【摘要】：人胚胎干細(xì)胞(embryonic stem cell, ESC)由于自身所具有的多向分化潛能和無限增殖的能力使它擁有廣闊的基礎(chǔ)及臨床應(yīng)用前景。但是,人ES細(xì)胞建系過程中需要破壞受精卵或囊胚,因此,倫理問題成為ES細(xì)胞研究無法逾越的障礙；此外,從ES細(xì)胞中獲得的功能細(xì)胞對患者來說屬于異體細(xì)胞,細(xì)胞治療時的免疫排斥問題嚴(yán)重阻礙了它的臨床研究進(jìn)展。在這種背景下,通過在分化的體細(xì)胞中表達(dá)特定的幾個轉(zhuǎn)錄因子,以誘導(dǎo)體細(xì)胞的重編程而獲得的可不斷自我更新且具有多向分化潛能的誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cell, iPSC)誕生了,由于它具有跟成體細(xì)胞完全相同的遺傳背景,又不涉及倫理道德問題,因此在應(yīng)用潛能方面顯示出無比優(yōu)越性。但是,iPS細(xì)胞是否真正具備與ES細(xì)胞相同的分化能力和分化效率,是否能夠為將來的臨床移植提供具備相應(yīng)功能的供體材料,近年來一直是干細(xì)胞領(lǐng)域中所被高度關(guān)注的焦點。 干細(xì)胞中的旁系群體(side population, SP)也一直是干細(xì)胞研究的熱點之一。對于眾多干/祖細(xì)胞而言,一個非常顯著的共性就是跨膜蛋白ATP綁定結(jié)構(gòu)(ATP-Binding cassette, ABC)超家族成員之一,半轉(zhuǎn)運(yùn)子ABCG2的表達(dá)。它們介導(dǎo)著包括細(xì)胞毒性藥物、類固醇、多肽、磷脂等在內(nèi)的多類物質(zhì)的轉(zhuǎn)運(yùn)。ABCG2對熒光染料Hoechst33342的特異性排除,被認(rèn)為是干細(xì)胞中SP分選的分子基礎(chǔ),因此被用做這類細(xì)胞的特異性標(biāo)記。然而ABCG2是否在人ES細(xì)胞和iPS細(xì)胞中表達(dá)?干細(xì)胞SP分選的“黃金法則”是否也同樣適用于這些細(xì)胞呢? 帶著這些問題,開始了我們的課題研究。整個課題是由三大研究部分所構(gòu)成：第一部分-“人誘導(dǎo)多能干細(xì)胞(iPSCs)的建立”,第二部分-“人誘導(dǎo)多能干細(xì)胞(iPSCs)的多項分化和第三部分-“剖析在人胚胎干細(xì)胞(hESCs)和誘導(dǎo)多能分化干細(xì)胞(iPSCs)中膜轉(zhuǎn)運(yùn)子ABCG2的表達(dá)與意義”。 第一部分主要為iPS實驗技術(shù)平臺的建立。我們分別采用慢病毒轉(zhuǎn)染系統(tǒng)和逆轉(zhuǎn)錄病毒轉(zhuǎn)染系統(tǒng),將不同的轉(zhuǎn)錄因子組合(Oct4,Sox2, c-Myc和K1f4或Oct4, Sox2, Nonog, c-Myc, Klf4和Lin28)導(dǎo)入人胎肺成纖維細(xì)胞(IMR-90)中,成功將其誘導(dǎo)成為具有完全相同遺傳背景的iPS細(xì)胞株。經(jīng)相關(guān)檢測,發(fā)現(xiàn)這些iPS細(xì)胞均表達(dá)特異性多能分化標(biāo)記,且能在小鼠體內(nèi)形成包含三個胚層的畸胎瘤,提示它們具備跟ES細(xì)胞相似的多向分化潛能。 第二部分主要為包括造血、神經(jīng)以及滋養(yǎng)層細(xì)胞在內(nèi)的iPS細(xì)胞體外誘導(dǎo)分化。我們根據(jù)在人ES細(xì)胞的培養(yǎng)和分化的經(jīng)驗,分別將我們所建立的iPS細(xì)胞系分化成造血細(xì)胞、神經(jīng)上皮細(xì)胞、各類終末神經(jīng)元以及滋養(yǎng)層細(xì)胞,并特別比較了人的ES細(xì)胞和iPS細(xì)胞的神經(jīng)分化效率。結(jié)果表明,我們所得到的iPS細(xì)胞均具備向上述各類細(xì)胞分化的能力,而且經(jīng)功能學(xué)檢測,發(fā)現(xiàn)將其誘導(dǎo)生成的運(yùn)動神經(jīng)元被激發(fā)后可產(chǎn)生與ES細(xì)胞來源運(yùn)動神經(jīng)元相似的動作電位,提示這類神經(jīng)元是具備相關(guān)感觸和傳導(dǎo)功能的,有望用于將來的臨床神經(jīng)退行性病變的治療,但是不同iPS細(xì)胞系在神經(jīng)分化的效率上存在著差異,其深層次的機(jī)制目前尚不可知。 第三部分是一項發(fā)現(xiàn)導(dǎo)向型課題。我們在對人ES細(xì)胞及iPS細(xì)胞的研究過程中發(fā)現(xiàn)了一個非常奇特的現(xiàn)象,那就是區(qū)別于其他類型的干/祖細(xì)胞,人的ES細(xì)胞和iPS細(xì)胞胞核能夠被熒光染料Hoechst所染色,通過進(jìn)一步實驗證明這種現(xiàn)象是由于干細(xì)胞SP的分選標(biāo)記一ABCG2在人ES細(xì)胞和iPS細(xì)胞中的缺失所導(dǎo)致的。并且恰恰相反,通過常規(guī)方法Hoechst染色對人ES細(xì)胞進(jìn)行流式細(xì)胞術(shù)分選,得到拒染Hoechst的“SP”細(xì)胞實際上是多能分化基因已經(jīng)下調(diào)的分化細(xì)胞,證明經(jīng)典的干細(xì)胞“SP”的定義與分選并不適用于人的多能分化干細(xì)胞(ES細(xì)胞和iPS細(xì)胞)。而有趣的是,來源于人ES細(xì)胞的滋養(yǎng)層細(xì)胞和神經(jīng)祖細(xì)胞卻能夠表達(dá)ABCG2因而可以拒染Hoechst。與之相對,我們在小鼠的ES細(xì)胞中檢測到ABCG2的表達(dá)以及對Hoechst的拒染,而在小鼠的滋養(yǎng)層細(xì)胞中卻未發(fā)現(xiàn)其表達(dá)。在人ES細(xì)胞中強(qiáng)行表達(dá)ABCG2則可以顯著增強(qiáng)它們對熒光染料Hoechst和化療藥物米托葸醌(mitoxanthrone, MTX)的抵抗性,并且大部分人ES細(xì)胞在撤去生長因bFGF(3天)后仍能維持其自我更新(self renew)狀態(tài)。我們在表達(dá)ABCG2的人ES細(xì)胞中還發(fā)現(xiàn)磷酸化的AKT表達(dá)水平顯著升高,提示ABCG2可能通過激活FGF信號通路的重要分支PI3K/AKT而降低人ES細(xì)胞對于子bFGF的依賴性,但是這其中的具體機(jī)制還有待進(jìn)一步探討。 綜上所述,我們成功地通過不同的病毒轉(zhuǎn)染技術(shù)建立了iPS技術(shù)平臺,并進(jìn)一步將所建立的iPS細(xì)胞系分化成造血干/祖細(xì)胞,神經(jīng)上皮細(xì)胞和區(qū)域神經(jīng)元,以及滋養(yǎng)層細(xì)胞,比較它們神經(jīng)分化能力的差異并進(jìn)行部分功能檢測,初步探討其做為不同細(xì)胞種類的移植候選物的可能性。同時我們首次系統(tǒng)性地闡述了干細(xì)胞SP分選標(biāo)記ABCG2在人多能分化干細(xì)胞(ES細(xì)胞和iPS細(xì)胞)中的缺失,對于重新認(rèn)識干細(xì)胞中的旁系群體、ABCG2與干細(xì)胞多能性的相關(guān)性、ES細(xì)胞的自我更新與物種差異有著積極意義。
[Abstract]:Human embryonic stem cell (ESC) has a broad foundation and clinical application prospects because of its multi-directional differentiation potential and unlimited proliferation ability. However, the establishment of human ES cell lines requires the destruction of fertilized eggs or blastocysts, so ethical issues become an insurmountable obstacle to ES cell research. Functional cells obtained in cells are allogeneic to patients, and immunological rejection during cell therapy seriously hampers its clinical advances. In this context, the self-renewal and abundance of these cells can be achieved by inducing reprogramming of somatic cells by expressing specific transcription factors in differentiated somatic cells Induced pluripotent stem cells (iPSC) with differentiated potentials were born. Because of their identical genetic background with adult cells and no ethical issues, iPSC has shown tremendous superiority in application potential. However, whether iPS cells really have the same differentiation ability as ES cells or not Differentiation efficiency and the ability to provide functional donor materials for future clinical transplantation have been the focus of attention in the field of stem cells in recent years.
Side population (SP) in stem cells has also been one of the hotspots in stem cell research. For many stem/progenitor cells, one of the most remarkable similarities is the expression of ABCG2, a semitransporter, a member of the ATP-Binding cassette (ABC) superfamily. The specific exclusion of Hoechst 33342 by ABCG2 is considered to be the molecular basis for SP sorting in stem cells and is therefore used as a specific marker for such cells. The same applies to these cells?
With these problems, we began our research. The whole project is composed of three parts: the first part - "the establishment of human induced pluripotent stem cells (iPSCs)," the second part - "the multiple differentiation of human induced pluripotent stem cells (iPSCs) and the third part -"the analysis of human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs)". Expression and significance of ABCG2 in cell membrane (iPSCs).
The first part is the establishment of an experimental platform for iPS. Different combinations of transcription factors (Oct4, Sox2, c-Myc and K1f4 or Oct4, Sox2, Nonog, c-Myc, Klf4 and Lin28) were successfully induced into human fetal lung fibroblasts (IMR-90) using lentiviral and retroviral transfection systems. These iPS cells expressed specific pluripotent markers and could form teratomas containing three embryonic layers in mice, suggesting that they had similar pluripotent potential to ES cells.
The second part is the induction and differentiation of iPS cells, including hematopoietic, neurotrophoblastic and neurotrophoblast cells in vitro. Based on our experience in the culture and differentiation of human ES cells, we differentiated our iPS cell lines into hematopoietic, neuroepithelial, terminal neurons and trophoblast cells, and compared human cells in particular. The results showed that all the iPS cells we obtained possessed the ability to differentiate into the above-mentioned types of cells, and functional tests showed that the motor neurons induced by these cells could produce action potentials similar to those of ES-derived motor neurons. It is expected to be used in the treatment of neurodegenerative diseases in the future because of its related sensory and conductive functions. However, there are differences in the efficiency of neural differentiation among different iPS cell lines, and the underlying mechanism is still unknown.
The third part is a discovery-oriented project. We have found a very strange phenomenon in the study of human ES cells and iPS cells, which is different from other types of stem/progenitor cells. The nuclei of human ES cells and iPS cells can be stained by fluorescent dye Hoechst. Further experiments show that this phenomenon is due to The absence of ABCG2 in human ES cells and iPS cells, a marker for stem cell SP sorting, was caused by the absence of ABCG2. On the contrary, Hoechst staining was used for flow cytometry sorting of human ES cells to obtain Hoechst-resistant "SP" cells, which were actually pluripotent differentiated cells whose genes had been down-regulated. The definition and sorting of "P" does not apply to human pluripotent stem cells (ES cells and iPS cells). Interestingly, trophoblast cells and neural progenitor cells derived from human ES cells can express ABCG2 and therefore can resist Hoechst. In contrast, we detected the expression of ABCG2 and the resistance to Hoechst in mouse ES cells. The expression of ABCG2 in human ES cells significantly enhanced their resistance to fluorescent dye Hoechst and the chemotherapeutic drug mitoxanthrone (MTX), and most human ES cells maintained their self-renew state after the removal of bFGF (3 days). We also found a significant increase in phosphorylated AKT expression in human ES cells expressing ABCG2, suggesting that ABCG2 may reduce the dependence of human ES cells on bFGF by activating PI3K/AKT, an important branch of the FGF signaling pathway, but the specific mechanism remains to be further explored.
To sum up, we have successfully established an iPS technology platform through different virus transfection techniques, and further differentiated the established iPS cell lines into hematopoietic stem/progenitor cells, neuroepithelial cells, regional neurons, and trophoblast cells, and compared their differences in neural differentiation ability and carried out some functional tests to preliminarily explore their role. For the first time, we systematically elucidate the deletion of stem cell SP sorting marker ABCG2 in human pluripotent stem cells (ES cells and iPS cells), the relationship between ABCG2 and stem cell pluripotency, the self-renewal and species of ES cells for the purpose of reconsidering the collateral population in stem cells. The difference is of positive significance.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 徐大鵬;嬰幼兒關(guān)節(jié)軟骨細(xì)胞的體外培養(yǎng)和表型研究及逆向誘導(dǎo)分化初探[D];蘇州大學(xué);2013年

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