本文選題：人胚胎干細(xì)胞　切入點：建系　出處：《浙江大學(xué)》2009年博士論文

【摘要】：人胚胎干細(xì)胞(hESC)是一類來源于植入前囊胚內(nèi)細(xì)胞團(ICM),具有體外無限增殖潛能且能在特定條件下分化為多類成體組織或胚外組織的細(xì)胞群體。它作為體外研究人類胚胎發(fā)育的優(yōu)勢模型,進行藥物篩選的良好平臺,參與細(xì)胞治療的無限資源,越來越受到全世界科研工作者的關(guān)注。 目前,建立能廣泛適用于臨床的hES細(xì)胞系成為世界關(guān)注的焦點。然而大部分已經(jīng)建立的hES細(xì)胞系,包括NIH認(rèn)可的在內(nèi),均在建系之初以及后續(xù)的培養(yǎng)過程中直接或間接地接觸到非人源化物質(zhì),這將限制其未來的臨床應(yīng)用前景。雖然人源飼養(yǎng)層培養(yǎng)體系,hESC自體來源的飼養(yǎng)層培養(yǎng)體系,以及多種無飼養(yǎng)層培養(yǎng)體系或許能安全有效地培養(yǎng)hESC,但是完全不接觸任何異源物質(zhì)的ICM分離過程也亟待建立。我們采用非接觸性激光輔助孵出系統(tǒng)結(jié)合連續(xù)的培養(yǎng)來獲得孵出囊胚,并以此為材料,不通過“免疫手術(shù)”等可能接觸異源物質(zhì)的操作,建立起一株屬于中國族群的hES細(xì)胞系ZJUhES-1。該細(xì)胞系符合hESC的相關(guān)標(biāo)準(zhǔn):具備典型的形態(tài)學(xué)特征;表達(dá)一系列hESC特異性標(biāo)志;能長期增殖并維持穩(wěn)定的二倍體核型;能在體內(nèi)體外分化為多個胚層的細(xì)胞。同時,DNA指紋圖譜鑒定證實ZJUhES-1具有特有的身份,不同于其它已報道細(xì)胞系。 成功建立干細(xì)胞治療需要具備多向分化潛能且免疫相容的干細(xì)胞群體,高效的定向分化技術(shù),以及能安全有效地擴增干細(xì)胞群體的培養(yǎng)體系。除了所熟知的一些成體組織外,hESC逐漸成為又一類能分離獲得間充質(zhì)樣細(xì)胞的組織來源。然而hESC單細(xì)胞消化后難以存活的特征,限制其目前的分化研究和未來的臨床應(yīng)用。因此我們采用ROCK抑制劑Y-27632提高h(yuǎn)ESC單細(xì)胞消化貼壁后的存活率,并以此單層系統(tǒng)為基礎(chǔ),利用無血清無血清替代物且成分明確的N2B27培養(yǎng)體系,將hESC分化為間充質(zhì)樣前體細(xì)胞(hESC-MP),整個過程不接觸任何動物源性物質(zhì)。分化所得的細(xì)胞呈現(xiàn)成纖維樣細(xì)胞形態(tài),符合MSC的評價標(biāo)準(zhǔn),如自我更新潛能,表達(dá)多種MSC特異性標(biāo)志,能分化為成骨細(xì)胞,脂肪細(xì)胞和軟骨細(xì)胞等間質(zhì)細(xì)胞。與其他文獻報道的hESC-MSC相比,我們所獲得hESC-MP還能分化為平滑肌細(xì)胞,心肌細(xì)胞,功能性肝細(xì)胞和表達(dá)不同神經(jīng)遞質(zhì)的神經(jīng)細(xì)胞,這使得它們更適合于未來的臨床應(yīng)用。 此外,我們還通過Y-27632協(xié)助的單層誘導(dǎo)系統(tǒng)在含血清的培養(yǎng)條件下將hESC分化為另一類MSC。與N2827培養(yǎng)體系中獲得的hESC-MP相比,這類hESC-MSC表達(dá)更高比例的MSC特異性分子標(biāo)志,并呈現(xiàn)更強的增殖潛能。我們將其移植入四氯化碳(CCl_4)誘導(dǎo)的小鼠急性肝損傷模型中,初步發(fā)現(xiàn)這類細(xì)胞能改善肝功能,并促進肝組織自身修復(fù)。
[Abstract]:Human embryonic stem cells (ESCs) are a group of cells derived from pre-implantation blastocysts, which have unlimited proliferative potential in vitro and can differentiate into many kinds of adult or extraembryonic tissues under certain conditions. It is used to study human beings in vitro. The dominant model of embryonic development, More and more researchers all over the world pay more and more attention to the excellent platform for drug screening and unlimited resources for cell therapy. At present, the establishment of hES cell lines, which can be widely used in clinical applications, has become the focus of attention in the world. However, most of the established hES cell lines, including those recognized by NIH, Both of them were exposed directly or indirectly to non-humanized substances at the beginning of the establishment and the subsequent culturing process, which will limit their future clinical application prospects, although the human feeder layer culture system has autologous feeding layer culture system of hESC. And several culture systems without feeding layer may be able to culture hESCs safely and effectively, but the separation process of ICM without exposure to any heterogenous substance is urgent. We use non-contact laser-assisted hatching system combined with continuous culture. To get hatched blastocysts, Using this as material, a hES cell line ZJUhES-1, which belongs to Chinese population, was established without possible exposure to heterogenous substances such as "immune surgery". The cell line conforms to the relevant criteria of hESC: it has typical morphological characteristics; Expression of a series of hESC specific markers; long-term proliferation and maintenance of stable diploid karyotype; in vitro and in vivo differentiation into multiple embryonic layers of cells. At the same time, the identification of ZJUhES-1 fingerprinting confirmed that ZJUhES-1 has a unique identity. Different from other reported cell lines. The successful establishment of stem cell therapy requires a multi-directional differentiation potential and immune compatible stem cell population, efficient directional differentiation technology, In addition to some familiar adult tissues, hESC has gradually become the source of another kind of tissue that can isolate and obtain mesenchymal cells. However, hESC single cells are difficult to survive after digestion. Therefore, we use Y-27632, a ROCK inhibitor, to improve the survival rate of hESC after digesting and sticking, and base on this monolayer system. HESC was differentiated into mesenchymal precursor cells (hESC-MPN) by using serum-free and serum-free N2B27 culture system with clear components, and no contact with any animal-derived substances was observed in the whole process. The differentiated cells were fibroblast-like cells. According to the evaluation criteria of MSC, such as self-renewal potential, expression of many specific markers of MSC, differentiation into interstitial cells such as osteoblasts, adipocytes and chondrocytes. Our hESC-MP can also differentiate into smooth muscle cells, cardiomyocytes, functional hepatocytes and nerve cells expressing different neurotransmitters, which makes them more suitable for future clinical applications. In addition, we also differentiated hESC into another kind of MSCs in serum-containing culture by a monolayer induction system assisted by Y-27632. Compared with the hESC-MP obtained in N2827 culture system, these hESC-MSC expressed a higher proportion of MSC specific molecular markers. We transplanted it into CCl4)-induced acute liver injury model in mice and found that this kind of cells could improve liver function and promote liver self-repair.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R329

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相關(guān)期刊論文 前2條

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本文編號：1664543