本文選題：生物信息學(xué)　切入點(diǎn)：骨髓源干細(xì)胞　出處：《第三軍醫(yī)大學(xué)》2009年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 干細(xì)胞自發(fā)現(xiàn)以來(lái)一直是研究的焦點(diǎn),它為許多疾病的治療提供了新的思路和方法,具有重要意義。由于干細(xì)胞表面缺乏明確的標(biāo)志,很難分離純化單一克隆的細(xì)胞種群,這就導(dǎo)致我們對(duì)干細(xì)胞的認(rèn)識(shí)存在非常大的局限性。正是由于上述這些原因,干細(xì)胞研究既是當(dāng)前生物醫(yī)學(xué)領(lǐng)域研究的熱點(diǎn)之一,更是研究的難點(diǎn)之一。 從干細(xì)胞本身發(fā)育的特點(diǎn)來(lái)看,具有高度的自我復(fù)制能力是其重要的特征之一,因此有學(xué)者推測(cè):在成體組織中可能殘留有胚胎發(fā)育過(guò)程中不同分化階段的干細(xì)胞群,其中可能就有具有胚胎時(shí)期干細(xì)胞特性的干細(xì)胞亞群。 在目前研究條件下,從成體組織中分離獲得的干細(xì)胞通常是多種干細(xì)胞的混合物,具有多種表面分化抗原。在傳統(tǒng)的觀念中,細(xì)胞表面分化抗原通常標(biāo)志著細(xì)胞具有不同的分化成熟特性。干細(xì)胞表面存在多種不同分化抗原是否代表著成體組織中殘留有多種不同發(fā)育階段的干細(xì)胞亞群?目前還不是很清楚,需要進(jìn)行深入的研究。 近年來(lái)在成體組織中發(fā)現(xiàn)一類(lèi)具有向多胚層發(fā)育潛能的多能干細(xì)胞。這些研究結(jié)果支持了成體組織中可能殘留有胚胎發(fā)育過(guò)程中的、具有不同分化階段的干細(xì)胞群。這些從成體組織中獲得的干細(xì)胞中,是否存在具有胚胎早期干細(xì)胞特征的細(xì)胞亞群,目前還缺乏足夠的證據(jù),但有個(gè)別的研究表明這個(gè)情況是可能存在的,Ratajczak等將這類(lèi)細(xì)胞命名為“類(lèi)胚胎干細(xì)胞”。 要證實(shí)成體干細(xì)胞中殘留有胚胎發(fā)育早期的干細(xì)胞群,還必需找到足夠多的實(shí)驗(yàn)證據(jù)來(lái)支持上述觀點(diǎn)�；谏鲜稣J(rèn)識(shí)和分析,我們通過(guò)以下幾個(gè)部分的研究來(lái)證實(shí)可能在成體組織中殘留有“類(lèi)胚胎干細(xì)胞”。 1.“類(lèi)胚胎干細(xì)胞”標(biāo)志物的篩選: 采用生物信息學(xué)技術(shù),對(duì)美國(guó)國(guó)立圖書(shū)館(NCBI)的數(shù)據(jù)庫(kù)進(jìn)行DDD (Digital Differential Display)分析,結(jié)果顯示,成體組織細(xì)胞與胚胎干細(xì)胞基因表達(dá)譜差異分子是顆粒酶B(Granzyme B,GRB);然后進(jìn)一步復(fù)習(xí)文獻(xiàn),發(fā)現(xiàn)階段性胚胎抗原-1(Stage specific embronic antigen-1,SSEA-1)、胚胎干細(xì)胞關(guān)鍵蛋白-4(Octamer-4,Oct-4)這兩種蛋白在胚胎干細(xì)胞發(fā)育中具有重要作用。最后我們得出結(jié)論:這三種蛋白質(zhì)可以作為標(biāo)志物鑒定成體干細(xì)胞中是否存在具有胚胎發(fā)育早期特性的干細(xì)胞亞群。 2.SSEA-1、Oct-4、GRB在骨髓、脂肪、皮膚來(lái)源的干細(xì)胞中的表達(dá): 為了證實(shí)成體組織中可能殘留有“類(lèi)胚胎干細(xì)胞”,我們采用不同的分離方法,分別從骨髓、脂肪、皮膚提取成體干細(xì)胞,然后采用免疫熒光化學(xué)的方法,觀察上述選定的三種標(biāo)記物在這三種不同組織來(lái)源的干細(xì)胞中表達(dá)的情況。 結(jié)果顯示,骨髓、脂肪、皮膚來(lái)源的成體干細(xì)胞均能不同程度地表達(dá)SSEA-1、Oct-4、GRB。皮膚來(lái)源的成體干細(xì)胞(Dermal-derived adult stem cells,DSCs)三種標(biāo)記物表達(dá)最弱,而其它兩種組織來(lái)源的干細(xì)胞中表達(dá)Oct-4、GRB蛋白的情況接近,骨髓來(lái)源的成體干細(xì)胞(Bone marrow-derived stem cells,BMSCs)表達(dá)SSEA-1最強(qiáng)。 3.SSEA-1表達(dá)陽(yáng)性骨髓成體干細(xì)胞的免疫磁珠分選及其生物學(xué)特性: 應(yīng)用SSEA-1抗體,通過(guò)免疫磁珠分選法,收獲了SSEA-1呈陽(yáng)性表達(dá)的骨髓來(lái)源的干細(xì)胞亞群(SSEA-1+BMSCs),SSEA-1的表達(dá)細(xì)胞接近80%;可以進(jìn)行成脂、成神經(jīng)和成胰島誘導(dǎo)分化,表明這類(lèi)干細(xì)胞具有多向分化潛能,其中可能存在“類(lèi)胚胎干細(xì)胞”。骨髓中“類(lèi)胚胎干細(xì)胞”的存在可能是其可塑性分化的另一條途徑。 4.骨髓來(lái)源的成體干細(xì)胞在創(chuàng)傷修復(fù)中的動(dòng)員及募集作用: 將Brdu標(biāo)記的第三代骨髓來(lái)源的成體干細(xì)胞輸入到5Gy放射性損傷的C57小鼠體內(nèi),建立骨髓移植模型;利用骨髓移植模型動(dòng)物分別建立皮膚缺損和Ccl4肝臟中毒損傷兩種實(shí)驗(yàn)動(dòng)物模型,研究骨髓成體干細(xì)胞在組織損傷中的作用。 結(jié)果顯示,嵌合于骨髓中的Brdu標(biāo)記的干細(xì)胞可以通過(guò)動(dòng)員及募集作用參與創(chuàng)傷的修復(fù)。
[Abstract]:Stem cells have been the focus of research since the discovery, provides new ideas and methods for the treatment of many diseases, which is of great significance. Because of the lack of clear stem cell surface markers, it is difficult to purify single clone cell population, which leads to our understanding of stem cells exist great limitations. It is precisely because of these reasons, the stem is one of the hot topics in the field of biomedical cell research, is one of the most difficult research.
From the characteristic of stem cells and development of itself, having the ability of self replication is one of its important features, so some scholars speculate that in adult tissue may be residual stem cells at different stages of differentiation during embryonic development, which may have characteristics of embryonic stem cells with stem cell subsets.
In the present study conditions, isolated stem cells are usually a mixture of different types of stem cells from adult tissues, with a variety of surface antigen. In the traditional concept, cell surface differentiation antigen usually marked with different differentiation cells. Stem cells have different surface differentiation antigens represents a there are a variety of tissue residues in different developmental stages of stem cell subsets? Is not clear, the need for in-depth research.
In recent years, found a developmental potential to embryonic pluripotent stem cells in adult tissues. These findings support adult tissues may remain in the process of embryonic development, with different stages of differentiation. These stem cells obtained from adult tissue stem cells, whether there is a cell the characteristics of early embryonic stem cell subsets, there is not enough evidence, but there are a few studies show that this situation is likely to exist, such as the Ratajczak of these cells named "embryonic stem cells".
To prove that adult stem cells are embryonic stem cells remaining in the early group, also must find experimental evidence enough to support this view. The understanding and analysis based on our study of the following several parts to identified in adult tissue residue "embryonic stem cells".
1. screening of the markers of "embryonic stem cell like cells":
閲囩敤鐢熺墿淇℃伅瀛︽妧鏈，

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