誘導(dǎo)神經(jīng)干細(xì)胞的研究進(jìn)展及應(yīng)用前景

綜述與專論

誘導(dǎo)神經(jīng)干細(xì)胞的研究進(jìn)展及應(yīng)用前景

唐璽和，陳志國(guó)，張愚

(首都醫(yī)科大學(xué)宣武醫(yī)院細(xì)胞治療中心，北京100053)

【摘要】誘導(dǎo)神經(jīng)干細(xì)胞(induced neural stem cells,iNSCs)是通過(guò)在成體細(xì)胞內(nèi)轉(zhuǎn)入特定外源因子而獲得。目前主要有兩種途徑獲得iNSCs,即直接誘導(dǎo)法和間接誘導(dǎo)法(需經(jīng)歷重編程過(guò)程中不穩(wěn)定的中間狀態(tài))。與誘導(dǎo)多能干細(xì)胞(induced pluripotent stem cells, iPSCs)、胚胎干細(xì)胞(embryonic stem cells,ESCs)比較，iNSCs直接將成體細(xì)胞重編程為神經(jīng)干細(xì)胞，而不經(jīng)過(guò)iPSCs再分化為神經(jīng)干細(xì)胞，可減少其致瘤性，縮短誘導(dǎo)周期,提高轉(zhuǎn)分化效率，這將使其更適于臨床應(yīng)用。本文就iNSCs的研究進(jìn)展，應(yīng)用前景加以綜述。

【關(guān)鍵詞】誘導(dǎo)神經(jīng)干細(xì)胞；體細(xì)胞重編程；誘導(dǎo)多能干細(xì)胞；治療

[基金項(xiàng)目]973計(jì)劃重點(diǎn)項(xiàng)目(2012CBA01307)帕金森氏病干細(xì)胞治療的研究。

[作者簡(jiǎn)介]唐璽和(1981-)男，博士研究生，研究方向：誘導(dǎo)神經(jīng)干細(xì)胞治療帕金森氏病。

[通訊作者]陳志國(guó)(1977-)，研究員，博士生導(dǎo)師，研究方向：神經(jīng)退行性疾病的干細(xì)胞治療。Email：chenzhiguo2999@yahoo.cn；張愚(1967-)，研究員，博士生導(dǎo)師，研究方向：帕金森氏病的神經(jīng)干細(xì)胞治療。Email:yaz@bjsap.org。

【中圖分類號(hào)】R33【文獻(xiàn)標(biāo)識(shí)碼】 A

doi:10.3969.j.issn.1671.7856. 2015.002.016

Advances and application of induced neural stem cells

TANG Xi-he, CHEN Zhi-guo, ZHANG Yu

(Cell Therapy Center, Xuanwu Hospital, Capital Medical University, Beijing 100053, China)

Abstract【】It has been reported that induced neural stem cells(iNSCs) can be obtained from rodent and human somatic cells through the forced expression of defined factors. Two different approaches have been successfully used to obtain iNSCs:a direct method and an indirect method which involves an unstable intermediate state.Compared with induced pluripotent stem cells (iPSCs) and embryonic stem cells(ESCs)，iNSCs are committed towards neural lineage, enabling them with a lower risk of tumorigenicity and more efficient transdifferentiation which makes it suitable for clinical use in the future.

【Key words】Induced neural stem cells; Somatic cell reprogramming;Induced pluripotent stem cells; Therapy

干細(xì)胞為許多神經(jīng)系統(tǒng)疾病治療帶來(lái)希望，比如帕金森氏病[1]，肌萎縮性脊髓側(cè)索硬化癥[2]，老年性癡呆[3]及脊髓外傷性[4-6]疾病。長(zhǎng)期以來(lái)，科研人員試圖使用外科技術(shù)或者立體定向技術(shù)將ESCs(胚胎干細(xì)胞)或者由其分化到某一階段的神經(jīng)細(xì)胞移植到病變部位，使其定植并產(chǎn)生功能，達(dá)到治療作用。大量研究表明，使用胚胎干細(xì)胞治療帕金森病、脊髓損傷及糖尿病有一定作用，但胚胎干細(xì)胞的致瘤性，異體移植的免疫排斥以及倫理學(xué)爭(zhēng)議限制其應(yīng)用。2006年，Yamanaka利用小鼠成纖維細(xì)胞成功獲得iPSCs，開(kāi)創(chuàng)了干細(xì)胞研究的新局面[7]，該研究使自體干細(xì)胞移植成為可能。2007年，Yamanaka[8]及Wernig[9]報(bào)道了由人成纖維細(xì)胞獲得iPSCs，之后iPSCs治療相關(guān)疾病的細(xì)胞模型及動(dòng)物模型也成功建立，為iPSCs向臨床應(yīng)用做了大量前期工作。但是，研究發(fā)現(xiàn)，iPSCs用于臨床治療還面臨很多問(wèn)題，如致瘤性，定向分化效率低，安全性問(wèn)題。因此，將體細(xì)胞直接誘導(dǎo)為成體干細(xì)胞，而不經(jīng)過(guò)iPSCs階段，以減少其致瘤性，提高分化效率成為研究熱點(diǎn)。

1體外獲得iNSCs的方法

目前誘導(dǎo)神經(jīng)干細(xì)胞方法主要分為直接誘導(dǎo)法和間接誘導(dǎo)法。直接誘導(dǎo)即在成體細(xì)胞內(nèi)轉(zhuǎn)入神經(jīng)干細(xì)胞表達(dá)的特定外源基因，microRNAs或者小分子蛋白等，直接獲得iNSCs。間接誘導(dǎo)：在成體細(xì)胞重編程為iPSCs過(guò)程中(可以稱之為中間狀態(tài)或者不穩(wěn)定狀態(tài))，改變誘導(dǎo)環(huán)境，使其成為iNSCs。

1.1成體細(xì)胞直接重編程為神經(jīng)干細(xì)胞或者神經(jīng)前體細(xì)胞

該方法主要是在成體細(xì)胞中直接轉(zhuǎn)入神經(jīng)干細(xì)胞表達(dá)的某些相關(guān)基因，使其直接重編程為神經(jīng)干細(xì)胞。Lujan[10]等報(bào)道轉(zhuǎn)入11個(gè)神經(jīng)干細(xì)胞相關(guān)的外源基因可以將小鼠成纖維細(xì)胞誘導(dǎo)成神經(jīng)干細(xì)胞，該細(xì)胞在體外可以分化出星形膠質(zhì)細(xì)胞，少突膠質(zhì)細(xì)胞及成熟神經(jīng)元，并能在體內(nèi)存活。Sheng C等[11]轉(zhuǎn)入Ascl1、Ngn2、Hes5等9個(gè)神經(jīng)干細(xì)胞相關(guān)基因?qū)⑿∈蟛G丸支持細(xì)胞誘導(dǎo)成神經(jīng)干細(xì)胞，該細(xì)胞在體外能分化出多巴胺能神經(jīng)元，膽堿能神經(jīng)元及神經(jīng)膠質(zhì)細(xì)胞。Han等[12]報(bào)轉(zhuǎn)入Brn4、sox2、klf4、c-Myc、E47基因或轉(zhuǎn)入Brn4、sox2、klf4、c-Myc均將小鼠成纖維細(xì)胞誘導(dǎo)為神經(jīng)干細(xì)胞，誘導(dǎo)神經(jīng)干細(xì)胞能在體外在傳代130次以上，且與野生型神經(jīng)干細(xì)胞形態(tài)，基因表達(dá)，十分相似。研究還發(fā)現(xiàn)轉(zhuǎn)入4因子與5因子誘導(dǎo)的神經(jīng)干細(xì)胞與野生型神經(jīng)干比較，同樣有成熟的分化能力，可分化出星形膠質(zhì)細(xì)胞、神經(jīng)元、少突膠質(zhì)細(xì)胞。Ring等[13]用逆轉(zhuǎn)錄病毒載體攜帶sox2轉(zhuǎn)染小鼠成纖維細(xì)胞，6～10 d后就出現(xiàn)神經(jīng)干細(xì)胞克隆。而轉(zhuǎn)染人成纖維細(xì)胞后，5 d內(nèi)就出現(xiàn)了神經(jīng)干細(xì)胞克隆，將誘導(dǎo)神經(jīng)干細(xì)胞與野生型神經(jīng)干細(xì)胞同時(shí)移植免疫缺陷小鼠，沒(méi)有腫瘤形成，證明誘導(dǎo)神經(jīng)干細(xì)胞的安全性。Saiyong Zhu[14]等只使用OCT4單個(gè)基因聯(lián)合A83-01、CHIR99021、NaB、LPA、rolipram、SP600125成功的將人成纖維細(xì)胞誘導(dǎo)為神經(jīng)干細(xì)胞，經(jīng)過(guò)4周體外自然分化，能分化出成熟的神經(jīng)元。移植小鼠腦內(nèi)后，該細(xì)胞能向神經(jīng)元分化，且無(wú)腫瘤形成。Kim SM 等利用逆轉(zhuǎn)錄病毒攜帶Klf4、c-Myc、Sox2、Brn4轉(zhuǎn)染小鼠成纖維細(xì)胞，4周后獲得誘導(dǎo)神經(jīng)干細(xì)胞，神經(jīng)干細(xì)胞無(wú)成瘤性[15]。

1.2成體細(xì)胞經(jīng)過(guò)iPSCs中間狀態(tài)誘導(dǎo)為神經(jīng)干細(xì)胞，即在成體細(xì)胞中轉(zhuǎn)入經(jīng)典Yamanaka 四因子(或者略加減)后，在iPSCs誘導(dǎo)的早期改變培養(yǎng)基，使其能向神經(jīng)干細(xì)胞方向發(fā)展

Kim等[16]使用Dox載體系統(tǒng)在小鼠成纖維細(xì)胞中轉(zhuǎn)入oct4、sox2、klf4、c-myc基因，在含有LIF的MEF上培養(yǎng)3～6 d后繼續(xù)在神經(jīng)干細(xì)胞培養(yǎng)基中培養(yǎng)8～9 d，將會(huì)有表達(dá)plzf、pax6等神經(jīng)干細(xì)胞標(biāo)志的克隆出現(xiàn)，這種“類似神經(jīng)干”細(xì)胞，可以繼續(xù)分化出星形膠質(zhì)細(xì)胞、神經(jīng)元，但是此種干細(xì)胞在體外只能擴(kuò)增3～5代。裴端卿等[17]使用非整合載體oriP/EBNA episomal攜帶oct4、sox2、sv40lt、klf4和microRNA302-367 電轉(zhuǎn)人尿液中提取的活細(xì)胞，然后在神經(jīng)干細(xì)胞培養(yǎng)基中加入CHIR99021、PD0325901、A83-01、Thiazoviv、DMH1，電轉(zhuǎn)后12 d可見(jiàn)神經(jīng)干細(xì)胞克隆出現(xiàn)，且能表sox1、sox2、pax6等神經(jīng)干細(xì)胞標(biāo)志性基因，誘導(dǎo)的神經(jīng)干細(xì)胞能分化成星形膠質(zhì)細(xì)胞，多巴胺能神經(jīng)元，谷氨酸能神經(jīng)元，但是不能自然分化為少突膠質(zhì)細(xì)胞，在使用PDGF-AA、NT3等小分子刺激后，可以分化出少突膠質(zhì)細(xì)胞。同時(shí)，將誘導(dǎo)神經(jīng)干細(xì)胞移植入新生小鼠紋狀體后，神經(jīng)干細(xì)胞能夠存活，遷移，腦內(nèi)沒(méi)有形成腫瘤，證明誘導(dǎo)神經(jīng)干細(xì)胞的體外分化能力及安全性。張素春等[18]等使用仙臺(tái)病毒為載體，將oct4、sox2、klf4、c-Myc轉(zhuǎn)入人成纖維細(xì)胞，在神經(jīng)干細(xì)胞培養(yǎng)基中加入LIF、CHIR99021、SB431542三個(gè)小分子，之后通過(guò)升高培養(yǎng)溫度(39℃)使得仙臺(tái)病毒滅火，第13天可見(jiàn)神經(jīng)干細(xì)胞克隆形成。其表達(dá)nestin、sox1、sox2、fabp7、pax6、hes5、notch1等神經(jīng)干細(xì)胞的標(biāo)志性基因，并且在體外可以分化為星形細(xì)胞，神經(jīng)元及少突膠質(zhì)細(xì)胞，該類細(xì)胞還具有腦區(qū)特異性。目前經(jīng)iPSCs中間狀態(tài)誘導(dǎo)神經(jīng)干細(xì)胞的方法受到推崇，因?yàn)槠湔T導(dǎo)體系隨著誘導(dǎo)iPSCs體系變革而更加安全。此前誘導(dǎo)iPSCs方法的缺點(diǎn)主要是向供體細(xì)胞中引入外源基因，且病毒轉(zhuǎn)染時(shí)可能將外源基因整合到細(xì)胞基因組中，導(dǎo)致供體細(xì)胞基因突變，另外，c-Myc，Klf4是致癌基因，可以增加細(xì)胞的成瘤性，因此，提高iPSCs的安全性主要在轉(zhuǎn)入因子選擇及載體的進(jìn)一步改進(jìn)。Nakagawa M等[19]在無(wú)c-Myc下將體細(xì)胞重編程為iPSCs，降低了iPSCs的成瘤性。Anokye-Danso等[20]采用miRNAs在無(wú)轉(zhuǎn)錄因子作用下直接誘導(dǎo)iPSCs。鄧宏魁等[21]單純使用化學(xué)小分子成功誘導(dǎo)出iPSCs，提供了更加安全的誘導(dǎo)系統(tǒng)。另外，除了成纖維細(xì)胞做為供體細(xì)胞外，Loh Y.HStaerk,JDowey SN等[22-24]使用外周血中的T細(xì)胞或單個(gè)核細(xì)胞成功誘導(dǎo)出iPSCs，免去了獲取成纖維細(xì)胞的侵襲性操作，這也提示了使用外周血單核細(xì)胞誘導(dǎo)神經(jīng)干細(xì)胞的可能性。

2誘導(dǎo)神經(jīng)干細(xì)胞的優(yōu)點(diǎn)及應(yīng)用前景

2.1安全性與多能性的統(tǒng)一

神經(jīng)干細(xì)胞移植被認(rèn)為是治療很多神經(jīng)變性疾病及神經(jīng)退行性疾病的新方法，除了病變神經(jīng)細(xì)胞的功能替代，干細(xì)胞釋放的神經(jīng)保護(hù)因子及生長(zhǎng)因子對(duì)疾病治療也有作用。目前開(kāi)展的動(dòng)物實(shí)驗(yàn)?zāi)Ｐ陀信两鹕1, 25]，亨廷頓舞蹈癥[26]，多發(fā)性硬化[27]，腦卒中[28]及脊髓損傷[29]等，實(shí)驗(yàn)結(jié)果顯示干細(xì)胞治療效果明顯，但是其用于臨床治療的效果并不明顯,這可能與移植細(xì)胞的數(shù)量，移植方法等有關(guān)系[30-33]。ESCs細(xì)胞與iPSCs具有多向分化能力，移植后成瘤性強(qiáng)[34]，而誘導(dǎo)神經(jīng)干細(xì)胞具有分化的特異性，主要向星形膠質(zhì)細(xì)胞，少突膠質(zhì)細(xì)胞，神經(jīng)元方向分化，其致瘤性下降[35]。與成體細(xì)胞相比，神經(jīng)干細(xì)胞在移植后仍然能維持自我更新及多向分化能力，對(duì)于腦神經(jīng)損傷及脊髓損傷這種并非只是單一神經(jīng)細(xì)胞病變的疾病，使用誘導(dǎo)神經(jīng)干細(xì)胞治療更加合適[36, 37]。

2.2誘導(dǎo)自體神經(jīng)干細(xì)胞減少移植免疫排斥

同ESCs細(xì)胞及其他供體來(lái)源干細(xì)胞移植相比，iPSCs與誘導(dǎo)神經(jīng)干細(xì)胞可來(lái)自本體，能減少免疫排斥反應(yīng)。但是經(jīng)過(guò)細(xì)胞重編程，外源基因插入以及某些自體基因被修飾，使得自體iPSCs移植仍然存在免疫反應(yīng)[38]，iNSCs是否因?yàn)槟承┗虮磉_(dá)不同而較iPSCs引起移植后免疫反應(yīng)減弱，需要進(jìn)一步探討。由供體細(xì)胞獲得誘導(dǎo)神經(jīng)干細(xì)胞需要一定周期，這對(duì)一些慢性疾病影響較小，但對(duì)于存在治療時(shí)間窗的急性疾病，如腦卒中，將影響其臨床應(yīng)用。

2.3誘導(dǎo)周期縮短及分化效率提高

與iPSCs相比較，誘導(dǎo)神經(jīng)干細(xì)胞周期明顯縮短[10，12，13]，盡管誘導(dǎo)神經(jīng)干細(xì)胞效率不高，但其能在體外快速擴(kuò)增，數(shù)量能夠滿足臨床需求，而且更易分化為神經(jīng)細(xì)胞。而由iPSCs分化神經(jīng)干細(xì)胞，需要經(jīng)歷不同的分化階段，耗時(shí)，且所得細(xì)胞并非處同一分化階段，單一細(xì)胞純度不高[34]，這是iPSCs臨床應(yīng)用前要解決的問(wèn)題。

目前，誘導(dǎo)神經(jīng)干細(xì)胞研究主要集中在如何縮短體細(xì)胞轉(zhuǎn)分化的周期及提高轉(zhuǎn)分化效率，以及外源因子載體的安全性，移植治療的有效性。另外，病變部位細(xì)胞原位轉(zhuǎn)分化為神經(jīng)干細(xì)胞以起到治療作用也是很有意義的研究方向。

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〔修回日期〕2014-11-05