重編程及誘導(dǎo)性多能干細(xì)胞在再生醫(yī)學(xué)的研究進(jìn)展

1、·綜述·重編程及誘導(dǎo)性多能干細(xì)胞在再生醫(yī)學(xué)的研究進(jìn)展劉路韋曦凌均棨細(xì)胞分化是由多種特定的分化基因網(wǎng)絡(luò)相互作用、共同調(diào)控完成的生物學(xué)過(guò)程,如骨髓間充質(zhì)細(xì)胞礦化,是在TGF/BMP、Wnt、Notch和Cadherin等信號(hào)通路協(xié)同作用下,先分化為成骨細(xì)胞,隨后礦化完成的1。在一定條件下該程序可逆向編程,稱為細(xì)胞重編程(reprogramming或去分化(de-differentiation2-3。由于胚胎干細(xì)胞(embryonic stem cells,ESCs的應(yīng)用面臨倫理及免疫排斥等問(wèn)題4,而將體細(xì)胞去分化為多能性干細(xì)胞,不僅可彌補(bǔ)ESCs 的不足,而且符合法律和倫理規(guī)范

2、,因此具有廣闊的應(yīng)用前景,是當(dāng)前干細(xì)胞組織工程學(xué)和再生醫(yī)學(xué)研究的最前沿5-6。近年來(lái),在干細(xì)胞組織工程學(xué)領(lǐng)域出現(xiàn)了較大的突破和進(jìn)展,但許多問(wèn)題仍然未得到解答,如已分化細(xì)胞重編程為更早期更原始細(xì)胞的能力,干細(xì)胞微環(huán)境對(duì)分化、轉(zhuǎn)分化、重編程的作用和影響,分化、轉(zhuǎn)分化和重編程的關(guān)系等7。由于其復(fù)雜性,分化及再生中涉及的分子調(diào)控機(jī)制尚不清楚,使對(duì)該生物學(xué)行為的認(rèn)識(shí)以及干細(xì)胞治療的臨床應(yīng)用受到了限制。一、轉(zhuǎn)分化和去分化較早的研究認(rèn)為,只有未分化狀態(tài)的細(xì)胞具備多向分化潛能,而部分或完全分化細(xì)胞不具備該功能8。然而,去分化、轉(zhuǎn)分化(transdifferentiation、可塑性(plasticity概念的

3、提出打破了這一傳統(tǒng)觀念。去分化是分化的細(xì)胞類型轉(zhuǎn)化為另一較原始的具備更多分化潛能的細(xì)胞類型,分化的細(xì)胞狀態(tài)轉(zhuǎn)化為類似胚胎干細(xì)胞或前體細(xì)胞的狀態(tài)9-10。轉(zhuǎn)分化最初定義為已分化細(xì)胞通過(guò)出生后的細(xì)胞核基因編程轉(zhuǎn)化為另一分化的細(xì)胞類型的不可逆的過(guò)程11。隨后,轉(zhuǎn)分化概念被用于描述單功能分化的細(xì)胞類型轉(zhuǎn)化為可分化為多細(xì)胞器官的多種細(xì)胞類型,以及組織特異性干細(xì)胞跨胚層分化的過(guò)程3。轉(zhuǎn)分化屬于廣義的細(xì)胞類型轉(zhuǎn)換(metaplasias,是一種由核心調(diào)控基因(master switch gene表達(dá)改變而引起的細(xì)胞形態(tài)和性能改變?？伤苄灾赋审w干細(xì)胞不僅可以生成它們所在組織的成熟細(xì)胞,而且在特定環(huán)境下能跨系或

4、跨胚層轉(zhuǎn)化成其他組織類型細(xì)胞的能力,如基因標(biāo)記的骨骼肌細(xì)胞和神經(jīng)干細(xì)胞可在一定條件下分化為造血干細(xì)胞12。盡管存在爭(zhēng)議,許多關(guān)于低等脊椎動(dòng)物的報(bào)道表明轉(zhuǎn)分化和去分化在自然界中絕非偶然現(xiàn)象13-14。轉(zhuǎn)分化和去分化涉及數(shù)個(gè)關(guān)鍵基因及其網(wǎng)絡(luò)的調(diào)控,對(duì)該分子機(jī)制的研究有助于進(jìn)一步認(rèn)識(shí)細(xì)胞的發(fā)育生物學(xué),為干細(xì)胞治療的應(yīng)用提供理論依據(jù)。然而,鑒定轉(zhuǎn)分化和去分化的金標(biāo)準(zhǔn);轉(zhuǎn)分化的細(xì)胞轉(zhuǎn)換是否首先通過(guò)細(xì)胞去分化,回到一個(gè)較原始的狀態(tài),再向另一方向分化11;轉(zhuǎn)分化和去分化是否由相似的分子機(jī)制和不同程度的細(xì)胞核重編程完成,該過(guò)程是否為時(shí)空依賴性的生物學(xué)過(guò)程,受表達(dá)的基因和元件協(xié)同網(wǎng)絡(luò)調(diào)控15等問(wèn)題尚待探討?；?/p>

5、項(xiàng)目:國(guó)家自然科學(xué)基金(30872876;廣東省科技計(jì)劃項(xiàng)目(2008B030301075作者單位:510055廣州,中山大學(xué)光華口腔醫(yī)學(xué)院·附屬口腔醫(yī)院·口腔醫(yī)學(xué)研究所二、重編程及誘導(dǎo)性多能干細(xì)胞ESCs具備良好的干細(xì)胞潛能,但越來(lái)越多的研究表明ESCs涉及難以解決的倫理、免疫排斥等問(wèn)題。相對(duì)而言,眾多器官來(lái)源的成體干細(xì)胞易于獲得,不存在免疫排斥,逐漸成為細(xì)胞替代治療的研究熱點(diǎn)和種子細(xì)胞的來(lái)源。然而,大多數(shù)成體干細(xì)胞自我增殖和多向分化能力有限,且多為異質(zhì)性細(xì)胞群,干細(xì)胞比例極低,作為種子細(xì)胞應(yīng)用的可能性受到限制16,因此誘導(dǎo)成人體細(xì)胞成為多能干細(xì)胞將提供有效的解決途徑。2

6、006年,Takahashi等2將從ESCs中篩選獲得的24個(gè)候選基因分別插入逆轉(zhuǎn)錄病毒載體,感染小鼠胚胎成纖維細(xì)胞(mouse embryonic fibroblast,MEF,揭示Oct-4、Sox-2、c-m yc、Klf4四種基因組合轉(zhuǎn)染的MEF具備與ESCs相似的細(xì)胞形態(tài)、表面標(biāo)記、自我增殖和多向分化能力,從而將轉(zhuǎn)染細(xì)胞命名為誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cells,iPS。隨后,Yu 等17通過(guò)整合14個(gè)重編程候選重組基因入人類體細(xì)胞,證實(shí)Oct-4、Sox-2、Nanog及Lin28在重編程及iPS細(xì)胞形成中的作用最為突出,其中Oct-4和S

7、ox-2直接影響iPS細(xì)胞克隆形成,而Nanog及Lin28則可協(xié)助Oct-4/Sox-2作用,提高克隆形成率和iPS細(xì)胞存活率。重編程及誘導(dǎo)性多能干細(xì)胞是干細(xì)胞領(lǐng)域的重大發(fā)現(xiàn),對(duì)解決種子細(xì)胞來(lái)源稀少的問(wèn)題具有重要意義。然而,對(duì)于重編程及誘導(dǎo)性多能干細(xì)胞的認(rèn)識(shí)還停留在初級(jí)階段,其具體調(diào)控機(jī)制、信號(hào)通路、與微環(huán)境的關(guān)系以及iPS在體內(nèi)的成瘤性等問(wèn)題尚未解決,且人工誘導(dǎo)iPS細(xì)胞的方法及效率均有待提高6,18。ESCs全能性相關(guān)轉(zhuǎn)錄因子Oct-4蛋白由octamer連接轉(zhuǎn)錄因子4基因(Oct4、Oct3/4、Pou5F1編碼,屬于POU家族成員,是誘導(dǎo)、維持細(xì)胞多能性及未分化狀態(tài)的主要調(diào)節(jié)因子19

8、。大量研究發(fā)現(xiàn),Oct-4在維持ESCs多能性和重編程iPS細(xì)胞的基因調(diào)控網(wǎng)絡(luò)中具備重要作用,其表達(dá)的喪失可導(dǎo)致多能性的喪失、細(xì)胞分化及iPS細(xì)胞克隆形成能力的喪失2,17,20。隨后研究揭示人類Oct4兩個(gè)亞型呈現(xiàn)不同的表達(dá)模式,并在維持自我更新和多向分化潛能方面具備不同的功能21。Oct4A亞型位于細(xì)胞核內(nèi),與維持干細(xì)胞性能和轉(zhuǎn)錄因子功能相關(guān);而Oct4B主要表達(dá)于細(xì)胞質(zhì),對(duì)干細(xì)胞功能的維持未證實(shí)有明顯相關(guān)作用。此外,自我更新和胚胎干細(xì)胞系標(biāo)記Sox2(Sex determining region Y bos-2也被報(bào)道在誘導(dǎo)多能干細(xì)胞和胚胎干細(xì)胞中具備不可或缺的關(guān)鍵作用6-7。Sox-2

9、是SRY相關(guān)HMG盒轉(zhuǎn)錄因子家族成員,在早期胚胎發(fā)育中與Oct-4呈現(xiàn)相關(guān)的表達(dá)模式并協(xié)同調(diào)控早期胚胎發(fā)育相關(guān)基因的表達(dá)22。通過(guò)RNA干擾敲除Sox2可與Wnt信號(hào)通路核心元件-catenin反應(yīng),使Wnt通路靶基因CCND1和c-m yc下調(diào),從而抑制礦化23。Oct4和Sox2可共同作用啟動(dòng)多種轉(zhuǎn)錄因子(如c-m yc、Klf4、FGF4和Nanog的表達(dá),調(diào)控細(xì)胞多能性,提示了Oct4-Sox2復(fù)合體在細(xì)胞分化和重編程基因調(diào)控網(wǎng)絡(luò)中的核心地位。有學(xué)者提出,在人類和小鼠胚胎干細(xì)胞中Sox2均為Oct4的靶基因,直接受Oct4調(diào)控24。然而,近年研究提出Oct4對(duì)骨髓細(xì)胞和組織來(lái)源成體細(xì)胞

10、多能性的維持并非不可缺少25,Sox2在成人體細(xì)胞重編程的過(guò)程中具備獨(dú)立的作用,甚至可影響Oct4的表達(dá)26。三、重編程在口腔再生醫(yī)學(xué)的研究現(xiàn)狀在口腔干細(xì)胞組織工程和再生醫(yī)學(xué)領(lǐng)域,關(guān)于誘導(dǎo)性多能干細(xì)胞重編程核心調(diào)控因子的研究尚屬起步階段。2006年,Kerkis等27從脫落乳牙中分離到表達(dá)Oct-4、Nanog、SSEA-3、SSEA-4等胚胎干細(xì)胞標(biāo)志的牙髓干細(xì)胞。Cheng等28亦報(bào)道成年猩猩的牙髓組織中含有Oct-4、Sox-2、Nanog、Rex-1陽(yáng)性細(xì)胞。此外,Agata等29證實(shí)缺氧狀態(tài)可激活未分化的豬牙髓細(xì)胞中Oct-4和Sox-2的瞬間表達(dá),而對(duì)已礦化誘導(dǎo)的牙髓細(xì)胞無(wú)明顯作用

11、。Techawattanawisal 等30則發(fā)現(xiàn),體外誘導(dǎo)成體大鼠牙周韌帶細(xì)胞,可強(qiáng)表達(dá)Sox-2和弱表達(dá)干細(xì)胞標(biāo)記ABCG2,但不表達(dá)Oct-4。四、存在問(wèn)題及前景展望關(guān)于分化這一可逆性過(guò)程的研究目前仍存在許多未解答的問(wèn)題,如在非自然條件下的細(xì)胞類型轉(zhuǎn)換是否顛覆了傳統(tǒng)的發(fā)育和分化理論,細(xì)胞重編程及其最終的狀態(tài)是否由外源性環(huán)境信號(hào)、細(xì)胞內(nèi)在分子機(jī)制抑或兩者共同決定31。迄今,我們對(duì)組織再生和細(xì)胞重編程的分子調(diào)控機(jī)制知之甚少,轉(zhuǎn)染和細(xì)胞核移植是目前認(rèn)為最有效的誘導(dǎo)體細(xì)胞重編程的方法。然而,由于缺乏鑒定iPS的金標(biāo)準(zhǔn),該作法是完全的重編程抑或細(xì)胞融合的假象尚無(wú)定論3。在各種成體細(xì)胞中,重編程可能

12、存在一個(gè)共同的基因調(diào)控網(wǎng)絡(luò),或許通過(guò)組織特異性的網(wǎng)絡(luò)調(diào)控,導(dǎo)致不同組織來(lái)源細(xì)胞自我更新和多向分化能力。而如何高效的引入關(guān)鍵核心基因調(diào)控網(wǎng)絡(luò),以確保該基因網(wǎng)絡(luò)能在各種發(fā)育分化等生物學(xué)轉(zhuǎn)化過(guò)程中發(fā)揮穩(wěn)定作用尚不清楚29。有研究表明,干細(xì)胞遷移至新環(huán)境可分化為表達(dá)該組織特異性標(biāo)記的細(xì)胞,但它們依然無(wú)法完全具備該組織來(lái)源細(xì)胞的所有生物學(xué)特性32。有學(xué)者提出,組織特異性干細(xì)胞在體內(nèi)存在多潛能狀態(tài),該狀態(tài)由外源性和內(nèi)源性干細(xì)胞微環(huán)境信號(hào)共同調(diào)控,而不是單一由Oct4等幾個(gè)轉(zhuǎn)錄因子調(diào)控33。如果這是事實(shí),體內(nèi)和體外干細(xì)胞微環(huán)境可能對(duì)重編程過(guò)程有不同的作用。核心轉(zhuǎn)錄因子激活、出生后的基因修飾啟動(dòng),以及隨后的基

13、因表達(dá)模式的改變和重編程過(guò)程,可能由干細(xì)胞微環(huán)境啟動(dòng)和誘導(dǎo)。由此可見(jiàn),分子水平的細(xì)胞重編程過(guò)程仍需進(jìn)一步研究,才能為體內(nèi)實(shí)驗(yàn)以及將來(lái)的臨床應(yīng)用提供可靠的實(shí)驗(yàn)依據(jù)。綜上所述,細(xì)胞重編程及誘導(dǎo)性多能干細(xì)胞的發(fā)現(xiàn)為解決組織工程中種子細(xì)胞來(lái)源不足這一難題具有重要意義。雖然誘導(dǎo)性多能干細(xì)胞最終的臨床應(yīng)用仍面臨許多技術(shù)問(wèn)題,如重編程基因的穩(wěn)定持續(xù)表達(dá)、提高轉(zhuǎn)染效率、非病毒載體的應(yīng)用等,但體細(xì)胞重編程為多能干細(xì)胞仍可望為再生醫(yī)學(xué)和干細(xì)胞治療全身性疾病提供優(yōu)質(zhì)種子細(xì)胞,在不久的將來(lái)應(yīng)用于各種老年性相關(guān)疾病和損傷修復(fù)治療。參考文獻(xiàn)1Lian JB,Stein GS,Javed A,et al.Networks

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