疾病。骨质疏松症发生的影响因素有很多，而随着高原人口的迅速增加，高原医学的发展，高原缺氧对骨质疏松症发病的影响也越来越受到关注。研究表明高原缺氧可以促进骨质疏松症的发生，但具体的作用机理目前尚不清楚。本文拟通过体外细胞培养的方法研究缺氧对大鼠成骨细胞功能的影响以及可能的机制。第一部分大鼠成骨细胞的培养和鉴定目的：用酶消化法从新生24hWistar大鼠颅骨中分离成骨细胞，进行体外培养和功能鉴定，为进一步研究作准备。方法：用胰酶-胶原酶消化法从新生24h内Wistar大鼠颅骨中分离成骨细胞，用偶氮偶联法显示成骨细胞中碱性磷酸酶的表达，钙化结节用茜素红法、四环素标记法、Von Kossa改良法染色。结果：用酶消化法进行新生24h内Wistar大鼠成骨细胞培养，方便易行，可分离、培养大量高纯度大鼠成骨细胞，这些成骨细胞在体外传代后，仍保留了它们的表型特征，可供进一步研究使用。第二部分缺氧对大鼠成骨细胞增殖、分化及矿化功能的影响目的：探讨缺氧对体外培养成骨细胞增殖、分化及矿化功能的影响。方法：应用MTT法、对硝基苯磷酸盐法及茜素红染色方法检测成骨细胞的增殖率、AKP表达及矿化结节数量。结果：缺氧使成骨细胞增殖率、AKP活性及矿化结节形成数量明显降低，并随着缺氧时间的延长降低更加显著。结论：缺氧抑制了体外培养成骨细胞的增殖及分化成熟，延长了矿化时间，使骨形成能力降低，，骨形成与骨吸收失衡，这可能是缺氧促进骨质疏松症形成的一个重要的发病机制。第三部分缺氧对大鼠成骨细胞凋亡的影响目的：探讨缺氧对体外培养成骨细胞凋亡的影响。方法：应用透射电镜观察、流式细胞技术(FCM)及丫啶橙染色法检测成骨细胞的显微变化及凋亡率。结果：(1)缺氧促进了成骨细胞内凋亡小体的产生；(2)缺氧提高了成骨细胞的凋亡率，并与缺氧时间呈正相关。结论：缺氧促进了体外培养成骨细胞的凋亡，使成骨细胞数量明显降低，骨形成能力下降。这可能是缺氧促进骨质疏松症发生的一个重要机制。第四部分缺氧对大鼠成骨细胞基因表达的影响目的：观察缺氧对大鼠成骨细胞(ROB)表达Cbfa1、CoL1α_1、BGP、IGF-1的影响，探讨它们在调节骨代谢过程中的重要作用。方法：应用RT-PCR法检测Cbfa1、CoL1α_1、BGP、IGF-1基因表达情况。结果：(1)缺氧能下调大鼠成骨细胞中Cbfa1、CoL1α_1、BGP基因的表达，但不影响大鼠成骨细胞中IGF-1的表达。(2)缺氧下调大鼠成骨细胞中Cbfa1、CoL1α_1、BGP基因的表达呈时间依赖性，随缺氧时间的延长基因表达明显下降。结论：(1)缺氧使大鼠成骨细胞Cbfa1、CoL1α_1、BGP基因表达降低，从而使骨形成能力下降，成骨细胞分化成熟延缓。这可能是缺氧促进骨质疏松症发生的一个重要机制。(2)缺氧促进骨质疏松症发生的作用与IGF-1基因的表达无关。
[Abstract]:Study on the Mechanism of the Effect of Hypoxia on the Osteoblasts of Rats With the aging of human society, the incidence of osteoporosis is increasing, and it has become an aging The high incidence of osteoporosis has many factors, and with the rapid increase of the population of the plateau, the development of the high altitude medicine, the development of the high altitude medicine and the development of the plateau hypoxia on the pathogenesis of osteoporosis The response is also getting more and more attention. The study shows that the plateau hypoxia can promote the occurrence of osteoporosis, but the specific work The mechanism is not yet clear. In this paper, the function of hypoxia on the rat's osteoblast is studied by the method of in vitro cell culture. Impact and possible mechanisms. The purpose of culture and identification of the first part of rat osteoblasts: the use of an enzyme digestion method to remove the cranium of a 24-hour-old Wistar rat Methods: Osteoblasts were isolated from the cranium of Wistar rats by pancreatin-collagenase digestion. The expression of alkaline phosphatase, Calcification of calcified nodules were stained with the modified method of the red, tetracycline and Von Kossa. Culture a large number of high-purity rat osteoblasts, which are passaged in vitro After that, their phenotypic characteristics were retained for further study and use. The effects of second part of hypoxia on the proliferation, differentiation and mineralization of rat osteoblasts: to explore the effects of hypoxia on the proliferation, differentiation and mineralization of rat's osteoblasts. The effects of culture on the proliferation, differentiation and mineralization of osteoblasts were studied. The rate of bone formation, the expression of AKP and the number of mineralized nodules were detected by plain red staining. Results: The rate of osteoblast proliferation, AKP activity and the number of mineralized nodules were significantly reduced by hypoxia, and with the hypoxia The extension of time is more pronounced. Conclusion: Hypoxia inhibited the proliferation of cultured osteoblasts in vitro. The maturation of the colonization and differentiation, the time of mineralization is prolonged, the bone formation capacity is reduced, the bone formation and the bone resorption are out of balance, which may be hypoxia and osteoporosis The effect of the third part of hypoxia on the apoptosis of rat osteoblasts Objective: To study the effect of hypoxia on the apoptosis of cultured osteoblasts in vitro. Lens Observation, Flow Cytometry (FCM) and Lamma's Orange Staining The changes of osteoblast and the rate of apoptosis were measured. Results: (1) Hypoxia promoted the production of apoptotic bodies in osteoblasts, and (2) hypoxia increased the rate of apoptosis and was positively related to the time of hypoxia. Conclusion: Hypoxia promotes the apoptosis of cultured osteoblasts in vitro, and the number of osteoblasts is significantly reduced and the bone formation ability is decreased. This may be An important mechanism of hypoxia to promote the occurrence of osteoporosis. The effect of the fourth part of hypoxia on the expression of the rat osteoblast gene : To observe the effects of hypoxia on the expression of Cbfa1, CoL1, BGP and IGF-1 in rat osteoblasts (ROB), and to study the effect of hypoxia on the regulation of bone metabolism. Methods: The expression of Cbfa1, CoL1, BGP and IGF-1 was detected by RT-PCR. Results: (1) Hypoxia can lower the osteogenesis of rats Expression of Cbfa1, CoL1-1 and BGP in the cells, but not large
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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