本文選題：大鼠 + 氣管　； 參考：《中國醫(yī)科大學》2005年博士論文

【摘要】：前言 干細胞是指那些具有自我更新能力,在一定的條件下又能產(chǎn)生各種高度分化的子代細胞的增殖性細胞。干細胞大致可分為胚胎干細胞和成體干細胞,目前已發(fā)現(xiàn)的成體干細胞有造血干細胞、骨髓間充質(zhì)干細胞、神經(jīng)干細胞、肌肉干細胞、角膜干細胞、胰腺干細胞、皮膚干細胞、肝臟干細胞等,臨床已開始應用干細胞治療血液、心臟、肝臟、神經(jīng)等系統(tǒng)疾病。由于氣管干細胞缺乏特異性的表面標志物,以及沒有合適的研究氣管干細胞的在體模型,其體內(nèi)的增殖分化過程及調(diào)控機制一直沒有明確。 本研究通過應用氟尿嘧啶(5-FU)建立大鼠體內(nèi)氣管損傷修復模型,5-FU屬抗嘧啶類代謝藥,通過抑制胸腺嘧啶核苷酸合成酶而抑制DNA的合成,可造成細胞周期內(nèi)細胞的變性壞死,主要作用于S期,對增殖細胞的各期均有作用,但對G_0期細胞不敏感,故有利于研究處于G_0期的干細胞。Hoechst33342是屬于Bisbenzimid類的活細胞熒光色素,細胞通透性高,能透過胞膜完整的細胞,嵌入細胞核DNA,受紫外光激發(fā)后能發(fā)出波長405nm和675nm兩種熒光,在熒光顯微鏡下可看到明亮的藍色熒光,因此能觀察到Hoechst33342陽性細胞團。使用流式細胞儀進行分析時,兩個波長均弱著色或完全不著色的細胞團中有很高的干細胞活性,分離出的細胞稱為側群(SP)細胞。本研究采用光鏡、免疫組化、熒光染色等方法,觀察氣管上皮修復過程并對干細胞加以定位研究;ABC轉(zhuǎn)運蛋白家族成員ABCG2/Bcrp1基因在不同來源SP干細胞均呈高表達,從小鼠骨骼肌和恒河猴骨髓中分離的SP細胞均有ABCG2/Bcrp1表達,而非SP細胞則不表達,故ABCG2/Bcrp1可作為確定SP干細胞的表型標記,并與SP細胞群所呈現(xiàn)的Hoechst33342弱著色或不著色相關,本研究采用免疫熒光及RTPCR方法對氣管干細胞中ABCG2在蛋白及mRNA水平的表達加以研究。
[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.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R363

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