本文關(guān)鍵詞：膜聯(lián)蛋白A1基因在兔骨髓間充質(zhì)干細(xì)胞體外誘導(dǎo)成骨和成脂早期的表達(dá)變化與意義　出處：《廣西醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 體外誘導(dǎo) 成骨分化 成脂分化 膜聯(lián)蛋白A1(AnnexinA1 ANXA1) 骨髓間充質(zhì)干細(xì)胞 分化 熒光定量PT-PCR

【摘要】：背景：隨著干細(xì)胞在臨床上的廣泛應(yīng)用,骨髓間充質(zhì)干細(xì)胞(Bone mesenchymal stem cells, BMSCs)已經(jīng)成為眾多種子細(xì)胞研究的熱點(diǎn)。目前對(duì)骨髓間充質(zhì)干細(xì)胞的體外分化機(jī)制尚不明確,以往眾多研究集中在骨形態(tài)發(fā)生蛋白上,而對(duì)其他基因的研究尚少,而最近在細(xì)胞分化領(lǐng)域內(nèi)研究的熱點(diǎn)基因膜聯(lián)蛋白A1(Annexin A1)在骨髓間充質(zhì)干細(xì)胞分化中的研究甚少,且研究方法過(guò)于單一,無(wú)法全面的解釋該基因的細(xì)胞分化功能。我們的研究旨在綜合性的探討骨髓間充質(zhì)干細(xì)胞分化過(guò)程中膜聯(lián)蛋白A1基因表達(dá)的變化意義。 第一部分兔骨髓間充質(zhì)干細(xì)胞體外培養(yǎng)與鑒定 目的：體外分離并培養(yǎng)高純度和生長(zhǎng)狀態(tài)穩(wěn)定的骨髓間充質(zhì)干細(xì)胞。 方法：應(yīng)用全骨髓貼壁法分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞,傳代培養(yǎng)純化,并鑒定其特異性表面標(biāo)記物；分別用成骨誘導(dǎo)劑和成脂誘導(dǎo)劑誘導(dǎo)分化骨髓間充質(zhì)干細(xì)胞,檢測(cè)細(xì)胞分化活性。 結(jié)果：骨髓間充質(zhì)干細(xì)胞生長(zhǎng)狀態(tài)及分化活性良好,細(xì)胞表面標(biāo)記物CD44、CD105表達(dá)率達(dá)95%以上,而CD45陰性表達(dá)。 結(jié)論：全骨髓貼壁法培養(yǎng)的骨髓間充質(zhì)干細(xì)胞生長(zhǎng)狀態(tài)良好,易于誘導(dǎo)。 第二部分膜聯(lián)蛋白A1基因在BMSCs成骨分化和成脂分化早期的表達(dá) 目的：(1)研究兔骨髓間充質(zhì)干細(xì)胞(BMSCs)在體外誘導(dǎo)成骨和成脂過(guò)程中膜聯(lián)蛋白A1基因的表達(dá)變化。(2)監(jiān)測(cè)骨髓間充質(zhì)干細(xì)胞在誘導(dǎo)分化過(guò)程中的細(xì)胞生長(zhǎng)狀態(tài)。 方法：(1)實(shí)驗(yàn)組分別加入含成骨誘導(dǎo)劑和成脂誘導(dǎo)劑的培養(yǎng)基,對(duì)照組細(xì)胞以不添加任何誘導(dǎo)劑的培養(yǎng)基培養(yǎng)。(2)分別在3、7天提取細(xì)胞總mRNA,并應(yīng)用熒光定量RT-PCR技術(shù)檢測(cè)各組膜聯(lián)蛋白A1基因表達(dá)情況。(3)應(yīng)用MTT法檢測(cè)細(xì)胞生長(zhǎng)、細(xì)胞流式學(xué)檢測(cè)細(xì)胞增殖情況與凋亡情況。 結(jié)果：(1)膜聯(lián)蛋白A1基因在成骨誘導(dǎo)過(guò)程中與未誘導(dǎo)組相比有明顯下調(diào)趨勢(shì)(3天2-△△CT=0.643±0.076,7天2-△△CT=0.862±0.028,差異有統(tǒng)計(jì)學(xué)意義(P0.01),而在成脂誘導(dǎo)組中則為上升(3天2-△△CT=1.264±0.115,7天2-△△CT=1.860±0.045；(2)成骨誘導(dǎo)液對(duì)MSCs生長(zhǎng)存在抑制作用并增加細(xì)胞凋亡(P0.01),而成脂誘導(dǎo)劑對(duì)MSCs的生長(zhǎng)與凋亡作用甚小。 結(jié)論：(1)成骨誘導(dǎo)液對(duì)骨髓間充質(zhì)干細(xì)胞的生長(zhǎng)起到抑制作用,并可促進(jìn)細(xì)胞凋亡；(2)膜聯(lián)蛋白A1基因可能與骨髓間充質(zhì)干細(xì)胞的體外成脂分化存在一定關(guān)系,但尚不能肯定其在成骨分化中的作用。通過(guò)對(duì)膜聯(lián)蛋白A1基因的研究,有可能為骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞或脂肪細(xì)胞分化的機(jī)制提供線索和新的研究方向。
[Abstract]:Background: bone mesenchymal stem cells has been widely used in bone marrow mesenchymal stem cells. BMSCs has become the focus of many seed cells research. At present, the differentiation mechanism of bone marrow mesenchymal stem cells in vitro is not clear, and many previous studies focused on bone morphogenetic protein. However, there are few studies on other genes, and the recent research on the hot gene, Annexin A1) in the field of cell differentiation, is very few in the differentiation of bone marrow mesenchymal stem cells. And the research method is too single. The purpose of our study was to investigate the significance of the expression of integrin A1 gene in the differentiation of bone marrow mesenchymal stem cells. Part I Culture and Identification of Rabbit Bone Marrow Mesenchymal Stem cells in Vitro Objective: to isolate and culture bone marrow mesenchymal stem cells with high purity and stable growth in vitro. Methods: bone marrow mesenchymal stem cells were isolated and purified by whole bone marrow adherent method, and their specific surface markers were identified. Bone marrow mesenchymal stem cells were induced by osteogenic inducer and lipogenic inducer, respectively. Results: the growth and differentiation activity of bone marrow mesenchymal stem cells was good. The expression rate of CD44-pCD105 was more than 95%, but the expression of CD45 was negative. Conclusion: bone marrow mesenchymal stem cells cultured by whole bone marrow adherent method grow well and are easy to induce. The second part of the expression of annexin A1 gene in the early stage of BMSCs osteogenic differentiation and adipogenic differentiation Objective: to study the expression changes of integrin A1 gene in rabbit bone marrow mesenchymal stem cells (BMSCs) during osteogenesis and adipogenesis in vitro. To monitor the growth of bone marrow mesenchymal stem cells during differentiation. Methods 1) the experimental group was supplemented with osteogenic inducer and adipogenic inducer, and the control group cells were cultured in medium without any inducer. The total mRNAs of cells were extracted at 7 days and the expression of annexin A1 gene in each group was detected by fluorescence quantitative RT-PCR. The cell growth was detected by MTT method. Cell proliferation and apoptosis were detected by flow cytometry. Results compared with the control group, the expression of annexin A1 gene was significantly down-regulated at 2 CT=0.643 鹵0. 076 at 3 days after osteogenesis induction. On the 7th day, 2- CT=0.862 鹵0.028, the difference was statistically significant (P 0.01). In the adipogenic induction group, 2- CT=1.264 鹵0.115 CT=1.860 鹵0.045 on the 3rd day and 2- CT=1.860 鹵0.045 on the 7th day; (2) Osteogenic fluid inhibited the growth of MSCs and increased the apoptosis of P0.01G, but the lipid inducer had little effect on the growth and apoptosis of MSCs. Conclusion (1) Osteogenic fluid can inhibit the growth of bone marrow mesenchymal stem cells and promote the apoptosis of bone marrow mesenchymal stem cells. 2) there may be some relationship between the membrane binding protein A1 gene and adipogenic differentiation of bone marrow mesenchymal stem cells in vitro, but its role in osteogenic differentiation is uncertain. It may provide clues and new research directions for the differentiation of bone marrow mesenchymal stem cells into osteoblasts or adipocytes.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R622;R329

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