【摘要】：胚胎干細(xì)胞(embryonic stem cell,ES cell)是一種具有自我更新能力和多種分化潛能的細(xì)胞,可以分化為三個(gè)胚層的細(xì)胞。作為一種獨(dú)特的體外生物系統(tǒng),胚胎干細(xì)胞可以用來發(fā)現(xiàn)和闡明生物發(fā)育過程中的奧秘,也是疾病模擬、藥物篩選和細(xì)胞治療等非常有用的材料。 胚胎干細(xì)胞經(jīng)體外誘導(dǎo)可以分化為外胚層組織,并進(jìn)一步分化為神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞。利用全反式維甲酸(alltrans retinoic acid,RA/ATRA)作為神經(jīng)誘導(dǎo)劑,已經(jīng)成功從人或者小鼠擬胚體(embryoid bodies,EBs)中分化得到多種神經(jīng)細(xì)胞。然而,由于RA作用的廣泛性和復(fù)雜性及所用擬胚體分化系統(tǒng)的復(fù)雜性和難以調(diào)控性,RA對(duì)胚胎干細(xì)胞向神經(jīng)細(xì)胞方向誘導(dǎo)分化的作用機(jī)制尚未明確闡釋。為了簡(jiǎn)化誘導(dǎo)體系,我們采用了單層貼壁培養(yǎng)作為分化模式,以無血清培養(yǎng)的方式,用不同濃度的RA誘導(dǎo)胚胎干細(xì)胞,觀察了RA對(duì)胚胎干細(xì)胞定向分化為神經(jīng)細(xì)胞的作用,并通過干預(yù)與神經(jīng)細(xì)胞分化相關(guān)的多條信號(hào)通路,探討了RA誘導(dǎo)胚胎干細(xì)胞分化為神經(jīng)細(xì)胞的機(jī)制。實(shí)驗(yàn)表明,在無血清單層貼壁培養(yǎng)條件下,RA可以促進(jìn)神經(jīng)細(xì)胞的分化、加速胚胎干細(xì)胞標(biāo)志性分子的丟失;維甲酸相關(guān)核受體及代謝酶類也發(fā)生動(dòng)念變化;維甲酸核受體RAR alpha的特異性拮抗劑、ERK(extracellular signal-regulated protein kinase)信號(hào)通路的抑制劑、GSK3(glycogen synthase kinase 3)的抑制劑均可以有效阻斷RA的誘導(dǎo)作用;FGF(fibroblast growth factor)信號(hào)通路的抑制劑不能有效阻斷RA的作用。實(shí)驗(yàn)闡明了,RA促進(jìn)胚胎干細(xì)胞分化為神經(jīng)細(xì)胞的作用是不依賴FGF信號(hào)通路的;維甲酸核受體RAR alpha在誘導(dǎo)分化過程中扮演重要角色;RA通過與ERK及Wnt信號(hào)通路之間的聯(lián)系而發(fā)揮誘導(dǎo)分化作用。 維脯酰胺(N-all-trans-retinoyl-L-proline,ATRP)是基于全反式維甲酸和芬維A胺的結(jié)構(gòu)合成的新化合物。有研究表明該化合物具有抑制肝癌細(xì)胞侵襲的作用,而在胚胎干細(xì)胞定向分化為神經(jīng)細(xì)胞中的作用還缺少研究。我們的實(shí)驗(yàn)證明了ATRP可以促進(jìn)胚胎干細(xì)胞向神經(jīng)細(xì)胞的分化,其誘導(dǎo)分化得到的神經(jīng)前體細(xì)胞比率要高于RA;與此同時(shí),ATRP加速了胚胎干細(xì)胞標(biāo)志性分子的丟失;ATRP發(fā)揮誘導(dǎo)分化作用的機(jī)制與RA有一定相似之處。值得注意的是,先前的報(bào)道認(rèn)為ATRP不是通過與維甲酸相關(guān)核受體結(jié)合而發(fā)揮作用的,而本研究觀察到,阻斷維甲酸核受體RAR alpha的信號(hào)通路也可以有效阻斷ATRP的誘導(dǎo)分化作用。ATRP與維甲酸信號(hào)通路之間的關(guān)系還需要進(jìn)一步地闡釋;ATRP的代謝情況也需進(jìn)一步研究,其促進(jìn)分化的作用有可能是代謝過程中轉(zhuǎn)化為RA的結(jié)果。 阿爾茨海默病(Alzheimer's disease,AD)是一種進(jìn)行性發(fā)展的致死性神經(jīng)退行性疾病,臨床表現(xiàn)為認(rèn)知障礙和記憶功能不斷惡化,日常生活能力進(jìn)行性減退,并有各種神經(jīng)精神癥狀和行為異常。目前,阿爾茨海默病的藥物治療主要集中在減輕病人膽堿能系統(tǒng)障礙而出現(xiàn)的功能紊亂和疾病癥狀上,沒能從根本上解決問題,而且病人在服用相關(guān)藥物后還會(huì)產(chǎn)生多種不良反應(yīng)。我們應(yīng)用RA誘導(dǎo)胚胎干細(xì)胞定向分化得到神經(jīng)干細(xì)胞、以側(cè)腦室為移植靶位,進(jìn)行了胚胎干細(xì)胞來源的神經(jīng)干細(xì)胞治療AD模型小鼠(Mo/Hu APPswe PS1dE9雙轉(zhuǎn)基因小鼠)的初步探索。實(shí)驗(yàn)表明,經(jīng)RA誘導(dǎo)后,小鼠胚胎干細(xì)胞分化為表達(dá)特異性分子標(biāo)志Nestin且具有分化為神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞能力的神經(jīng)干細(xì)胞。該神經(jīng)干細(xì)胞經(jīng)體外純化、擴(kuò)增及標(biāo)記后,通過立體定位注射移植入雙轉(zhuǎn)基因AD模型小鼠的一側(cè)腦室。手術(shù)一周后,動(dòng)物側(cè)腦室壁可以見到移植的細(xì)胞;手術(shù)六周后,Morris水迷宮測(cè)試顯示治療動(dòng)物的空間記憶能力得到顯著提高;手術(shù)八周后,在治療動(dòng)物的皮層發(fā)現(xiàn)部分移植細(xì)胞呈神經(jīng)元特異性標(biāo)志MAP2陽性和膽堿能神經(jīng)元特異性標(biāo)志ChAT陽性。研究顯示,經(jīng)側(cè)腦室移植胚胎干細(xì)胞來源的外源性神經(jīng)干細(xì)胞可以有效提高AD模型動(dòng)物的認(rèn)知能力。這為臨床應(yīng)用胚胎干細(xì)胞治療AD等中樞神經(jīng)系統(tǒng)退行性疾病提供了一定理論和實(shí)踐基礎(chǔ)。
[Abstract]:Embryonic stem cell (ES cell) is a kind of cell with self-renewal ability and multiple differentiation potential, and can be divided into three germ cells. As a unique in vitro biological system, embryonic stem cells can be used to identify and elucidate the mysteries of the process of biological development, as well as very useful materials such as disease simulation, drug screening and cell therapy. In-vitro induction of embryonic stem cells can be differentiated into ectodermal tissue and further differentiated into neurons and glial cells. The use of all-trans retinoic acid (RA/ ATRA) as a nerve inducing agent has been successfully differentiated from human or mouse embryonic bodies (EBs) to obtain a variety of nerves. However, the mechanism of the mechanism of RA to induce differentiation of embryonic stem cells in the direction of nerve cells is not clear due to the wide and complex nature of RA and the complexity and difficulty of the differentiation system of the embryoid used. In order to simplify the induction system, a single-layer adherent culture was used as the differentiation mode, and the embryonic stem cells were induced with different concentrations of RA in the form of serum-free culture, and the orientation and differentiation of the embryonic stem cells into the nerve cells were observed. The effects of RA on the differentiation of embryonic stem cells into nerve cells were discussed by means of multiple signal pathways related to the differentiation of neural cells. Mechanism. The experiment shows that RA can promote the differentiation of nerve cells and accelerate the loss of the symbolic molecules of embryonic stem cells under the condition of serum-free monolayer adherent culture, and the related nuclear receptors and the metabolic enzymes of the retinoic acid also change; the specificity of the retinoic acid nuclear receptor RAR alpha The inhibitor of the signal pathway of the anti-agent, ERK, and the inhibitor of GSK3 can effectively block the induction of RA, and the inhibitor of the signal pathway of the fibroblast growth factor can not block the RA effectively. The role of RA in promoting the differentiation of embryonic stem cells into nerve cells is not dependent on the signaling pathway of the FGF, and the retinoic acid nuclear receptor RAR alpha plays an important role in the induction of differentiation, and RA induces differentiation through the association with the ERK and Wnt signaling pathways. The function of N-all-trans-retinol-L-proline (ATRP) is based on the structural synthesis of all-trans-retinoic acid and finiveramine. The compound has the effect of inhibiting the invasion of the liver cancer cells, and the directional differentiation of the embryonic stem cells into the nerve cells There is also a lack of research. Our experiments have shown that ATRP can promote the differentiation of embryonic stem cells into nerve cells, and the ratio of neural precursor cells induced by the induction of differentiation is higher than that of RA; at the same time, ATRP accelerates the signature of embryonic stem cells The mechanism of ATRP to induce differentiation and RA has one. The similarities are noted. It is worth noting that the previous report found that ATRP did not function by binding to the retinoic acid-related nuclear receptor, while the study observed that the signal path blocking the RAR alpha of the retinoic acid nuclear receptor could also effectively block the induction of the ATRP. The relationship between the ATRP and the signal pathway of the retinoic acid also needs to be further explained; the metabolism of the ATRP is further studied, and the effect of promoting differentiation is likely to be a transformation in the process of metabolism. As a result of RA, Alzheimer's disease (AD) is a progressive, fatal neurodegenerative disease characterized by a worsening of cognitive and memory functions, progressive deterioration of the ability of daily life, and various neuroses. The symptoms and behavior of the people are abnormal. At present, the drug treatment of Alzheimer's disease is mainly focused on the function disorder and the disease symptom which are caused by the disorder of the cholinergic system of the patient, and the problem can not be solved fundamentally, A variety of adverse reactions were produced. We applied RA to induce the directional differentiation of embryonic stem cells to obtain neural stem cells. The neural stem cells derived from embryonic stem cells were used to treat AD model mice (Mo/ Hu APPswe PS1dE9 double transgenes). The results indicated that after RA induction, the mouse embryonic stem cells were differentiated into the expression-specific molecular marker, Nestin, and differentiated into neurons and glial cells. the neural stem cell is purified, expanded and marked in vitro, and then transplanted into the double-transgenic AD mould through a three-dimensional positioning injection, One week after the operation, the wall of the lateral ventricle of the animal can see the transplanted cells; after 6 weeks of operation, the Morris water maze test shows that the spatial memory capacity of the treated animals is significant increased; after 8 weeks of operation, partial transplantation cells were found to be neuron-specific marker MAP2-positive and cholinergic neuron-specific in the cortex of the treated animals The marker ChAT is positive. The study shows that the exogenous neural stem cells derived from the embryonic stem cell in the lateral ventricle can effectively improve the AD model. The cognitive ability of a type of animal is provided for clinical application of embryonic stem cells to treat the degenerative diseases of the central nervous system such as AD.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Transplanted neuronal precursors migrate and differentiate in the devel-oping mouse brain[J];Cell Research;2002年Z1期

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本文編號(hào)：2446672