大鼠体内气管损伤修复过程中气管干细胞定位及Shh动态变化的研究

发布时间：2018-05-13 00:11

本文选题：大鼠 + 气管　；参考：《中国医科大学》2005年博士论文

【摘要】：前言干细胞是指那些具有自我更新能力,在一定的条件下又能产生各种高度分化的子代细胞的增殖性细胞。干细胞大致可分为胚胎干细胞和成体干细胞,目前已发现的成体干细胞有造血干细胞、骨髓间充质干细胞、神经干细胞、肌肉干细胞、角膜干细胞、胰腺干细胞、皮肤干细胞、肝脏干细胞等,临床已开始应用干细胞治疗血液、心脏、肝脏、神经等系统疾病。由于气管干细胞缺乏特异性的表面标志物,以及没有合适的研究气管干细胞的在体模型,其体内的增殖分化过程及调控机制一直没有明确。本研究通过应用氟尿嘧啶(5-FU)建立大鼠体内气管损伤修复模型,5-FU属抗嘧啶类代谢药,通过抑制胸腺嘧啶核苷酸合成酶而抑制DNA的合成,可造成细胞周期内细胞的变性坏死,主要作用于S期,对增殖细胞的各期均有作用,但对G_0期细胞不敏感,故有利于研究处于G_0期的干细胞。Hoechst33342是属于Bisbenzimid类的活细胞荧光色素,细胞通透性高,能透过胞膜完整的细胞,嵌入细胞核DNA,受紫外光激发后能发出波长405nm和675nm两种荧光,在荧光显微镜下可看到明亮的蓝色荧光,因此能观察到Hoechst33342阳性细胞团。使用流式细胞仪进行分析时,两个波长均弱着色或完全不着色的细胞团中有很高的干细胞活性,分离出的细胞称为侧群(SP)细胞。本研究采用光镜、免疫组化、荧光染色等方法,观察气管上皮修复过程并对干细胞加以定位研究;ABC转运蛋白家族成员ABCG2/Bcrp1基因在不同来源SP干细胞均呈高表达,从小鼠骨骼肌和恒河猴骨髓中分离的SP细胞均有ABCG2/Bcrp1表达,而非SP细胞则不表达,故ABCG2/Bcrp1可作为确定SP干细胞的表型标记,并与SP细胞群所呈现的Hoechst33342弱着色或不着色相关,本研究采用免疫荧光及RTPCR方法对气管干细胞中ABCG2在蛋白及mRNA水平的表达加以研究。
[Abstract]:Preface Stem cells are proliferative cells that have the ability of self-renewal and can produce all kinds of highly differentiated progenies under certain conditions. Stem cells can be roughly divided into embryonic stem cells and adult stem cells. At present, adult stem cells have been discovered as hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, muscle stem cells, corneal stem cells, pancreatic stem cells, skin stem cells, Liver stem cells, clinical use of stem cells have begun to treat blood, heart, liver, nerve and other systemic diseases. Due to the lack of specific surface markers of tracheal stem cells and the lack of suitable in vivo model of tracheal stem cells, the process of proliferation and differentiation in vivo and its regulatory mechanism have not been clear. In this study, 5-FU was used to establish a rat model of trachea injury repair. 5-FU was used to inhibit the synthesis of DNA by inhibiting thymidine nucleotide synthase, which resulted in degeneration and necrosis of cells in the cell cycle. The stem cells, which mainly act in S phase, have effects on all stages of proliferative cells, but they are not sensitive to G0 phase cells, so it is helpful to study that stem cells. Hoechst33342, which is in G _ 0 phase, is a kind of living cytofluorescence pigment belonging to Bisbenzimid, and the cell permeability is high. The cells which can penetrate the intact cell membrane are embedded into the nuclear DNA. After being excited by ultraviolet light, they can emit two kinds of fluorescence: 405nm and 675nm. The bright blue fluorescence can be seen under the fluorescence microscope, so the Hoechst33342 positive cell cluster can be observed. When using flow cytometry, the cells with weak or no staining at two wavelengths have very high stem cell activity, and the isolated cells are called lateral group (SPN) cells. In this study, the repair process of tracheal epithelium was observed by means of light microscopy, immunohistochemistry and fluorescence staining. The expression of ABCG2/Bcrp1 gene in SP stem cells from different sources was studied. Sp cells isolated from mouse skeletal muscle and rhesus monkey bone marrow expressed ABCG2/Bcrp1, but non-SP cells did not. Therefore, ABCG2/Bcrp1 could be used as a phenotypic marker to identify SP stem cells, which was related to the weak or non-staining of Hoechst33342 in SP cell group. In this study, immunofluorescence and RTPCR methods were used to study the expression of ABCG2 at protein and mRNA levels in tracheal stem cells.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R363

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