人骨髓间充质干细胞成骨分化特异性miRNA的筛选

发布时间：2018-03-03 05:01

本文选题：间充质干细胞　切入点：miRNA　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 背景microRNA(miRNA)是一类非编码蛋白并且参与转录后调节的单链小分子RNA(约20~25个碱基),对基因表达具有重要调控作用。近年来的研究表明在线虫、果蝇、小鼠、大鼠和人等多个物种中存在着一个庞大的miRNA家族,miRNA参与调控生物体的生长发育、信号转导、组织分化、疾病发生等,同时还决定了其它很多生物体发育和行为等的变化。骨髓间充质干细胞在不同诱导条件下具有多向分化潜能,可分化成多种类型的结缔组织,如骨、软骨、骨骼肌、肌腱、韧带、真皮、脂肪和骨髓基质,也可分化成神经系统的神经元和神经胶质细胞等,而且来源广泛,易于在体外扩增,是目前组织工程中一种重要的种子细胞。 miRNA作为一个广泛存在的可对基因表达进行微调的分子,参与生物体的生长、发育、衰老及死亡的调控,其在干细胞增殖及分化中的意义近年来开始受到关注。miRNA在多种干细胞中均有表达,并且通过负向调节干细胞中某些关键基因的表达,对干细胞的自我更新和多向分化发挥作用。目的通过检测骨髓间充质干细胞及其诱导分化的成骨细胞中miRNA基因的表达谱,筛选出诱导分化前后细胞表达的差异性miRNAs,寻找可能的人骨髓间充质干细胞成骨分化特异性的miRNAs,为进一步阐明miRNA在骨髓间充质干细胞定向分化中的作用和意义奠定基础。方法(1)采用密度梯度离心法自骨髓中分离培养人骨髓间充质干细胞,通过观察原代及传代MSCs的形态特点,流式细胞仪检测细胞表面标志物,对人骨髓间充质干细胞进行鉴定。(2)采用地塞米松、β-甘油磷酸钠和维生素C联合诱导人MSCs向成骨细胞方向分化,通过ALP染色、茜素红S法染色及电镜观察对诱导分化的成骨细胞进行鉴定。(3)利用miRNA基因芯片技术,检测人骨髓间充质干细胞及其诱导分化的成骨细胞中miRNAs的表达谱,并用SAM分析筛选出人MSCs和其诱导分化的成骨细胞表达的差异性miRNAs。(4)采用实时定量PCR和Northern blotting分析的方法,对miRNA基因芯片筛选出的人MSCs及其诱导分化的成骨细胞的部分差异miRNA基因进行验证。结果(1)经密度梯度离心法分离贴壁培养所得到的细胞,通过流式细胞仪检测的结果为:细胞表面标志物CD29、CD44为阳性,CD34、CD45为阴性,符合骨髓间充质干细胞特征。(2)经地塞米松、β-甘油磷酸钠和维生素C联合诱导14天后得到的细胞:ALP染色及茜素红S法染色均阳性;扫描电镜显示细胞多为多角形,伸突细长且多,细胞相互交联,局部产生絮丝状基质成分;透射电镜显示细胞分裂相多见,核仁明显,大量的粗面内质网、高尔基体及线粒体,并可见部分溶酶体及分泌颗粒,细胞外基质中出现胶原纤维及钙盐颗粒。符合成骨细胞的特征。(3)用miRNA基因芯片检测了三组样品中人骨髓间充质干细胞及其诱导分化的成骨细胞miRNAs的表达谱,并筛选出了在人MSCs和其诱导分化的成骨细胞表达的差异性的miRNAs。其中在每组样品中均筛选出了人MSCs和其诱导分化的成骨细胞的差异的miRNAs,如成骨细胞较MSCs高表达的miRNAs在三组样品中分别为:36个、14个、8个;成骨细胞较MSCs低表达的miRNAs在三组样品中分别为:24个、27个、20个。但miRNAs存在很大的个体差异性,在三组样品中有共同存在趋势的MSCs和成骨细胞的差异miRNAs较少,如hsa-miR-30a-5p、hsa-miR-30c、hsa-miR-210、hsa-miR-31等。(4)针对hsa-miR-30a-5p和hsa-miR-210分别用Northern blotting和实时定量PCR的方法在第1组样品中进行验证,结果均与芯片检测的结果一致。结论(1)用密度梯度离心法可以从骨髓中分离得到人骨髓间充质干细胞,并可在体外培养将其诱导分化为成骨细胞。(2)在人骨髓间充质干细胞及其诱导分化的成骨细胞中均有大量miRNAs的表达,但其表达在不同个体来源的细胞存在明显的差异性。(3)在人骨髓间充质干细胞和其诱导分化的成骨细胞中存在表达差异的miRNAs,它们可能对人骨髓间充质干细胞的成骨诱导分化起着重要的调控作用,即所谓的人骨髓间充质干细胞成骨分化特异性miRNA。
[Abstract]:Background microRNA (miRNA) is a small single stranded RNA for a class of non protein encoding and participate in the post transcriptional regulation of the (about 20~25 nucleotides), plays an important role in regulating gene expression. Recent studies showed that C. elegans, Drosophila, mouse, rat and human and other species in the existence of a huge miRNA the miRNA family, is involved in the regulation of growth and development of organism, signal transduction, tissue differentiation, occurrence of disease, but also decide the change of many other organism development and behavior. Bone marrow mesenchymal stem cells with multi-directional differentiation potential under different induction conditions, can differentiate into various types of connective tissue, such as bone, cartilage, skeletal muscle, tendon, ligament, leather, fat and bone marrow, can differentiate into neurons and glial cells, and a wide range of sources, easy in vitro amplification, is an important seed in tissue engineering at present Cells.
MiRNA as a widespread of gene expression in fine-tuning the molecule, involved in organism growth, development, aging and death in the regulation of stem cell proliferation and differentiation in significance in recent years, researchers have begun to focus on.MiRNA in a variety of stem cells expressed, and through the negative regulation of stem cell gene expression of some key the role of stem cell self-renewal and differentiation.
The spectrum of miRNA expression in osteoblasts in gene detection of bone marrow mesenchymal stem cells and their differentiation, screened before and after differentiation of miRNAs cells the expression of differences, to find possible human bone marrow mesenchymal stem cells differentiation of bone specific miRNAs, to further clarify the role of bone marrow and miRNA the significance of cell differentiation in mesenchymal stem lay the foundation.
Methods (1) isolated from bone marrow cultured human bone marrow mesenchymal stem cells by density gradient centrifugation, the morphological characteristics of primary and passage MSCs, flow cytometry was used to detect cell surface markers and identification of human bone marrow mesenchymal stem cells. (2) using dexamethasone, beta sodium glycerophosphate and vitamin C and induce MSCs to differentiate into osteoblasts by ALP staining, alizarin red S staining and electron microscopy were used to identify the osteoblast differentiation. (3) using miRNA gene chip technology, detection of human bone marrow mesenchymal stem cells and differentiation into bone miRNAs expression cells in the spectrum, and SAM analysis showed that MSCs and the differentiation of osteoblasts in different expression of miRNAs. (4) by using the method of real-time quantitative analysis of PCR and Northern blotting, screened by miRNA gene chip MSCs and its differentiation into The partial difference of bone cells was verified by miRNA gene.
Results (1) the adherent cells obtained by density gradient centrifugation, by flow cytometry results: cell surface markers CD29, CD34, CD44 positive, CD45 negative with bone marrow mesenchymal stem cells. (2) by dexamethasone, beta glycerol phosphate sodium and vitamin C combined with 14 days after the induction of the cells: ALP staining and alizarin red S staining were positive; scanning electron microscopy showed that the cells were polygonal, slender and stretching process, cells interconnected, local flocculation of filamentous matrix components; transmission electron microscopy revealed that the cell division phase, obvious nucleolus, a lot of rough the endoplasmic reticulum, Golgi apparatus and mitochondria and secretory granules, and the visible portion of lysosomes, collagen fibrils and calcium salt particles in the extracellular matrix. In line with the characteristics of osteoblasts. (3) in the three groups of samples of human bone marrow mesenchymal stem cells and its detection by miRNA gene chip Spectrum miRNAs expression of osteoblast differentiation, and screened in MSCs and the differentiation of osteoblasts difference expression of miRNAs. in each sample were screened out MSCs and its differentiation into different bone cells of miRNAs, such as osteoblasts with high expression of MSCs the miRNAs samples in three groups respectively: 36, 14, 8; low expression of bone cells than MSCs miRNAs in the three groups of samples were 24, 27, 20. But the miRNAs are difference, there are differences in common trend MSCs and osteogenesis the miRNAs cell is less, the samples in three groups such as hsa-miR-30a-5p, hsa-miR-30c, hsa-miR-210, hsa-miR-31 et al. (4) according to the method of hsa-miR-30a-5p and hsa-miR-210 respectively by Northern blotting and real-time quantitative PCR in first samples to verify the results with the chip detection results.
Conclusion (1) human bone marrow mesenchymal stem cells isolated from bone marrow by density gradient centrifugation, and were cultured in vitro and differentiate into bone cells. (2) were expressed in bone cells in a large number of miRNAs into human bone marrow mesenchymal stem cells and their differentiation, but the there are obvious differences in the expression of different individual cells. (3) in human bone marrow mesenchymal stem cells and their differentiation into the differential expression of miRNAs in bone cells, they may be important for regulation of human bone marrow mesenchymal stem cells osteoblast differentiation plays, the so-called human bone marrow mesenchymal stem cells into bone cells specific miRNA.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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