小鼠胚胎DA区MSC样干细胞的分离及鉴定
发布时间：2018-10-23 20:19
【摘要】： 间充质干细胞(mesenchymal stem cells,MSCs)是一类具有多向分化潜能的干细胞,可分化为多种间质细胞。有研究表明,造血发育位点中存在着多种干细胞。小鼠胚胎AGM区是永久造血的发育位点,DA(dorsal aorta)区是其重要组成部分,有研究表明造血干细胞(hemotopoietic stem cells,HSC)定位于此。包括间充质细胞在内的基质微环境对DA区HSC的发育发挥着重要的作用,研究基质微环境中的干细胞有助于阐明胚胎造血发育调控机制。 为了从小鼠胚胎DA区中分离获得MSC和比MSC具有更多分化潜能的干细胞。本研究从11.5dpc小鼠胚胎分离获取DA区细胞,转染质粒pSV3neo-SV40,筛选具有G418抗性的阳性细胞单克隆,检测其细胞增殖能力、表型和多向分化潜能。然后将所获得mDAF3细胞,照射后作为基质层去培养10.5dpc小鼠胚胎DA区细胞,挑取克隆,检测细胞增殖能力、表型和分化潜能。主要观察和检测的内容包括:细胞形态、细胞增殖能力、细胞表面标志、多向分化潜能、RT-PCR检测特异性表达产物、免疫荧光染色、吞噬实验、Matrigel成管实验、电镜观察向内皮细胞诱导后的W-P小体,集落细胞培养及与骨髓贴壁细胞共培养向造血细胞诱导分化等。 研究结果显示,获得两个永生化的成纤维样细胞克隆mDAF3和mDAF18,细胞形态均一,倍增时间约为24h,具有MSC的表型特征(CD29+、CD44+、CD105+和Sca-1+),还表达造血和内皮细胞表面标志(CD31、CD34等)。mDAF3和mDAF18在特定诱导条件下可分化为多种间质细胞,如向脂肪细胞分化,细胞内有脂肪滴的形成;向成骨细胞分化,碱性磷酸酶染色表达增加,培养4周,可见骨结节形成;向成软骨细胞分化,Collagen-Ⅱ表达阳性。RT-PCR检测特异性表达产物,进一步从分子水平证实所获得MSC样基质细胞具有多向分化潜能。mDAV6和mDAV8是在以mDAF3为基质层的基础上挑取出的克隆。mDAV6和mDAV8也为典型的成纤维样细胞,细胞形态均一,倍增时间约为22～24h,不仅具有MSC的表型特征,还表达造血和内皮细胞标志(CD31、CD34、CD45、CD11b、Flk-1和CD144)。它们在特定诱导条件下可向脂肪细胞、成骨细胞和成软骨细胞分化,RT-PCR可检测到特异性表达产物。功能上还可吞噬荧光颗粒和在Matrigel上形成血管网络状结构,向内皮细胞诱导后电镜下可见W-P小体。与骨髓贴壁细胞共培养后,造血相关表面标志CD11b和CD45表达升高,CD34表达下降。 我们从小鼠胚胎DA区获得了具有MSC分化潜能的单克隆细胞,提示小鼠胚胎DA区存在MSC。另外我们还从小鼠胚胎DA区获得比MSC具有更多分化潜能的干细胞,提示小鼠胚胎DA区可能存在MSC的前体细胞,为进一步探讨基质微环境对胚胎造血发育调控奠定基础。
[Abstract]:Mesenchymal stem cell (mesenchymal stem cells,MSCs) is a kind of stem cells with multiple differentiation potential and can be differentiated into many kinds of mesenchymal cells. Studies have shown that there are a variety of stem cells in hematopoietic development sites. The AGM region of mouse embryo is an important component of the, DA (dorsal aorta) region, which is the developmental site of permanent hematopoiesis. Some studies have shown that the (hemotopoietic stem cells,HSC of hematopoietic stem cells is located in this region. The stromal microenvironment including mesenchymal cells plays an important role in the development of HSC in the DA region. The study of stem cells in the stromal microenvironment is helpful to elucidate the regulatory mechanism of embryonic hematopoietic development. In order to isolate MSC and stem cells with more differentiation potential than MSC from mouse embryonic DA region. In this study, DA region cells were isolated from 11.5dpc mouse embryos and transfected with plasmid pSV3neo-SV40, to screen G418 resistant positive cells. The proliferation, phenotypic and multidirectional differentiation potential of G418 positive cells were detected. Then, the mDAF3 cells were irradiated as matrix layer to culture 10.5dpc mouse embryonic DA region cells, then cloned and detected the proliferation ability, phenotype and differentiation potential of the cells. The main contents of observation and detection include: cell morphology, cell proliferation ability, cell surface markers, multidirectional differentiation potential, RT-PCR detection of specific expression products, immunofluorescence staining, phagocytosis test, Matrigel tube forming test. The W-P corpuscles induced by endothelial cells, colony cell culture and hematopoietic differentiation with bone marrow adherent cells were observed by electron microscope. The results showed that two immortalized fibroblast clones, mDAF3 and mDAF18, were homogeneous in morphology. The doubling time is about 24 h, which has the phenotypic characteristics of MSC (CD29, CD44, CD105 and Sca-1), and also expresses the surface markers of hematopoietic and endothelial cells (CD31,CD34, etc.). MDAF3 and mDAF18 can differentiate into many kinds of interstitial cells, such as adipocytes, under certain induction conditions. There were fat droplets in the cells, differentiation into osteoblasts, increased expression of alkaline phosphatase, bone nodules formed after 4 weeks of culture, differentiation to chondroblasts, positive expression of Collagen- 鈪，
本文编号：2290356
[Abstract]:Mesenchymal stem cell (mesenchymal stem cells,MSCs) is a kind of stem cells with multiple differentiation potential and can be differentiated into many kinds of mesenchymal cells. Studies have shown that there are a variety of stem cells in hematopoietic development sites. The AGM region of mouse embryo is an important component of the, DA (dorsal aorta) region, which is the developmental site of permanent hematopoiesis. Some studies have shown that the (hemotopoietic stem cells,HSC of hematopoietic stem cells is located in this region. The stromal microenvironment including mesenchymal cells plays an important role in the development of HSC in the DA region. The study of stem cells in the stromal microenvironment is helpful to elucidate the regulatory mechanism of embryonic hematopoietic development. In order to isolate MSC and stem cells with more differentiation potential than MSC from mouse embryonic DA region. In this study, DA region cells were isolated from 11.5dpc mouse embryos and transfected with plasmid pSV3neo-SV40, to screen G418 resistant positive cells. The proliferation, phenotypic and multidirectional differentiation potential of G418 positive cells were detected. Then, the mDAF3 cells were irradiated as matrix layer to culture 10.5dpc mouse embryonic DA region cells, then cloned and detected the proliferation ability, phenotype and differentiation potential of the cells. The main contents of observation and detection include: cell morphology, cell proliferation ability, cell surface markers, multidirectional differentiation potential, RT-PCR detection of specific expression products, immunofluorescence staining, phagocytosis test, Matrigel tube forming test. The W-P corpuscles induced by endothelial cells, colony cell culture and hematopoietic differentiation with bone marrow adherent cells were observed by electron microscope. The results showed that two immortalized fibroblast clones, mDAF3 and mDAF18, were homogeneous in morphology. The doubling time is about 24 h, which has the phenotypic characteristics of MSC (CD29, CD44, CD105 and Sca-1), and also expresses the surface markers of hematopoietic and endothelial cells (CD31,CD34, etc.). MDAF3 and mDAF18 can differentiate into many kinds of interstitial cells, such as adipocytes, under certain induction conditions. There were fat droplets in the cells, differentiation into osteoblasts, increased expression of alkaline phosphatase, bone nodules formed after 4 weeks of culture, differentiation to chondroblasts, positive expression of Collagen- 鈪，
本文编号：2290356
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