【摘要】： 間充質(zhì)干細胞(mesenchymal stem cells,MSCs)是一類具有多向分化潛能的干細胞,可分化為多種間質(zhì)細胞。有研究表明,造血發(fā)育位點中存在著多種干細胞。小鼠胚胎AGM區(qū)是永久造血的發(fā)育位點,DA(dorsal aorta)區(qū)是其重要組成部分,有研究表明造血干細胞(hemotopoietic stem cells,HSC)定位于此。包括間充質(zhì)細胞在內(nèi)的基質(zhì)微環(huán)境對DA區(qū)HSC的發(fā)育發(fā)揮著重要的作用,研究基質(zhì)微環(huán)境中的干細胞有助于闡明胚胎造血發(fā)育調(diào)控機制。 為了從小鼠胚胎DA區(qū)中分離獲得MSC和比MSC具有更多分化潛能的干細胞。本研究從11.5dpc小鼠胚胎分離獲取DA區(qū)細胞,轉(zhuǎn)染質(zhì)粒pSV3neo-SV40,篩選具有G418抗性的陽性細胞單克隆,檢測其細胞增殖能力、表型和多向分化潛能。然后將所獲得mDAF3細胞,照射后作為基質(zhì)層去培養(yǎng)10.5dpc小鼠胚胎DA區(qū)細胞,挑取克隆,檢測細胞增殖能力、表型和分化潛能。主要觀察和檢測的內(nèi)容包括:細胞形態(tài)、細胞增殖能力、細胞表面標志、多向分化潛能、RT-PCR檢測特異性表達產(chǎn)物、免疫熒光染色、吞噬實驗、Matrigel成管實驗、電鏡觀察向內(nèi)皮細胞誘導(dǎo)后的W-P小體,集落細胞培養(yǎng)及與骨髓貼壁細胞共培養(yǎng)向造血細胞誘導(dǎo)分化等。 研究結(jié)果顯示,獲得兩個永生化的成纖維樣細胞克隆mDAF3和mDAF18,細胞形態(tài)均一,倍增時間約為24h,具有MSC的表型特征(CD29+、CD44+、CD105+和Sca-1+),還表達造血和內(nèi)皮細胞表面標志(CD31、CD34等)。mDAF3和mDAF18在特定誘導(dǎo)條件下可分化為多種間質(zhì)細胞,如向脂肪細胞分化,細胞內(nèi)有脂肪滴的形成;向成骨細胞分化,堿性磷酸酶染色表達增加,培養(yǎng)4周,可見骨結(jié)節(jié)形成;向成軟骨細胞分化,Collagen-Ⅱ表達陽性。RT-PCR檢測特異性表達產(chǎn)物,進一步從分子水平證實所獲得MSC樣基質(zhì)細胞具有多向分化潛能。mDAV6和mDAV8是在以mDAF3為基質(zhì)層的基礎(chǔ)上挑取出的克隆。mDAV6和mDAV8也為典型的成纖維樣細胞,細胞形態(tài)均一,倍增時間約為22～24h,不僅具有MSC的表型特征,還表達造血和內(nèi)皮細胞標志(CD31、CD34、CD45、CD11b、Flk-1和CD144)。它們在特定誘導(dǎo)條件下可向脂肪細胞、成骨細胞和成軟骨細胞分化,RT-PCR可檢測到特異性表達產(chǎn)物。功能上還可吞噬熒光顆粒和在Matrigel上形成血管網(wǎng)絡(luò)狀結(jié)構(gòu),向內(nèi)皮細胞誘導(dǎo)后電鏡下可見W-P小體。與骨髓貼壁細胞共培養(yǎng)后,造血相關(guān)表面標志CD11b和CD45表達升高,CD34表達下降。 我們從小鼠胚胎DA區(qū)獲得了具有MSC分化潛能的單克隆細胞,提示小鼠胚胎DA區(qū)存在MSC。另外我們還從小鼠胚胎DA區(qū)獲得比MSC具有更多分化潛能的干細胞,提示小鼠胚胎DA區(qū)可能存在MSC的前體細胞,為進一步探討基質(zhì)微環(huán)境對胚胎造血發(fā)育調(diào)控奠定基礎(chǔ)。
[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，

本文編號：2290356