【摘要】： 缺氧對(duì)大鼠成骨細(xì)胞作用機(jī)制的研究 隨著人類社會(huì)的老齡化，骨質(zhì)疏松癥的發(fā)病率逐漸增高，已經(jīng)成為老齡化社會(huì)的高發(fā)病率疾病。骨質(zhì)疏松癥發(fā)生的影響因素有很多，而隨著高原人口的迅速增加，高原醫(yī)學(xué)的發(fā)展，高原缺氧對(duì)骨質(zhì)疏松癥發(fā)病的影響也越來(lái)越受到關(guān)注。研究表明高原缺氧可以促進(jìn)骨質(zhì)疏松癥的發(fā)生，但具體的作用機(jī)理目前尚不清楚。本文擬通過(guò)體外細(xì)胞培養(yǎng)的方法研究缺氧對(duì)大鼠成骨細(xì)胞功能的影響以及可能的機(jī)制。 第一部分大鼠成骨細(xì)胞的培養(yǎng)和鑒定 目的：用酶消化法從新生24hWistar大鼠顱骨中分離成骨細(xì)胞，進(jìn)行體外培養(yǎng)和功能鑒定，為進(jìn)一步研究作準(zhǔn)備。 方法：用胰酶-膠原酶消化法從新生24h內(nèi)Wistar大鼠顱骨中分離成骨細(xì)胞，用偶氮偶聯(lián)法顯示成骨細(xì)胞中堿性磷酸酶的表達(dá)，鈣化結(jié)節(jié)用茜素紅法、四環(huán)素標(biāo)記法、Von Kossa改良法染色。 結(jié)果：用酶消化法進(jìn)行新生24h內(nèi)Wistar大鼠成骨細(xì)胞培養(yǎng)，方便易行，可分離、培養(yǎng)大量高純度大鼠成骨細(xì)胞，這些成骨細(xì)胞在體外傳代后，仍保留了它們的表型特征，可供進(jìn)一步研究使用。 第二部分缺氧對(duì)大鼠成骨細(xì)胞增殖、分化及礦化功能的影響 目的：探討缺氧對(duì)體外培養(yǎng)成骨細(xì)胞增殖、分化及礦化功能的影響。 方法：應(yīng)用MTT法、對(duì)硝基苯磷酸鹽法及茜素紅染色方法檢測(cè)成骨細(xì)胞的增殖率、AKP表達(dá)及礦化結(jié)節(jié)數(shù)量。 結(jié)果：缺氧使成骨細(xì)胞增殖率、AKP活性及礦化結(jié)節(jié)形成數(shù)量明顯降低，并隨著缺氧時(shí)間的延長(zhǎng)降低更加顯著。 結(jié)論：缺氧抑制了體外培養(yǎng)成骨細(xì)胞的增殖及分化成熟，延長(zhǎng)了礦化時(shí)間，使骨形成能力降低，，骨形成與骨吸收失衡，這可能是缺氧促進(jìn)骨質(zhì)疏松癥形成的一個(gè)重要的發(fā)病機(jī)制。 第三部分缺氧對(duì)大鼠成骨細(xì)胞凋亡的影響 目的：探討缺氧對(duì)體外培養(yǎng)成骨細(xì)胞凋亡的影響。 方法：應(yīng)用透射電鏡觀察、流式細(xì)胞技術(shù)(FCM)及丫啶橙染色法檢測(cè)成骨細(xì)胞的顯微變化及凋亡率。 結(jié)果：(1)缺氧促進(jìn)了成骨細(xì)胞內(nèi)凋亡小體的產(chǎn)生；(2)缺氧提高了成骨細(xì)胞的凋亡率，并與缺氧時(shí)間呈正相關(guān)。 結(jié)論：缺氧促進(jìn)了體外培養(yǎng)成骨細(xì)胞的凋亡，使成骨細(xì)胞數(shù)量明顯降低，骨形成能力下降。這可能是缺氧促進(jìn)骨質(zhì)疏松癥發(fā)生的一個(gè)重要機(jī)制。 第四部分缺氧對(duì)大鼠成骨細(xì)胞基因表達(dá)的影響 目的：觀察缺氧對(duì)大鼠成骨細(xì)胞(ROB)表達(dá)Cbfa1、CoL1α_1、BGP、IGF-1的影響，探討它們?cè)谡{(diào)節(jié)骨代謝過(guò)程中的重要作用。 方法：應(yīng)用RT-PCR法檢測(cè)Cbfa1、CoL1α_1、BGP、IGF-1基因表達(dá)情況。 結(jié)果：(1)缺氧能下調(diào)大鼠成骨細(xì)胞中Cbfa1、CoL1α_1、BGP基因的表達(dá)，但不影響大鼠成骨細(xì)胞中IGF-1的表達(dá)。(2)缺氧下調(diào)大鼠成骨細(xì)胞中Cbfa1、CoL1α_1、BGP基因的表達(dá)呈時(shí)間依賴性，隨缺氧時(shí)間的延長(zhǎng)基因表達(dá)明顯下降。 結(jié)論：(1)缺氧使大鼠成骨細(xì)胞Cbfa1、CoL1α_1、BGP基因表達(dá)降低，從而使骨形成能力下降，成骨細(xì)胞分化成熟延緩。這可能是缺氧促進(jìn)骨質(zhì)疏松癥發(fā)生的一個(gè)重要機(jī)制。(2)缺氧促進(jìn)骨質(zhì)疏松癥發(fā)生的作用與IGF-1基因的表達(dá)無(wú)關(guān)。
[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
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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