龐希寧

中國(guó)醫(yī)科大學(xué)細(xì)胞生物學(xué)衛(wèi)生部重點(diǎn)實(shí)驗(yàn)室干細(xì)胞與再生醫(yī)學(xué)研究室,沈陽(yáng) 110001

細(xì)胞發(fā)育和分化的內(nèi)部編程是遵照細(xì)胞內(nèi)基因 表達(dá)規(guī)律進(jìn)行的。每個(gè)細(xì)胞都有全能的細(xì)胞核及相同的分化潛能。在胚胎發(fā)育不同階段,由于細(xì)胞所處微環(huán)境不同及細(xì)胞定向分化內(nèi)部編程不同,基因表達(dá)就存在差異,即開(kāi)放某些基因,關(guān)閉某些基因,以使細(xì)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(xì)胞分化過(guò)程中不是全部表達(dá),而是基因的差異表達(dá),即奢侈基因按一定程序有選擇地相繼活化表達(dá)。調(diào)控細(xì)胞分化的基因編程是由不同信號(hào)分子在特定時(shí)間和空間作用于細(xì)胞,產(chǎn)生基因表達(dá)的內(nèi)在規(guī)律。總之,如能改變基因的表達(dá)就能改變細(xì)胞的編程,從而改變細(xì)胞的分化方向。

細(xì)胞重編程是在一定條件下成體細(xì)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(xì)胞的命運(yùn)發(fā)生改變。細(xì)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(dá)水平。對(duì)于細(xì)胞重編程的深入研究有助于掌握機(jī)體細(xì)胞的發(fā)生發(fā)育機(jī)制,為解決再生醫(yī)學(xué)種子細(xì)胞來(lái)源問(wèn)題提供理論基礎(chǔ)[1]。

細(xì)胞重編程的種類

細(xì)胞重編程主要分為兩類:(1)分化的成體細(xì)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(xì)胞[2],如誘導(dǎo)多潛能干細(xì)胞 (induced pluripotent stem cells,iPSCs)。近年來(lái),iPSCs研究為基因調(diào)控去分化和轉(zhuǎn)分化提供了分子實(shí)驗(yàn)依據(jù),使有目的地調(diào)控細(xì)胞基因的表達(dá),改變細(xì)胞內(nèi)部編程來(lái)改變?cè)瓉?lái)分化方向成為現(xiàn)實(shí)。(2)一種胚層來(lái)源的細(xì)胞或多能性干細(xì)胞向同胚層或不同胚層來(lái)源的另一種成體細(xì)胞或多能性干細(xì)胞轉(zhuǎn)化,也稱轉(zhuǎn)分化 (transdifferentiation)[3]。

影響細(xì)胞重編程的因素

研究表明,基因表達(dá)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?；?、印記基因表達(dá)、端粒長(zhǎng)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(fù)著基因表達(dá)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(dá),是調(diào)控細(xì)胞內(nèi)基因表達(dá)的基本機(jī)制之一。ncRNA不僅在干細(xì)胞的多能性維持過(guò)程中有重要作用,在成體細(xì)胞重編程中也發(fā)揮重要作用[6],還參與了干細(xì)胞分化的調(diào)節(jié)[7]。

細(xì)胞重編程主要針對(duì)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(dá),對(duì)細(xì)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(guò)與調(diào)控基因啟動(dòng)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(dòng)子區(qū)域無(wú)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(gè)與神經(jīng)細(xì)胞分化相關(guān)的基因,其抑制的解除是神經(jīng)細(xì)胞分化的必要條件[8]。最近研究表明,胰島 β細(xì)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(duì)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(xì)胞中表達(dá),是促進(jìn)早期胰腺發(fā)育以及胰島 β細(xì)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(dòng)子中的TAAT序列結(jié)合,從而在啟動(dòng)胰島內(nèi)分泌細(xì)胞分化過(guò)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(xì)胞的發(fā)育機(jī)制研究不斷深入,提供了大量細(xì)胞分化的分子生物學(xué)信息,為未來(lái)改變細(xì)胞的內(nèi)部編程提供了理論依據(jù)。通過(guò)調(diào)控轉(zhuǎn)錄因子和DNA、組蛋白表觀遺傳學(xué)修飾和miRNA來(lái)抑制或促進(jìn)不同基因的表達(dá)已成為可能。miRNA可在轉(zhuǎn)錄后水平調(diào)節(jié)靶基因的表達(dá),即通過(guò)對(duì)mRNA特異序列的抑制,批量調(diào)節(jié)基因的活性而改變細(xì)胞的編程。

研究細(xì)胞重編程的方法

目前主要通過(guò)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(duì)應(yīng)的基因或者小分子導(dǎo)入成體細(xì)胞,將其進(jìn)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(xì)胞時(shí),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(xì)胞基因組中保持整合狀態(tài),并傳給宿主細(xì)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(diǎn)是轉(zhuǎn)入基因可以長(zhǎng)期穩(wěn)定表達(dá),并且對(duì)大部分哺乳動(dòng)物細(xì)胞,包括神經(jīng)元、干細(xì)胞等難轉(zhuǎn)染的細(xì)胞,特別是體外懸浮生長(zhǎng)的細(xì)胞,都有很好的轉(zhuǎn)染效率。缺點(diǎn)是逆轉(zhuǎn)錄病毒整合到宿主細(xì)胞基因組的位置是隨機(jī)的,這也就意味著有引起基因突變、激活癌基因的風(fēng)險(xiǎn)[1,13]。

腺病毒轉(zhuǎn)導(dǎo) 腺病毒從腺樣組織分離出來(lái),其遺傳物質(zhì)為線型雙股DNA,全長(zhǎng)約30000～42000 bp。腺病毒的優(yōu)點(diǎn)是幾乎在所有已知細(xì)胞中都不整合到染色體中,因此不會(huì)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(xì)胞性,因此對(duì)大多數(shù)細(xì)胞特別是上皮細(xì)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(dá)到1013VP/ml,這一特點(diǎn)使其非常適用于基因治療。腺病毒的缺點(diǎn)是由于其不能整合到宿主細(xì)胞基因組中,因此不能長(zhǎng)期穩(wěn)定表達(dá)[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(jìn)入目的細(xì)胞中表達(dá)。優(yōu)點(diǎn)是細(xì)胞中不再留存有任何外源的DNA,不易使基因癌化。缺點(diǎn)是瞬時(shí)表達(dá),轉(zhuǎn)染成功細(xì)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(gè)自主因子,遵循 “剪切—黏貼”機(jī)制,在生物體染色體中特征性的TTAA四核苷酸序列位點(diǎn)準(zhǔn)確地切入和轉(zhuǎn)座,并可以在作用一段時(shí)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(diǎn)是需要多次使用轉(zhuǎn)座酶去除轉(zhuǎn)入序列,但仍然可能會(huì)留下一些痕跡[17-18]。

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

RNA干擾 通過(guò)RNA干擾抑制某個(gè)或某幾個(gè)基因的方法來(lái)對(duì)細(xì)胞基因表達(dá)進(jìn)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(xì)胞技術(shù)的出現(xiàn)[20],將有助于推動(dòng)RNA干擾方法對(duì)成體細(xì)胞的重編程。

重編程細(xì)胞的來(lái)源及其轉(zhuǎn)化方向的最新進(jìn)展

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

表1 各不同種屬不同胚層發(fā)育來(lái)源的細(xì)胞誘導(dǎo)的誘導(dǎo)多潛能干細(xì)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(xì)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(xì)胞可以通過(guò)重編程向胰島內(nèi)分泌細(xì)胞 β細(xì)胞轉(zhuǎn)化[77],不同器官中內(nèi)胚層來(lái)源的肝細(xì)胞也可通過(guò)重編程向胰島 β細(xì)胞轉(zhuǎn)化[78]。而胰腺外分泌腺泡細(xì)胞還可通過(guò)重編程向肝細(xì)胞轉(zhuǎn)化[79]。此外,內(nèi)胚層來(lái)源的肝細(xì)胞可通過(guò)重編程向外胚層來(lái)源的神經(jīng)細(xì)胞轉(zhuǎn)化[80],中胚層來(lái)源的皮膚成纖維細(xì)胞可通過(guò)重編程向外胚層來(lái)源的神經(jīng)細(xì)胞[81]及內(nèi)胚層來(lái)源的肝樣細(xì)胞轉(zhuǎn)化[82],中胚層來(lái)源的骨髓間充質(zhì)干細(xì)胞重編程向內(nèi)胚層來(lái)源的胰島β細(xì)胞轉(zhuǎn)化,中胚層來(lái)源的成纖維細(xì)胞重編程向中胚層來(lái)源的心肌細(xì)胞轉(zhuǎn)化[83],外胚層來(lái)源的表皮黑色素細(xì)胞重編程為外胚層來(lái)源神經(jīng)嵴干細(xì)胞樣細(xì)胞[84](表2)。綜上,各不同胚層發(fā)育來(lái)源的成體細(xì)胞都有可能通過(guò)重編程擦去原來(lái)的記憶重新轉(zhuǎn)分化為同一胚層或其他胚層發(fā)育來(lái)源的成體細(xì)胞,這同樣證實(shí)改變細(xì)胞基因表達(dá)程序 (時(shí)空和差異)就能改變細(xì)胞的分化方向。

總之,目前正在進(jìn)入一個(gè)可以通過(guò)調(diào)節(jié)關(guān)鍵基因?qū)?xì)胞進(jìn)行重新編程的時(shí)代,人類未來(lái)將由此從許多分化的細(xì)胞獲得更多所需要的另一些分化細(xì)胞,這對(duì)疾病的細(xì)胞替代治療和細(xì)胞分化機(jī)制研究具有劃時(shí)代的意義。

表2 不同胚層發(fā)育來(lái)源體細(xì)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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