楊旭瓊，吳珍芳，李紫聰

哺乳動(dòng)物體細(xì)胞核移植表觀遺傳重編程研究進(jìn)展

楊旭瓊，吳珍芳，李紫聰

華南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)學(xué)院，國(guó)家生豬種業(yè)工程技術(shù)研究中心，廣州 510642

體細(xì)胞核移植(somatic cell nuclear transfer, SCNT)是唯一能賦予體細(xì)胞基因組全能性的生殖工程技術(shù)，對(duì)動(dòng)物種質(zhì)資源保存、畜牧業(yè)發(fā)展和生物醫(yī)學(xué)研究等具有重大意義。盡管該技術(shù)已經(jīng)取得了許多研究進(jìn)展，但哺乳動(dòng)物克隆胚胎的發(fā)育效率依然很低，嚴(yán)重限制其在畜牧業(yè)和生物醫(yī)學(xué)上的應(yīng)用。導(dǎo)致克隆胚胎發(fā)育效率低的主要原因是體細(xì)胞重編程錯(cuò)誤或重編程不完全，主要表現(xiàn)為：印記基因表達(dá)異常、DNA甲基化異常，組蛋白修飾異常等。本文簡(jiǎn)要介紹了體細(xì)胞核移植技術(shù)，系統(tǒng)總結(jié)了哺乳動(dòng)物克隆胚胎發(fā)育效率低的主要影響因素，以期為提升體細(xì)胞克隆效率相關(guān)研究與實(shí)踐提供理論參考。

體細(xì)胞核移植；；DNA甲基化；組蛋白修飾

體細(xì)胞核移植(somatic cell nuclear transfer, SCNT)又稱(chēng)體細(xì)胞克隆，指利用顯微操作技術(shù)，以去核卵母細(xì)胞為受體，單細(xì)胞核為供體，將體細(xì)胞核移入成熟的去核卵母細(xì)胞中，激活形成克隆胚胎，進(jìn)而培育出基因型與供體體細(xì)胞相同的克隆動(dòng)物(圖1)。體細(xì)胞核移植技術(shù)是當(dāng)代生命科學(xué)研究和應(yīng)用的關(guān)鍵技術(shù)之一，是生命科學(xué)研究高水平發(fā)展的體現(xiàn)，在農(nóng)業(yè)動(dòng)物生產(chǎn)、藥物生產(chǎn)、再生醫(yī)學(xué)和保護(hù)寶貴遺傳資源等方面具有廣泛的應(yīng)用價(jià)值。然而，克隆胚胎的發(fā)育效率遠(yuǎn)遠(yuǎn)低于體外受精胚的發(fā)育效率，哺乳動(dòng)物的克隆胚胎發(fā)育效率只有1%~5%[1]，嚴(yán)重限制了克隆技術(shù)的發(fā)展。導(dǎo)致體細(xì)胞克隆效率低下的主要原因是體細(xì)胞重編程錯(cuò)誤或重編程不完全[2,3]。鑒于此，研究人員希望通過(guò)一些有效的技術(shù)手段來(lái)提高克隆胚胎的發(fā)育能力，如采用不同的細(xì)胞系作為供體細(xì)胞，同時(shí)優(yōu)化克隆操作的融合參數(shù)[4~7]、敲除印記基因(X-inactive specific trans-cript)[8]、RNA干擾技術(shù)(RNA interference, RNAi)抑制基因的異常表達(dá)[9]、注射Kdm4b或Kdm5b去甲基化酶[10,11]等。這些研究雖然取得了一定的效果，但大多數(shù)研究結(jié)果表明克隆效率并沒(méi)有大幅度的提高，且克隆動(dòng)物后代存在諸多異常。因此，如何克服克隆效率低下和解決克隆動(dòng)物異常成為體細(xì)胞克隆研究的熱點(diǎn)。

體細(xì)胞克隆的供體細(xì)胞是高度分化的體細(xì)胞，具有高度特異的DNA修飾和組蛋白修飾，以此來(lái)維持體細(xì)胞的細(xì)胞特性。由于克隆胚胎的發(fā)育依賴(lài)于體細(xì)胞的細(xì)胞核，所以當(dāng)供體細(xì)胞核被放置到成熟的去核卵母細(xì)胞中時(shí)，供體細(xì)胞核必須經(jīng)過(guò)重編程，抹去分化狀態(tài)的細(xì)胞記憶，激活對(duì)早期胚胎發(fā)育具有重要作用的基因，如多能性基因、抑制與分化相關(guān)的基因，從而使得體細(xì)胞獲得發(fā)育的全能性[12]。由于重編程發(fā)生在一個(gè)相對(duì)較短的時(shí)間框架內(nèi)，若克隆胚胎的發(fā)育與正常的胚胎發(fā)育不一致，克隆胚胎的發(fā)育狀態(tài)會(huì)出現(xiàn)一系列的錯(cuò)誤。越來(lái)越多的數(shù)據(jù)表明，錯(cuò)誤的重編程模式會(huì)使克隆動(dòng)物出現(xiàn)表觀遺傳修飾的偏差。如X染色體失活[13]、印記基因與非印記基因的表達(dá)[3,12~14]、DNA甲基化[15~17]和染色體修飾[18]等。本文對(duì)體細(xì)胞核移植技術(shù)的發(fā)展以及影響克隆胚胎發(fā)育效率低的主要原因進(jìn)行了總結(jié)，以期為未來(lái)提高哺乳動(dòng)物克隆發(fā)育效率的研究提供參考。

1 體細(xì)胞核移植技術(shù)

1.1 體細(xì)胞核移植技術(shù)的發(fā)展歷程

SCNT技術(shù)實(shí)現(xiàn)了體細(xì)胞的全能性。早在1952年，英國(guó)遺傳學(xué)家Briggs和King將青蛙()胚胎卵裂球細(xì)胞的細(xì)胞核移植到去核的卵母細(xì)胞中，以此來(lái)研究胚胎干細(xì)胞的細(xì)胞核是否發(fā)生分化。這是首次利用兩棲動(dòng)物的胚胎干細(xì)胞實(shí)現(xiàn)胚胎細(xì)胞核移植技術(shù)，但是當(dāng)時(shí)并未成功克隆出青蛙[19]。1962年，英國(guó)生物學(xué)家Gurdon首次在兩棲動(dòng)物上利用SCNT技術(shù)成功地將分化的青蛙體細(xì)胞克隆出小蝌蚪[20]。直到1997年第一頭克隆羊“多莉”(Dolly)誕生[21]，這是世界上第一個(gè)克隆成功的哺乳動(dòng)物。隨后，奶牛()[22]、小鼠()[23]、山羊()[24]、豬()[25,26]、兔子()[27]、貓()[28]、騾子()[29]、馬()[30]、大鼠()[31]、獵犬(Canislupus familiaris)[32]和駱駝()[33]等成功克隆的20多種哺乳動(dòng)物相繼問(wèn)世。2017年，我國(guó)成功克隆出世界上第一批體細(xì)胞克隆猴()[34]。

圖1 體細(xì)胞核移植的流程

1.2 體細(xì)胞核移植技術(shù)的應(yīng)用和存在問(wèn)題

SCNT技術(shù)能夠培育優(yōu)良畜種，如選育高品質(zhì)家畜和擴(kuò)大繁殖高性能產(chǎn)量個(gè)體；此外還可以培育抗病物種、制備異種器官移植供體動(dòng)物、制備人類(lèi)疾病動(dòng)物模型和轉(zhuǎn)基因動(dòng)物生物反應(yīng)器等。除了克隆動(dòng)物外，SCNT在干細(xì)胞生物學(xué)和人類(lèi)疾病治療等方面具有巨大的潛力。受精卵發(fā)育到囊胚期分化形成內(nèi)細(xì)胞團(tuán)(inner cell mass, ICM)和滋養(yǎng)層細(xì)胞(trophoblast, TE)，其中ICM可分離培養(yǎng)出胚胎干細(xì)胞(embryonic stem cells, ESCs)?？寺∨咛グl(fā)育到囊胚期，ICM也可分離培養(yǎng)出多能干細(xì)胞(pluripotent stem cells, PSCs)或者稱(chēng)核移植胚胎干細(xì)胞(nuclear transfer embryonic stem cells, ntESCs)[35]。

在生物醫(yī)學(xué)疾病治療方面，治療性克隆能夠通過(guò)培養(yǎng)人的ntESCs，建立并保存每個(gè)個(gè)體自身的ntESCs，用于組織和器官替代療法?；颊叩膎tESCs和患者本身具有相同的基因組，可避免排斥反應(yīng)等不適問(wèn)題[36,37]。2001年，Wakayama等[38]在成年小鼠體細(xì)胞克隆胚胎中分離出了具備多能性特征的核移植ESCs，這也是第一例ntESCs，為人ntESCs的研究提供了實(shí)驗(yàn)基礎(chǔ)。Rideout等[39]通過(guò)同源重組的方法實(shí)現(xiàn)了突變等位基因的遺傳固定，并獲得ntESCs細(xì)胞系用作治療免疫缺陷小鼠。2007年，Byrne等[40]成功獲得猴子的ntESCs。2013年，Tachibana等[35]獲得了第一例人的ntESCs。隨后，更多的實(shí)驗(yàn)室相繼報(bào)道獲得了健康成年人[41]、糖尿病[37]及老年性黃斑變性病[36]人體細(xì)胞來(lái)源的ntESCs。眾所周知，ntESCs在人類(lèi)中的研究能為組織和器官功能失調(diào)或受損的患者提供干細(xì)胞新來(lái)源。這種干細(xì)胞可以更新和替換損壞了的細(xì)胞或組織，可為上百萬(wàn)的患者減緩病情。在臨床上用患有線粒體疾病患者的卵細(xì)胞核移植到另一個(gè)健康去核卵細(xì)胞中，從而阻斷線粒體疾病的下一代遺傳。2017年張進(jìn)等[42]利用Leigh氏綜合征攜帶者卵細(xì)胞紡錘體移植獲得了健康嬰兒，這是第一例線粒體替代嬰兒。當(dāng)然，線粒體疾病的替代治療存在人們關(guān)心的倫理問(wèn)題，這也是阻礙其臨床廣泛推廣的主要原因。

目前，SCNT技術(shù)已經(jīng)成熟，但是依然存在一些問(wèn)題，嚴(yán)重限制其在生產(chǎn)實(shí)際中的應(yīng)用和發(fā)展。總的來(lái)說(shuō)，哺乳動(dòng)物的克隆效率都比較低下，主要表現(xiàn)在：克隆胚胎體外發(fā)育效率低，如猴SCNT單個(gè)卵母細(xì)胞的孵化效率僅為0.7%[40]；體內(nèi)發(fā)育至出生效率低，例如豬的出生率大約在0.5%~1%左右[43,44]。在克隆胚胎中，由TE分化形成的胎盤(pán)經(jīng)常存在異常狀態(tài)[45]，胎盤(pán)異常幾乎是所有克隆哺乳動(dòng)物的一個(gè)共有特征，例如胎盤(pán)增生、胎盤(pán)血管缺陷、臍帶畸形[46]等。此外，克隆動(dòng)物的健康狀況也存在一定的異常，包括肥胖、免疫及呼吸缺陷和早期死亡等[45,47,48]。由于SCNT技術(shù)受到卵母細(xì)胞、供體細(xì)胞的質(zhì)量以及代孕母體等個(gè)體差異的影響，因此很難從統(tǒng)計(jì)學(xué)的數(shù)據(jù)分析上確定影響因素[49]。

2 表觀遺傳重編程對(duì)體細(xì)胞核移植胚胎發(fā)育效率的影響

生物體的大多數(shù)細(xì)胞具有相同的遺傳物質(zhì)，SCNT重編程主要通過(guò)表觀遺傳重編程來(lái)實(shí)現(xiàn)。在克隆胚胎發(fā)育早期，存在體細(xì)胞標(biāo)記和細(xì)胞類(lèi)型特異性分化記憶。無(wú)論是體細(xì)胞標(biāo)記還是細(xì)胞特異性分化記憶都可能導(dǎo)致特定的重編程錯(cuò)誤，致使在克隆胚胎發(fā)育過(guò)程中出現(xiàn)各種異常。若要使其正常發(fā)育，克隆胚胎應(yīng)該以某種方式克服這兩個(gè)表觀遺傳障礙。因此，當(dāng)供體細(xì)胞核與去核的卵母細(xì)胞融合后，供體細(xì)胞核需要對(duì)核內(nèi)已有的表觀遺傳修飾進(jìn)行重編程，即擦除供體細(xì)胞表觀遺傳模式，激活與胚胎發(fā)育相關(guān)的基因，抑制與細(xì)胞分化相關(guān)的基因，重新獲得發(fā)育的全能性。當(dāng)胚胎附植于子宮后，胚胎從全能狀態(tài)再分化，用于組織生成及器官發(fā)生[50]。而在克隆胚胎重編程的過(guò)程中，由于體細(xì)胞的表觀遺傳修飾去除不完全，未能建立起正確的表觀遺傳修飾來(lái)調(diào)控胚胎的正常發(fā)育，致使其出現(xiàn)各種異常。表觀遺傳重編程主要包括基因組印記、X染色體失活、DNA甲基化和組蛋白修飾等(表1)。

2.1 抑制印記基因Xist表達(dá)可顯著提高體細(xì)胞核移植胚胎發(fā)育效率

XY型哺乳動(dòng)物，其X和Y染色體是由同源常染色體進(jìn)化而來(lái)。由于雄性和雌性X染色體上的基因數(shù)目不同，兩者之間存在大規(guī)模的遺傳失衡，為平衡這種劑量差異，在雌性胚胎發(fā)育過(guò)程中會(huì)選擇失活其中一條X染色體[51]?；蚴荴染色體上順式調(diào)控X染色體失活的印記基因，其轉(zhuǎn)錄產(chǎn)物是在X染色體失活中心(X-chromosome inactivation, XCI)開(kāi)始轉(zhuǎn)錄的長(zhǎng)鏈非編碼RNA(long non-coding RNA, lncRNA)，轉(zhuǎn)錄產(chǎn)物lncRNA通過(guò)包圍整條X染色體使得X染色體失活[52]。

表1 小鼠和豬SCNT胚胎發(fā)育效率的表觀遺傳重編程影響因素及對(duì)策

哺乳動(dòng)物X染色體存在兩種形式的失活方式：印記失活和隨機(jī)失活。XCI在胚胎發(fā)育早期建立，受胚胎發(fā)育調(diào)控，且調(diào)控方式與物種種類(lèi)密切相關(guān)[53]。雌性小鼠胚胎發(fā)育過(guò)程中，在胚胎2~4細(xì)胞期基因從父源X染色體上開(kāi)始轉(zhuǎn)錄啟動(dòng)表達(dá)，致使父方來(lái)源X染色體失活，此印記表達(dá)模式，在胚胎外組織中始終維持[54]。當(dāng)胚胎發(fā)育到囊胚期時(shí)，這種印記形式的X染色體失活會(huì)在ICM中經(jīng)歷再活化，直至胚胎著床期，ICM中會(huì)隨機(jī)失活一條X染色體[55]。但是，人類(lèi)胚胎發(fā)育中表達(dá)模式并沒(méi)有像小鼠一樣，而是在胚胎發(fā)育后期隨機(jī)失活[56]。

動(dòng)物克隆胚胎發(fā)育過(guò)程中存在許多表達(dá)異常的基因，是其中之一[57]。印記基因的表達(dá)與表觀遺傳修飾乙酰化、甲基化密切相關(guān)，包括組蛋白H3和H4低乙?；?、H3-lysine 4(H3K4)低甲基化、H3-lysine 9 (H3K9)甲基化和多梳沉默復(fù)合體(poly-comb repressive complex 2, PRC2)依賴(lài)的H3-lysine 27 (H3K27)甲基化等[58,59]。比較克隆胚胎和受精胚胎的轉(zhuǎn)錄產(chǎn)物，發(fā)現(xiàn)無(wú)論雌性或雄性小鼠克隆胚胎中基因都異常表達(dá)，其X染色體連鎖基因都受到持續(xù)地特異性抑制，導(dǎo)致染色體水平的基因大面積下調(diào)[8]。同樣，F(xiàn)ukuda等[60]發(fā)現(xiàn)小鼠克隆胚胎中異常表達(dá)，X染色體異常失活。研究表明，在小鼠克隆胚胎桑葚胚期開(kāi)始異常高表達(dá)，結(jié)果導(dǎo)致了染色體水平的大面積基因下調(diào)，利用基因缺陷型供體細(xì)胞用于克隆實(shí)驗(yàn)，可顯著提高小鼠克隆效率，克隆效率提高到8~9倍[8]。在雄性小鼠克隆胚胎中注入抗的小干擾RNA (siRNA)，也觀察到了類(lèi)似的效果，同時(shí)也表明克隆胚胎植入前的異常表達(dá)嚴(yán)重影響克隆胚胎的發(fā)育能力[9]。敲除供體細(xì)胞的基因或干擾克隆胚胎中的基因顯著的提高了小鼠的克隆效率，這說(shuō)明糾正基因的錯(cuò)誤表達(dá)對(duì)克隆胚胎的發(fā)育效率有顯著作用。

對(duì)豬而言，發(fā)育異常的克隆胎兒同樣存在的異常表達(dá)，且這種異常始于桑椹胚期[61]。通過(guò)RNAi的方式，在克隆胚胎1-細(xì)胞期注射siRNA，結(jié)果表明豬克隆胚的表達(dá)異常升高，小幅度提高了豬克隆胚胎發(fā)育效率[57]。一方面，由于克隆所用供體細(xì)胞是豬腎髓質(zhì)部細(xì)胞，該細(xì)胞易老化，易發(fā)生癌變，致使表達(dá)異常升高；另一方面，因?yàn)閟iRNA作用時(shí)間太短，當(dāng)豬克隆胚胎發(fā)育到桑椹胚時(shí)，siRNA已經(jīng)失去它的干擾作用，所以通過(guò)注射siRNA提高豬克隆胚胎的發(fā)育效率似乎并不可行。此外，通過(guò)RNAi的方式干擾基因，對(duì)提高豬孤雌胚胎發(fā)育效率也有顯著效果[62]。Yang等[63]利用TALEN技術(shù)突變豬供體細(xì)胞的基因，結(jié)果表明X染色體部分再活化，并沒(méi)有提高豬克隆效率。Ruan等[64]利用TALEN技術(shù)在豬供體細(xì)胞基因第一外顯子重復(fù)序列前以插入大片段的方式破壞重復(fù)序列，進(jìn)而失活基因，大幅度提高了豬的克隆發(fā)育效率。但是，出生豬只數(shù)較少，共移植530個(gè)豬克隆胚胎，獲得健康克隆胎兒11只。

2.2 DNA去甲基化水平影響體細(xì)胞核移植胚胎發(fā)育效率

哺乳動(dòng)物DNA甲基化是指在DNA甲基化轉(zhuǎn)移酶(DNA methyltransferase, DNMT)的幫助下，將DNA分子中S-腺苷蛋氨酸的甲基轉(zhuǎn)移至胞嘧啶殘基的第5位碳原子上的過(guò)程[65~67]。DNA甲基化由DNMT建立和維持，相反地，TET蛋白酶(ten-eleven translocation, TET)可以催化5-mC (5-methylcyto-sine, 5-mC)轉(zhuǎn)化為5-羥甲基胞嘧啶(5-hydroxymeth-ylcytosine, 5-hmC)，進(jìn)而啟動(dòng)DNA去甲基化程序[68,69]。哺乳動(dòng)物早期胚胎發(fā)育，基因組中的DNA甲基化修飾會(huì)發(fā)生重編程過(guò)程，廣泛進(jìn)行去甲基化，以此在囊胚期達(dá)到最低水平。牛、鼠、豬等動(dòng)物受精后，父本和母本經(jīng)歷不同的去甲基化方式，前者主動(dòng)去甲基化，后者被動(dòng)去甲基化[16,70]。DNA去甲基化是細(xì)胞多能性建立和維持的關(guān)鍵步驟，是重編程的第一步，同時(shí)也是核移植后早期胚胎發(fā)育正常啟動(dòng)和維持的重要環(huán)節(jié)[71]，對(duì)表觀遺傳修飾起到關(guān)鍵作用。體細(xì)胞基因組的CpG島大多數(shù)處于高度甲基化狀態(tài)，全面去甲基化則是SCNT重編程的必須步驟[72]。相比正常的體外受精胚胎，克隆胚胎基因組去甲基化也發(fā)生在卵裂時(shí)期，但是克隆胚胎去甲基化不完全，其基因組甲基化水平更接近體細(xì)胞的狀態(tài)[50]。Inoue等[73]研究發(fā)現(xiàn)，在小鼠受精胚胎中，除印記基因外，新合成的DNA大多未被甲基化。然而，Matoba等[74]研究發(fā)現(xiàn)在小鼠克隆胚胎囊胚期，某些基因的啟動(dòng)子部位具有高水平的DNA甲基化。Gao等[75]研究表明異常的DNA再甲基化阻礙了合子基因組激活，是影響SCNT胚胎發(fā)育的重要表觀遺傳障礙。DNMT的抑制能夠克服DNA再甲基化的缺陷，同時(shí)提高植入后SCNT胚胎發(fā)育效率及克隆效率，抑制DNMT和過(guò)表達(dá)組蛋白賴(lài)氨酸去甲基化酶(K-demethylases, Kdms)相結(jié)合的方法可以進(jìn)一步提高克隆效率。以上研究表明，DNA甲基化程度是重要的表觀遺傳障礙之一。

2.3 組蛋白修飾可改善體細(xì)胞核移植胚胎發(fā)育效率

組蛋白脫乙酰酶(histone deacetylase, HDAC)可調(diào)節(jié)組蛋白的乙酰化水平，從而實(shí)現(xiàn)對(duì)基因表達(dá)的表觀遺傳調(diào)控[76]。組蛋白脫乙酰酶抑制劑(histone deacetylase inhibitor, HDACi)通過(guò)其功能基團(tuán)與HDAC的Zn2+形成螯合物，抑制HDAC的活性，增加細(xì)胞內(nèi)組蛋白的乙?；潭?，從而提高靶基因的表達(dá)水平[76]。HDACi在動(dòng)物克隆胚胎發(fā)育中被廣泛用來(lái)改善不同物種胚胎發(fā)育的重編程[77]。早在2006年Kishigami等[78]和Rybouchkin等[79]發(fā)現(xiàn)HDACi能使小鼠克隆胚胎效率從1%提高到6%。曲古抑菌素A (trichostatin A, TSA)是一種有效的HDACi。Inoue等[80]通過(guò)添加TSA藥物處理小鼠克隆胚胎，顯著提高了克隆胚胎2-細(xì)胞期后的發(fā)育效率，從而將克隆效率提高了5~10倍，但是TSA對(duì)克隆胚胎中異常表達(dá)的基因數(shù)量以及表達(dá)模式?jīng)]有影響。同樣，HDACi藥物治療使得豬[31,81,82]、牛[83~85]的克隆胚胎發(fā)育效率均有所提高，但其對(duì)SCNT重編程的機(jī)制仍不清楚。此外，也有研究表明用HDACi處理豬克隆胚胎，H3K14、H4K5和H4K8等賴(lài)氨酸殘基出現(xiàn)乙?；F(xiàn)象[86,87]。

哺乳動(dòng)物的卵母細(xì)胞和精子本身處于轉(zhuǎn)錄沉默，受精后，受精卵則恢復(fù)轉(zhuǎn)錄，該過(guò)程稱(chēng)為合子基因組激活(zygotic genome activation, ZGA)。不同物種的ZGA時(shí)間不同，小鼠和人的ZGA時(shí)間分別在胚胎2-細(xì)胞期和胚胎8-細(xì)胞期。當(dāng)ZGA啟動(dòng)時(shí)，受精胚胎中母系儲(chǔ)存的RNA迅速降解，并被新合成的合子RNA取代。同樣地，克隆胚胎的ZGA也存在類(lèi)似機(jī)制，且克隆胚胎早期發(fā)育過(guò)程中出現(xiàn)發(fā)育停滯的時(shí)間與ZGA時(shí)間高度相關(guān)。有研究表明，在小鼠克隆胚胎中大約有1000個(gè)基因組區(qū)域或基因未能在ZGA時(shí)間內(nèi)激活[11]。有趣的是，在重編程抵抗區(qū)(reprogramming-resistant regions, RRRs)富含轉(zhuǎn)錄抑制標(biāo)記物H3K9me3，這說(shuō)明了供體細(xì)胞中組蛋白H3K9me3可能是阻止克隆胚胎ZGA的屏障。Matoba等[11]通過(guò)注射H3K9me3特異性去甲基酶Kdm4d的mRNAs不僅克服了ZGA缺陷，而且解決了植入前胚胎發(fā)育停滯的問(wèn)題，使得幼崽出生率提高了8%以上。

Wang等[88]揭示了異染色質(zhì)組蛋白修飾H3K9me3在配子細(xì)胞以及受精后和早期胚胎發(fā)育過(guò)程中的重編程與其在逆轉(zhuǎn)座子沉默中的作用及調(diào)控機(jī)制。相關(guān)研究表明，供體細(xì)胞和2-細(xì)胞期克隆胚胎中在某些區(qū)域都富含異染色質(zhì)組蛋白H3K9me3標(biāo)記[89]，且在小鼠克隆胚胎2-細(xì)胞期，存在一些區(qū)域沒(méi)有去甲基化[10]。這一觀察結(jié)果也證實(shí)了供體細(xì)胞中H3K9me3是SCNT重編程的表觀遺傳屏障[11,36,44]。研究表明在小鼠克隆胚胎2-細(xì)胞期和4-細(xì)胞期注射Kdm4b和Kdm5b去甲基化酶，針對(duì)組蛋白H3K9me3和H3K4me3去甲基化，顯著提高了囊胚發(fā)育率，且從克隆胚胎中成功分離培養(yǎng)出ntESCs[10]。Matoba等[74]采用敲除(KO-)供體細(xì)胞與Kdm4d- mRNA注射相結(jié)合的方法，以支持細(xì)胞作為供體細(xì)胞克隆小鼠，使得克隆效率顯著提高到24%。盡管如此，小鼠克隆效率依然低于體外受精發(fā)育效率。最近研究表明，通過(guò)注射Kdm4b也可以提高豬[64]、牛[90]以及猴[34]的克隆效率。以上結(jié)果說(shuō)明，H3K9me3去甲基化是克隆胚胎正常發(fā)育過(guò)程中重編程所必需的組蛋白修飾，同時(shí)也是克隆胚胎成功重編程的限制因素之一[91]。此外，母源印記H3K27me3組蛋白修飾同樣是影響克隆胚胎重編程的重要因素。研究發(fā)現(xiàn)，受精胚胎調(diào)控印記基因的母源H3K27me3結(jié)構(gòu)域并未在克隆胚胎中建立[92,93]，致使H3K27me3依賴(lài)性印記基因大部分失去其印記狀態(tài)，成為雙等位基因表達(dá)[74]。Inoue等[93]表示印記基因也受母源H3K27me3的調(diào)控，由于供體細(xì)胞的位點(diǎn)缺少H3K27me3標(biāo)記，克隆胚胎中H3K27me3的重編程不完全，進(jìn)而導(dǎo)致克隆胚胎異常激活。因此，為解決供體細(xì)胞中H3K27me3的缺失問(wèn)題，在供體細(xì)胞母源等位基因中靶向沉積H3K27me3可能是一個(gè)必要的策略。

3 結(jié)語(yǔ)與展望

SCNT的成功是生命科學(xué)領(lǐng)域的一次重大突破，其在優(yōu)良種畜擴(kuò)繁、瀕危物種保護(hù)、克隆性治療等方面具有廣闊的應(yīng)用前景。然而，運(yùn)用克隆技術(shù)成功克隆出青蛙距今已有50余年，克隆胚胎發(fā)育至成體的成功率仍保持在一個(gè)很低水平。盡管，自Dolly羊誕生20年來(lái)，科學(xué)家致力于SCNT操作過(guò)程中影響克隆胚胎發(fā)育效率的各種條件和參數(shù)的研究，但克隆效率并未得到顯著提高?？寺⌒实偷母驹蚴枪w細(xì)胞核的表觀重編程異常[50]。對(duì)此，人們需要對(duì)重編程過(guò)程中染色質(zhì)和表觀基因組的變化進(jìn)行系統(tǒng)和詳細(xì)的分析。隨著測(cè)序技術(shù)的更新?lián)Q代，轉(zhuǎn)錄組測(cè)序及相關(guān)的表觀遺傳學(xué)研究，使得對(duì)SCNT的重編程研究成為可能[96]。從技術(shù)上來(lái)說(shuō)，獲取足夠的克隆樣本用于此類(lèi)分析仍然具有較高的難度，但近些年的相關(guān)研究證明，利用早期胚胎進(jìn)行此類(lèi)研究具有一定的可行性[57,97,98]。SCNT可將分化的體細(xì)胞重編程為全能性胚胎，但在克隆胚胎早期發(fā)育過(guò)程中，大多數(shù)克隆胚胎會(huì)出現(xiàn)停滯現(xiàn)象，其潛在的分子機(jī)制尚未明了?？蒲腥藛T對(duì)提高克隆效率的研究，使得表觀遺傳障礙與其特定的重編程錯(cuò)誤兩者之間的關(guān)系變得更加清晰，從而更加準(zhǔn)確地理解在細(xì)胞分化和克隆胚胎植入過(guò)程中，表觀遺傳調(diào)節(jié)機(jī)制的作用。此外，通過(guò)比較分析不同重編程系統(tǒng)之間的異同，來(lái)探究克隆胚胎的重編程機(jī)制也是一種可行的方案。例如，H3K9me3組蛋白修飾、染色質(zhì)組裝因子(CAF1)蛋白質(zhì)復(fù)合物、異染色質(zhì)蛋白1 (HP1)是誘導(dǎo)多能干細(xì)胞(induced pluripotent stem cells, iPSCs)重編程的障礙[99~101]，而iPSCs重編程與SCNT的重編程機(jī)制類(lèi)似。對(duì)此，在今后的研究中，進(jìn)一步探究這些重編程障礙是否也在SCNT重編程中起作用，可以作為體細(xì)胞核移植潛在的研究方向。

總之，供體核的表觀重編程異常修復(fù)依然是體細(xì)胞核移植研究及發(fā)展的重點(diǎn)。利用新型技術(shù)，如高通量測(cè)序[102,103]、CRISPR/Cas9[104,105]等，將更加快速準(zhǔn)確地解析體細(xì)胞表觀重編程機(jī)制，從而大幅度提高克隆效率，降低克隆動(dòng)物異常表型的發(fā)生率，最終將SCNT技術(shù)應(yīng)用于更多領(lǐng)域。

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Advances in epigenetic reprogramming of somatic cells nuclear transfer in mammals

Xuqiong Yang, Zhenfang Wu, Zicong Li

Somatic cell nuclear transfer (SCNT) is the only reproductive engineering technique that can confer genomic totipotency on somatic cell. SCNT is of great significance for animal germplasm conservation, animal husbandry development, and biomedical research. Although many research advances have been made in this technology, the developmental rate of SCNT mammalian embryos is very low, which seriously limits the application of SCNT in animal husbandry and biomedicine. The primary reason for the low efficiency of cloned embryos is somatic cell reprogramming errors or incomplete reprogramming. These errors or incompleteness present as the abnormal expression of imprinted gene, abnormal DNA methylation, and abnormal histone modification. In this review, we summarize the main factors that influence the low development efficiency of mammalian cloned embryos to provide theoretical reference for the research and practice of improving somatic cell cloning efficiency.

somatic cell nuclear transfer (SCNT);; DNA methylation; histone modification

2019-07-03;

2019-10-07

國(guó)家自然科學(xué)基金面上項(xiàng)目(編號(hào)：31772554)資助[Supported by the National Natural Science Foundation of China(No. 31772554)

楊旭瓊，碩士研究生，專(zhuān)業(yè)方向：動(dòng)物遺傳育種與繁殖。E-mail: 1814639793@qq.com

李紫聰，教授，博士生導(dǎo)師，研究方向：動(dòng)物遺傳育種與繁殖。E-mail: lizicongcong@163.com

10.16288/j.yczz.19-193

2019/11/19 13:16:00

URI: http://kns.cnki.net/kcms/detail/11.1913.r.20191118.1633.002.html

(責(zé)任編委: 高紹榮)