膜联蛋白A1基因在兔骨髓间充质干细胞体外诱导成骨和成脂早期的表达变化与意义

发布时间：2018-01-04 00:25

本文关键词：膜联蛋白A1基因在兔骨髓间充质干细胞体外诱导成骨和成脂早期的表达变化与意义　出处：《广西医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景：随着干细胞在临床上的广泛应用,骨髓间充质干细胞(Bone mesenchymal stem cells, BMSCs)已经成为众多种子细胞研究的热点。目前对骨髓间充质干细胞的体外分化机制尚不明确,以往众多研究集中在骨形态发生蛋白上,而对其他基因的研究尚少,而最近在细胞分化领域内研究的热点基因膜联蛋白A1(Annexin A1)在骨髓间充质干细胞分化中的研究甚少,且研究方法过于单一,无法全面的解释该基因的细胞分化功能。我们的研究旨在综合性的探讨骨髓间充质干细胞分化过程中膜联蛋白A1基因表达的变化意义。第一部分兔骨髓间充质干细胞体外培养与鉴定目的：体外分离并培养高纯度和生长状态稳定的骨髓间充质干细胞。方法：应用全骨髓贴壁法分离培养骨髓间充质干细胞,传代培养纯化,并鉴定其特异性表面标记物；分别用成骨诱导剂和成脂诱导剂诱导分化骨髓间充质干细胞,检测细胞分化活性。结果：骨髓间充质干细胞生长状态及分化活性良好,细胞表面标记物CD44、CD105表达率达95%以上,而CD45阴性表达。结论：全骨髓贴壁法培养的骨髓间充质干细胞生长状态良好,易于诱导。第二部分膜联蛋白A1基因在BMSCs成骨分化和成脂分化早期的表达目的：(1)研究兔骨髓间充质干细胞(BMSCs)在体外诱导成骨和成脂过程中膜联蛋白A1基因的表达变化。(2)监测骨髓间充质干细胞在诱导分化过程中的细胞生长状态。方法：(1)实验组分别加入含成骨诱导剂和成脂诱导剂的培养基,对照组细胞以不添加任何诱导剂的培养基培养。(2)分别在3、7天提取细胞总mRNA,并应用荧光定量RT-PCR技术检测各组膜联蛋白A1基因表达情况。(3)应用MTT法检测细胞生长、细胞流式学检测细胞增殖情况与凋亡情况。结果：(1)膜联蛋白A1基因在成骨诱导过程中与未诱导组相比有明显下调趋势(3天2-△△CT=0.643±0.076,7天2-△△CT=0.862±0.028,差异有统计学意义(P0.01),而在成脂诱导组中则为上升(3天2-△△CT=1.264±0.115,7天2-△△CT=1.860±0.045；(2)成骨诱导液对MSCs生长存在抑制作用并增加细胞凋亡(P0.01),而成脂诱导剂对MSCs的生长与凋亡作用甚小。结论：(1)成骨诱导液对骨髓间充质干细胞的生长起到抑制作用,并可促进细胞凋亡；(2)膜联蛋白A1基因可能与骨髓间充质干细胞的体外成脂分化存在一定关系,但尚不能肯定其在成骨分化中的作用。通过对膜联蛋白A1基因的研究,有可能为骨髓间充质干细胞向成骨细胞或脂肪细胞分化的机制提供线索和新的研究方向。
[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 卤0. 076 at 3 days after osteogenesis induction. On the 7th day, 2- CT=0.862 卤0.028, the difference was statistically significant (P 0.01). In the adipogenic induction group, 2- CT=1.264 卤0.115 CT=1.860 卤0.045 on the 3rd day and 2- CT=1.860 卤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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R622;R329

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