龐希寧

中國醫(yī)科大學(xué)細胞生物學(xué)衛(wèi)生部重點實驗室干細胞與再生醫(yī)學(xué)研究室,沈陽 110001

細胞發(fā)育和分化的內(nèi)部編程是遵照細胞內(nèi)基因 表達規(guī)律進行的。每個細胞都有全能的細胞核及相同的分化潛能。在胚胎發(fā)育不同階段,由于細胞所處微環(huán)境不同及細胞定向分化內(nèi)部編程不同,基因表達就存在差異,即開放某些基因,關(guān)閉某些基因,以使細胞合成特異性的蛋白質(zhì),產(chǎn)生不同的結(jié)構(gòu)、功能及表型?；蚪MDNA在細胞分化過程中不是全部表達,而是基因的差異表達,即奢侈基因按一定程序有選擇地相繼活化表達。調(diào)控細胞分化的基因編程是由不同信號分子在特定時間和空間作用于細胞,產(chǎn)生基因表達的內(nèi)在規(guī)律?？傊?如能改變基因的表達就能改變細胞的編程,從而改變細胞的分化方向。

細胞重編程是在一定條件下成體細胞的記憶被擦除,重新程序化產(chǎn)生新的表型和功能,導(dǎo)致細胞的命運發(fā)生改變。細胞重編程主要發(fā)生在不涉及基因組DNA序列改變的基因表達水平。對于細胞重編程的深入研究有助于掌握機體細胞的發(fā)生發(fā)育機制,為解決再生醫(yī)學(xué)種子細胞來源問題提供理論基礎(chǔ)[1]。

細胞重編程的種類

細胞重編程主要分為兩類:(1)分化的成體細胞在特定條件下被逆轉(zhuǎn)后恢復(fù)到全能性或多能性狀態(tài),形成新的多能性干細胞[2],如誘導(dǎo)多潛能干細胞 (induced pluripotent stem cells,iPSCs)。近年來,iPSCs研究為基因調(diào)控去分化和轉(zhuǎn)分化提供了分子實驗依據(jù),使有目的地調(diào)控細胞基因的表達,改變細胞內(nèi)部編程來改變原來分化方向成為現(xiàn)實。(2)一種胚層來源的細胞或多能性干細胞向同胚層或不同胚層來源的另一種成體細胞或多能性干細胞轉(zhuǎn)化,也稱轉(zhuǎn)分化 (transdifferentiation)[3]。

影響細胞重編程的因素

研究表明,基因表達不但受轉(zhuǎn)錄因子的調(diào)控,還與其DNA和組蛋白表觀遺傳學(xué)修飾有關(guān)[4],包括DNA甲基化、組蛋白乙酰化、印記基因表達、端粒長度恢復(fù)、X染色體失活等[5]。在真核生物基因組的非蛋白質(zhì)編碼區(qū)存在大量非編碼RNA(non-coding RNA,ncRNA)基因,這些非編碼區(qū)域擔(dān)負著基因表達調(diào)控等重要功能,其編碼產(chǎn)物可在轉(zhuǎn)錄后水平調(diào)節(jié)靶基因的表達,是調(diào)控細胞內(nèi)基因表達的基本機制之一。ncRNA不僅在干細胞的多能性維持過程中有重要作用,在成體細胞重編程中也發(fā)揮重要作用[6],還參與了干細胞分化的調(diào)節(jié)[7]。

細胞重編程主要針對定向分化的某些關(guān)鍵調(diào)節(jié)基因,特別是近年發(fā)現(xiàn)的某些因子能調(diào)控大量基因的表達,對細胞編程起著重要作用。例如:神經(jīng)元限制性沉默因子 (RE1-silencing transcription factor/neuron restrictive silencer factor,REST/NRSF)通過與調(diào)控基因啟動子的一段21nt的DNA保守序列—神經(jīng)元限制性沉默元件 (neuron restrictive silencer element/repressor element 1,NRSE/RE-1)結(jié)合,經(jīng)一系列反應(yīng)使組蛋白發(fā)生甲基化和去乙?；揎?使染色質(zhì)呈凝縮狀態(tài),啟動子區(qū)域無法和轉(zhuǎn)錄因子及RNA聚合酶結(jié)合,而抑制其轉(zhuǎn)錄活性。這種表觀遺傳學(xué)修飾可批量抑制上千個與神經(jīng)細胞分化相關(guān)的基因,其抑制的解除是神經(jīng)細胞分化的必要條件[8]。最近研究表明,胰島 β細胞分化基因胰十二指腸同源框1(Pdx1)、胰島素和神經(jīng)原質(zhì)蛋白3(Ngn3)、神經(jīng)源性分化蛋白1(NeuroD1)和成對盒4(Pax4)均因具有NRSE序列而受REST/NRSF調(diào)節(jié)[9-11]。Pdx1主要在胰腺前體細胞中表達,是促進早期胰腺發(fā)育以及胰島 β細胞成熟的關(guān)鍵基因,是最受關(guān)注的具有正向調(diào)節(jié)作用的轉(zhuǎn)錄激活因子,能和眾多與胰島分化有關(guān)的靶基因啟動子中的TAAT序列結(jié)合,從而在啟動胰島內(nèi)分泌細胞分化過程中發(fā)揮重要的作用[12]。

隨著發(fā)育分子生物學(xué)研究的深入,許多組織細胞的發(fā)育機制研究不斷深入,提供了大量細胞分化的分子生物學(xué)信息,為未來改變細胞的內(nèi)部編程提供了理論依據(jù)。通過調(diào)控轉(zhuǎn)錄因子和DNA、組蛋白表觀遺傳學(xué)修飾和miRNA來抑制或促進不同基因的表達已成為可能。miRNA可在轉(zhuǎn)錄后水平調(diào)節(jié)靶基因的表達,即通過對mRNA特異序列的抑制,批量調(diào)節(jié)基因的活性而改變細胞的編程。

研究細胞重編程的方法

目前主要通過逆轉(zhuǎn)錄病毒 (主要是慢病毒)、腺病毒、質(zhì)粒和轉(zhuǎn)座子等介導(dǎo)的方式將轉(zhuǎn)錄因子對應(yīng)的基因或者小分子導(dǎo)入成體細胞,將其進行重編程。

逆轉(zhuǎn)錄病毒轉(zhuǎn)導(dǎo) 逆轉(zhuǎn)錄病毒又名反轉(zhuǎn)錄病毒,是一組RNA病毒,其病毒科下包括慢病毒在內(nèi)共7屬病毒。病毒感染宿主細胞時,在逆轉(zhuǎn)錄酶作用下,逆轉(zhuǎn)錄病毒首先將其RNA逆轉(zhuǎn)錄為DNA,然后將這段逆轉(zhuǎn)錄的基因插入細胞基因組中保持整合狀態(tài),并傳給宿主細胞后代。慢病毒作為目前應(yīng)用最廣泛的逆轉(zhuǎn)錄病毒,其優(yōu)點是轉(zhuǎn)入基因可以長期穩(wěn)定表達,并且對大部分哺乳動物細胞,包括神經(jīng)元、干細胞等難轉(zhuǎn)染的細胞,特別是體外懸浮生長的細胞,都有很好的轉(zhuǎn)染效率。缺點是逆轉(zhuǎn)錄病毒整合到宿主細胞基因組的位置是隨機的,這也就意味著有引起基因突變、激活癌基因的風(fēng)險[1,13]。

腺病毒轉(zhuǎn)導(dǎo) 腺病毒從腺樣組織分離出來,其遺傳物質(zhì)為線型雙股DNA,全長約30000～42000 bp。腺病毒的優(yōu)點是幾乎在所有已知細胞中都不整合到染色體中,因此不會干擾其他宿主基因,并且人類感染野生型腺病毒后僅產(chǎn)生輕微的自限性癥狀。腺病毒具有嗜上皮細胞性,因此對大多數(shù)細胞特別是上皮細胞有幾乎100%的感染效率。腺病毒系統(tǒng)包裝的病毒顆粒滴度高,濃縮后可以達到1013VP/ml,這一特點使其非常適用于基因治療。腺病毒的缺點是由于其不能整合到宿主細胞基因組中,因此不能長期穩(wěn)定表達[14-15]。

質(zhì)粒轉(zhuǎn)染轉(zhuǎn)導(dǎo) 采用脂質(zhì)體轉(zhuǎn)染的方法,將外源性質(zhì)粒轉(zhuǎn)導(dǎo)進入目的細胞中表達。優(yōu)點是細胞中不再留存有任何外源的DNA,不易使基因癌化。缺點是瞬時表達,轉(zhuǎn)染成功細胞的獲得率尚待提高[16]。

piggyBac轉(zhuǎn)座子轉(zhuǎn)導(dǎo) piggyBac轉(zhuǎn)座子是一個自主因子,遵循 “剪切—黏貼”機制,在生物體染色體中特征性的TTAA四核苷酸序列位點準確地切入和轉(zhuǎn)座,并可以在作用一段時間后采用轉(zhuǎn)座酶切除外源性插入序列。piggyBac轉(zhuǎn)座子受生物體種類的限制較少,適用范圍較廣,轉(zhuǎn)座頻率較高。其作為非病毒體系提高了安全性,不易使基因癌化。缺點是需要多次使用轉(zhuǎn)座酶去除轉(zhuǎn)入序列,但仍然可能會留下一些痕跡[17-18]。

蛋白直接誘導(dǎo) 可以通過4個蛋白 (Oct4、Sox2、Klf4和c-Myc)誘導(dǎo)成體細胞重編程為iPSCs,優(yōu)點是誘導(dǎo)過程不存在外源基因,缺點是誘導(dǎo)效率沒有病毒載體誘導(dǎo)的效率高[19]。

RNA干擾 通過RNA干擾抑制某個或某幾個基因的方法來對細胞基因表達進行重新編程,用十四烷基聚精氨酸肽鏈 [myristoylated polyarginine peptides,Myr-Ala-(Arg)7-Cys-CONH2,MPAP]將小干擾RNA導(dǎo)入細胞技術(shù)的出現(xiàn)[20],將有助于推動RNA干擾方法對成體細胞的重編程。

重編程細胞的來源及其轉(zhuǎn)化方向的最新進展

重編程細胞產(chǎn)生iPSCs 不同胚層發(fā)育來源的成體細胞甚至胚外組織均有很多重編程產(chǎn)生iPSCs的報道 (表1),說明分化成熟的細胞都有可能通過重編程擦去原來的記憶去分化為胚胎干細胞。這些研究證實改變細胞基因表達程序 (時空和差異)就能改變細胞的分化方向。

表1 各不同種屬不同胚層發(fā)育來源的細胞誘導(dǎo)的誘導(dǎo)多潛能干細胞Table 1 Indceced pluripotent stem cells induced from cells of various species and germ layers

重編程后轉(zhuǎn)分化的細胞 不但同一器官中發(fā)育于同一內(nèi)胚層的胰腺外分泌腺泡細胞可以通過重編程向胰島內(nèi)分泌細胞 β細胞轉(zhuǎn)化[77],不同器官中內(nèi)胚層來源的肝細胞也可通過重編程向胰島 β細胞轉(zhuǎn)化[78]。而胰腺外分泌腺泡細胞還可通過重編程向肝細胞轉(zhuǎn)化[79]。此外,內(nèi)胚層來源的肝細胞可通過重編程向外胚層來源的神經(jīng)細胞轉(zhuǎn)化[80],中胚層來源的皮膚成纖維細胞可通過重編程向外胚層來源的神經(jīng)細胞[81]及內(nèi)胚層來源的肝樣細胞轉(zhuǎn)化[82],中胚層來源的骨髓間充質(zhì)干細胞重編程向內(nèi)胚層來源的胰島β細胞轉(zhuǎn)化,中胚層來源的成纖維細胞重編程向中胚層來源的心肌細胞轉(zhuǎn)化[83],外胚層來源的表皮黑色素細胞重編程為外胚層來源神經(jīng)嵴干細胞樣細胞[84](表2)。綜上,各不同胚層發(fā)育來源的成體細胞都有可能通過重編程擦去原來的記憶重新轉(zhuǎn)分化為同一胚層或其他胚層發(fā)育來源的成體細胞,這同樣證實改變細胞基因表達程序 (時空和差異)就能改變細胞的分化方向。

總之,目前正在進入一個可以通過調(diào)節(jié)關(guān)鍵基因?qū)毎M行重新編程的時代,人類未來將由此從許多分化的細胞獲得更多所需要的另一些分化細胞,這對疾病的細胞替代治療和細胞分化機制研究具有劃時代的意義。

表2 不同胚層發(fā)育來源體細胞的重編程轉(zhuǎn)分化Table 2 Reprogramming of adult cells from developmental original of various germ layers

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