家畜胚胎干細胞多能性候選信號通路及分子標志

趙云程，陳博，周川，張秀華，黃俊成

1 新疆維吾爾自治區(qū)動物生物技術重點開放實驗室，烏魯木齊 830000

2 農(nóng)業(yè)部草食家畜繁育生物技術重點開放實驗室，烏魯木齊 830000

干細胞專欄

家畜胚胎干細胞多能性候選信號通路及分子標志

趙云程1,2，陳博1,2，周川1,2，張秀華1,2，黃俊成1,2

1 新疆維吾爾自治區(qū)動物生物技術重點開放實驗室，烏魯木齊 830000

2 農(nóng)業(yè)部草食家畜繁育生物技術重點開放實驗室，烏魯木齊 830000

家畜胚胎干細胞具有重要的生物學意義和廣闊的應用前景。以下對比了小鼠、人胚胎干細胞多能性調(diào)控信號通路的異同，闡述了小鼠、人胚胎干細胞與家畜胚胎干細胞在多能性分子標志上的差異，并結(jié)合本實驗室開展綿羊胚胎干細胞研究的實際經(jīng)驗，對目前家畜胚胎干細胞建系中可能存在的多能性候選信號通路及分子標志進行了探討。

家畜，胚胎干細胞，多能性，信號通路，候選基因

Abstract:Domesticated ungulates embryonic stem (ES) cells have great significances in biology and wide application prospects.This review compared the key signaling pathways related with pluripotency between mouse and human ES cells, and the difference of transcription factors in mouse, human and domesticated ungulates ES cells were elaborated. Finally the pluripotency candidate signaling network and transcription factors related in the derivation of domesticated ungulates ES cell were discussed combined with practical experience of ovine embryonic stem cell derivation in our laboratory.

Keywords:domesticated ungulates, embryonic stem cells, pluripotency, signaling pathway, candidate genes

胚胎干細胞 (Embryonic stem cell，ESC)，是由哺乳動物著床前囊胚期內(nèi)細胞團 (Inner cell mass，ICM) 經(jīng)體外特定培養(yǎng)環(huán)境選擇、適應后，獲得的具有無限增殖能力和多向分化潛能的細胞系，其特定的生物學特性能夠使其與ICM重新整合，并參與到胚胎發(fā)育的全部過程中。自1989年采用ESC技術獲得了第一個轉(zhuǎn)基因小鼠以來[1]，ESC技術在家畜遺傳育種中展現(xiàn)出巨大前景。采用ESC克隆技術，其整合效率遠遠高于傳統(tǒng)核移植技術，可在短期內(nèi)生產(chǎn)較多的具有遺傳同質(zhì)性的動物，免去后裔測定，大幅度提高良種家畜的繁殖效率。

然而，自 1981年小鼠胚胎干細胞成功建系以來，僅有小鼠[2]、大鼠[3-4]獲得了具備生殖系傳遞能力的ES細胞，極大地制約了ESC技術在家畜遺傳育種中的研究與應用。目前，國內(nèi)外在家畜ESC的研究中，普遍借助小鼠胚胎干細胞 (Mouse embryonic stem cell，mESC)、人胚胎干細胞 (Human embryonic stem cell，hESC) 的成功經(jīng)驗，將影響mESC、hESC增殖、分化的因素，如飼養(yǎng)層細胞、條件培養(yǎng)基、細胞生長因子、激素、胎牛血清和血清提取物等進行有機組合，依照mESC、hESC建系標準，直接應用到家畜ESC建系中，從而試圖篩選出適合家畜ESC多能性維持的培養(yǎng)條件，然而卻收效甚微。導致家畜ESC建系失敗的原因之一是因為mESC和hESC多能性維持機制的差別，使得優(yōu)化組合各種因素變得十分困難；比如，mESC分子調(diào)控網(wǎng)絡中 BMP4 (Bone morphogenetic proteins 4) 與LIF協(xié)同維持其多能性，而在hESC中，BMP4會誘導hESC發(fā)生分化[5-6]。另外，家畜ESC多能性標志的匱乏，是導致建系失敗的另一原因；研究表明mESC、hESC的多能性標志，如SSEA-1、SSEA-4、POU5F1、Nanog、堿性磷酸酶活性等，不僅在牛、豬、山羊囊胚的ICM中表達，同時在其囊胚的滋養(yǎng)層上有所表達[7-11]。針對上述原因，就使得我們不得不考慮家畜 ESC至今未能建系，是因為所借鑒的mESC、hESC添加因子受體本身就不處于家畜ESC的分子調(diào)控網(wǎng)中？還是這些因子在激活家畜ESC自我更新通路的同時，又激活了另一個與之分化有關的通路？或是因為多能性分子標志的模棱兩可性，使得我們一再與家畜 ESC“擦肩而過”？因此，目前許多研究者普遍達成共識：家畜ESC的研究必須從多能性標志和信號通路入手[12-14]，借鑒 mESC、hESC多能性維持的分子機制，了解對比家畜 ESC與 mESC、hESC多能性調(diào)控機制的異同，對家畜ESC研究具有十分重要的作用。

1 ESC多能性維持機制中的關鍵信號轉(zhuǎn)導通路

近年來，隨著對mESC、hESC研究的逐步深入，mESC、hESC多能性維持的分子調(diào)控機制已逐步揭示。在小鼠中，mESC多能性的維持需要通過LIF-LIFR/gp130-Jak-STAT3途徑和 BMP-Id協(xié)同來完成，與之不同的是，hESC多能性的維持則需要 Activin/Nodal和 FGF的共同參與。mESC與hESC二者在多能性維持的分子調(diào)控機制上存在顯著差異。

1.1 LIF-STAT3途徑

白血病抑制因子 (Leukemia inhibitor factor，LIF)屬于白介素6 (IL-6) 細胞因子家族中的一員，是一種多功能的細胞因子。目前研究證實，LIF-LIFR/gp130-Jak-STAT3途徑是 mESC自我更新的重要途徑。對mESC體外多潛能性的維持，保持未分化狀態(tài)發(fā)揮著重要作用[15-16]。研究發(fā)現(xiàn)，LIF與LIFR結(jié)合后，LIFR與gp130 (glycoprotein 130) 迅速聚合形成異源二聚體激活下游的 Janus 酪氨酸蛋白激酶(Janus-associated tyrosine kinase，JAK)、信號轉(zhuǎn)導子和轉(zhuǎn)錄激活子3 (Signal transducer and activation of transcription，STAT) 途徑。STAT3的激活是mESC細胞自我更新的關鍵[17-18]。缺少IL-6家族成員、撤去MEF，均會導致STAT3失活和mESC分化；外源LIF因子、血清，活化 STAT3，則足以維持 mESC的多能性[19]。大鼠胚胎干細胞 (rESC) 研究表明，外源hLIF能有效促進rESC集落形成率，因此Buehr推測，LIF-STAT3應該是“真正”意義上ESC的本質(zhì)特性[3]。

在hESC研究中，起初認為LIF對hESC沒有效果[20]，或是LIF有利于hESC多能性的維持[21]。隨著研究的進一步深入，研究表明，外源hLIF因子能有效使hESC-STAT3的Tyr (705)、Ser (727) 發(fā)生磷酸化 (p-STAT3)，但 p-STAT3并不能維持 hESC多能性[6,22-23]。Daheron等研究發(fā)現(xiàn)，雖然 mLIF與hLIF同源程度較高 (氨基酸序列一致性為78%)，但 mLIF卻表現(xiàn)出種屬特異性，無法使 hESC的STAT3發(fā)生磷酸化[23]。LIF-STAT3信號通路在mESC與 hESC上的顯著差異，可能與其各自在胚胎發(fā)育中的不同發(fā)育階段有關[24]。

近期，Intawicha 報道，mLIF能有效激活兔類胚胎干細胞 (ES-like cells) 的LIF-Jak-STAT3途徑，并提高其自我更新能力[25]；而牛 ES樣細胞研究表明，牛囊胚ICM、及ICM原代培養(yǎng)過程中的確存在LIFR及gp130信號轉(zhuǎn)導，但LIF對牛ES樣細胞增殖并無明顯作用[26]。另外，抑制 LIF-STAT3途徑，對豬外胚層細胞多能性并無顯著影響[27]；但 Brevini等研究發(fā)現(xiàn)，豬ES樣細胞中雖然并不存在LIFR，但LIF卻能有效抑制豬ES樣細胞類胚體的形成[28-29]。

1.2 MAPK/ERK途徑

研究發(fā)現(xiàn)，LIFR與gp130形成異源二聚體后，gp130除了能激活STAT3自我更新途徑以外，還能激活MAPK/ERK級聯(lián)反應[24]，而Erk-1/2級聯(lián)反應對mESC的分化具有十分重要的調(diào)控作用[19]，研究表明，Erk-1/2在不同品系小鼠早期胚胎發(fā)育過程中表現(xiàn)出應答水平的不一致性[30]。利用小分子化合物阻斷 ERK-1/2級聯(lián)反應，129Sv/ter、C57BL/6品系mESC建系率分別提高至76.5%和47%[31]，并且可從更多小鼠品系中建立 mESC (如 CBA、MF1、SCID、NOD品系)[32-34]。我們研究發(fā)現(xiàn)，添加FGFR、MEK 特異性抑制劑 (SU5402、PD0325901) 阻斷ERK-1/2分化級聯(lián)反應后，能有效獲得昆明鼠 ES(KM-ES) 細胞，建系率81.48% (22/27)，KM-ES集落生長穩(wěn)定，堿性磷酸酶活性顯著提高，分化得到明顯抑制 (圖 1)，有效解決了采用飼養(yǎng)層、血清替代物等常規(guī)培養(yǎng)體系中KM-ES集落分化率高、生長不穩(wěn)定的問題。與之類似的是，BMP4途徑的引入，正是依靠BMP4通過SMAD 1/5或SMAD 8途徑誘導產(chǎn)生Id蛋白 (Inhibitor of differentiation)，Id蛋白進而抑制MAPK級聯(lián)反應，并與LIF/STAT3協(xié)同維持mESC的多能性[35-37]。Li等采用FGF受體酪氨酸激酶和ERK-1/2級聯(lián)反應特異性抑制劑SU5402和PD0325901，阻斷MAPK/ERK途徑后，成功建立了rESC系[4]。STAT3與ERK-1/2途徑相互協(xié)調(diào)，對維持mESC、rESC細胞的自我更新和分化之間的平衡具有重要作用。

圖1 KM-ES堿性磷酸酶活性檢測Fig.1 Alkaline phosphatase staining for KM-ES.

與mESC不同的是，hESC未分化狀態(tài)下則保持了較高的ERK活性，MAPK/ERK途徑對hESC的凋亡、增殖和分化過程具有一定調(diào)節(jié)作用[38]。研究表明，堿性成纖維細胞生長因子 (Basic fibroblast growth factor，bFGF) 是hESC自我更新機制中的核心調(diào)控因子[39-40]，bFGF與細胞表面受體結(jié)合后，激活胞內(nèi)MAPK/ERK級聯(lián)反應，對hESC多能性維持起到十分重要的調(diào)控作用[41-43]。目前研究發(fā)現(xiàn)，在未分化的 hESC中均能檢測到 FGFR-1、FGFR-2、FGFR-3、FGFR-4的表達，而以 FGFR-1的表達量最高[44-47]。采用 FGFR特異性抑制劑SU5402研究發(fā)現(xiàn)，F(xiàn)GFR受到抑制的條件下，hESC將會迅速發(fā)生分化[41]。hESC多能性的維持表現(xiàn)出對FGF的量性需求：當bFGF添加量在4 ng/mL時，hESC的維持需要有滋養(yǎng)層細胞[20]；當bFGF添加量達到8～40 ng/mL時，培養(yǎng)系統(tǒng)中不再需要飼養(yǎng)層細胞，而需要Noggin的參與 (Noggin為BMP4的拮抗蛋白)[41,48-49]；當bFGF達到100 ng/mL時，hESC可處于不分化狀態(tài)[50]。采用MEK/ERK信號級聯(lián)反應抑制劑PD98059、U0126，會迅速導致hESC發(fā)生分化[51]，這與mESC、rESC多能性調(diào)控機制存在顯著差別。

目前研究發(fā)現(xiàn)，家畜ES樣細胞與hESC更為相似[14]，bFGF能有效促進豬ES樣細胞的原代集落形成率及自我更新[52-53]，并對兔ES樣細胞多能性的維持具有一定作用[54]。

1.3 Wnt途徑

近年來，研究表明Wnt途徑在mESC與hESC多能性維持中發(fā)揮著十分重要的作用，對ESC向皮膚、神經(jīng)系統(tǒng)、血液系統(tǒng)的分化起到調(diào)控作用[55]，與 LIF-STAT3、BMP4途徑不同的是，Wnt途徑在mESC與 hESC的自我更新中作用機理基本是一致的[56]，都能抑制糖原合成激酶3 (Glycogen synthase kinase 3，GSK-3) 的活性，解除對β連環(huán)蛋白的磷酸化，維持ESC的自我更新[57]。mESC、hESC本身可以自發(fā)激活Wnt途徑，而當分化通路啟動后，Wnt的表達量呈現(xiàn)下調(diào)趨勢[58]。采用GSK-3的特異性抑制劑 6-bromoindirubin-3′-oxime (BIO)，可以特異性抑制GSK-3的磷酸化活性，導致核內(nèi)β連環(huán)蛋白含量增高，從而激活 Wnt的下游信號轉(zhuǎn)導途徑，最終使得 mESC、hESC的多能性狀態(tài)得到維持[59]。Wnt的激活能夠調(diào)高c-Myc的水平，而c-Myc則是STAT3的靶位基因，表明Wnt途徑與LIF-STAT3協(xié)同作用于c-Myc，共同維持mESC的多能性[60-62]。

雖然，Wnt途徑在mESC、hESC多能性維持過程中的作用基本一致，但家畜ESC研究發(fā)現(xiàn)，采用Wnt途徑激活劑 (BIO、Wnt3a) 并不能阻止外胚層細胞的分化[63]。

1.4 TGFβ/Activin/Nodal途徑

TGFβ是一個大的超家族，超過40個成員，包括 TGFβ、Activin、Nodal和 BMP 等[56,64]。BMPs是TGFβ超家族中最大的成員，它在促進ESC自我更新中作用不明顯，但是可以通過SMAD途徑激活Id的表達，而Id蛋白則能夠抑制神經(jīng)發(fā)生轉(zhuǎn)錄因子bHLH的表達，從而最終抑制向神經(jīng)系統(tǒng)的分化[65-66]；外源性Id蛋白將會模擬BMP4的生理學特性，維持mESC的多能性[65]。但是，BMP4在維持mESC細胞多能性的作用，需要在 LIF的存在下才能實現(xiàn)，這是因為BMP4能誘導ESC細胞向內(nèi)胚層和中胚層分化，而LIF通過LIF-STAT3途徑抑制了BMP4的這一促分化作用，但同時對BMP4誘導的Id蛋白表達沒有作用，從而與LIF-STAT3共同維持mESC細胞的多能性[65]。與mESC自我更新維持機制截然相反，BMP4并不能維持hESC的自我更新，相反會使得 hESC向滋養(yǎng)層細胞和原始內(nèi)胚層細胞分化[5]，hESC多能性需要 BMP4的拮抗物 Noggin與bFGF配合才能得以維持[49]。此外，TGFβ超家族成員Activin A、Nodal在未分化的hESC中表達量較高[67]。hESC研究表明，Smad 2/3參與了 TGFβ/Activin/Nodal的信號轉(zhuǎn)導，在未分化過程中 Smad 2/3被激活，而Smad 1/5處于抑制狀態(tài)，隨著分化發(fā)生，Smad 2/3信號減弱，而Smad 1/5信號增強[68-69]。特異性小分子抑制劑BIO，可以模擬Wnt途徑從而保持hESC的未分化狀態(tài)，而此時Smad 2/3磷酸化水平仍然處于較高水平[69]。

TGFβ家族成員對mESC、hESC多能性的維持具有十分重要的作用，但其對家畜ESC的調(diào)控作用卻并不十分清楚。Pant等研究發(fā)現(xiàn)，添加Noggin抑制BMP4途徑后，牛ICM原代集落Nanog表達量顯著上調(diào)[70]。同時，Alberio等研究發(fā)現(xiàn)，添加BMP4將會導致豬外胚層細胞向滋養(yǎng)層細胞與生殖細胞方向分化；抑制Activin/Nodal途徑，豬外胚層細胞迅速分化為神經(jīng)細胞，因此提出Activin/Nodal信號途徑是在哺乳動物細胞多能性維持過程中起調(diào)控作用的保守途徑[27]。而另有研究表明，添加 Activin及Noggin并不能阻止豬、馬外胚層細胞的分化，激活Activin途徑或抑制BMP4活性，對家畜ESC多能性的維持并無明顯作用[63]。

1.5 PI3K/AKT途徑

磷脂酰肌醇3激酶 (PI3K) 是一種脂質(zhì)激酶，對細胞的增殖、生長、發(fā)育、遷移及細胞周期等生理活動具有十分重要的調(diào)控作用[71-72]。PI3K/AKT途徑處于LIF、bFGF信號下游，在mESC中，當PI3K的負調(diào)控基因PTEN缺失后，將會促進細胞周期由G1期向S期的轉(zhuǎn)變，從而使得mESC增殖加速[72]。添加 PI3K特異性抑制劑，將會激活 LIF介導的MAPK/ERK途徑，降低LIF對mESC自我更新的調(diào)控作用，使得mESC增殖減慢[73-74]，同時編程性死亡發(fā)生率升高[75]。而hESC研究表明，PI3K/AKT與MAPK/ERK途徑，并不存在交匯作用，二者對hESC自我更新的維持存在疊加效應[51]；阻斷 PI3K/AKT途徑，將會影響 hESC增殖，使編程性死亡發(fā)生率升高[51]。此外，研究發(fā)現(xiàn)采用PI3K特異性抑制劑，阻斷PI3K途徑后，將會增加早期附植胚胎編程性死亡的發(fā)生率[75]。

同時研究表明，PI3K/AKT途徑對ESC多能性的維持作用顯著。在添加LIF及飼養(yǎng)層細胞條件下，抑制PI3K活性將會導致mESC、hESC發(fā)生分化，表明PI3K/AKT信號途徑對ESC (如：小鼠、猴、人)多能性狀態(tài)的維持具有十分重要的調(diào)控作用[73-74,76-78]。Storm等研究發(fā)現(xiàn)，抑制PI3K/AKT途徑將會導致包括Nanog和Zscan4家族在內(nèi)的646種基因的表達量發(fā)生改變，而其中Zscan4家族基因并未與Nanog基因關聯(lián)[79-81]；此外研究表明，二細胞早期胚胎及ESC具備較高水平的Zscan4，下調(diào)Zscan4基因?qū)е履遗邿o法附植[82]，因此 PI3K/AKT途徑可能通過Nanog與Zscan4兩種方式調(diào)控mESC的多能性[79]。

目前研究表明，PI3K/AKT途徑對兔ES樣細胞多能性的維持具有十分重要的調(diào)控作用，與 hESC類似，兔ES樣細胞自我更新過程中PI3K/AKT途徑與 MAPK/ERK 途徑并未存在交匯作用[83]；Brevini等研究發(fā)現(xiàn)，豬ES樣細胞雖然不存在LIFR，但LIF可能通過PI3K/AKT途徑，抑制豬ES樣細胞類胚體的形成，從而維持豬ES樣細胞的多能性[28-29,84]。

針對上述與mESC、hESC多能性密切相關的信號通路，我們在綿羊ESC研究過程中，通過對6～8 d囊胚ICM原代集落機械法傳代后，添加LIF-STAT3、Wnt與 Noggin信號途徑的有效激活因子 (mLIF、hLIF、Wnt3a、Noggin)，但結(jié)果表明綿羊ES樣集落周邊細胞呈彌散式生長，堿性磷酸酶活性降低，集落逐步呈平鋪式生長，界限模糊 (圖 2)，初步表明上述因子并不能有效阻止綿羊ES樣細胞發(fā)生分化。

圖2 mLIF、hLIF、Wnt3a、Noggin并不足以維持綿羊類胚胎干細胞的自我更新Fig.2 mLIF, hLIF, Wnt3a and Noggin fail to maintain self-renewal of ovine ES-like cells. (A) The 10 ng/mL mLIF and hLIF fail to maintain self-renewal of ovine ES-like cells(Alkaline phosphatase staining). (B) The 100 ng/mL Wnt3a fail to maintain self-renewal of ovine ES-like cells. (C) The 100 ng/mL noggin fail to inhibition of differentiation of ovine ES-like cells. Scale bars=50 μm.

2 家畜ESC多能性候選基因

目前，制約家畜ESC研究的一個核心關鍵問題是缺乏家畜ESC的多能性標志，這也直接導致研究過程中無法及時有效地篩選出“真正”意義上的家畜ESC。近年來，研究表明POU5F1(POU domain 5 transcript factor 1)、Sox2(SRY-box containing gene 2)和Nanog等轉(zhuǎn)錄因子對mESC、hESC的自我更新和分化具有十分重要的作用，當它們表達時，ESC的自我更新途徑被激活，分化途徑受到抑制[85-87]，三者彼此調(diào)控，同時對上述影響ESC自我更新和分化的外源信號分子作出應答，嚴格控制著ESC的自我更新與分化進程，最終形成了ESC多能性機制的調(diào)控中樞——POU5F1、Sox2、Nanog[88-89]。

POU5F1又稱為OCT-4[90]，是植入前胚胎發(fā)育的重要調(diào)節(jié)因子，特異性地表達于多種多能性細胞：卵母細胞、原始生殖細胞、早期植入前胚胎、原始外胚層、ICM和ESC細胞[91-92]，是mESC和hESC細胞的多能性分子標志[91-93]。目前，多種POU5F1的靶位基因已經(jīng)得到確認，包括Fgf4、Utfl、Opn、Rexl/Zfp42、Fbxl5和Sox2。其中，Sox2在小鼠早期胚胎發(fā)育過程中起到十分重要的作用，不僅在小鼠早期胚胎中表達，同時會在胚外外胚層的多能性細胞中表達，Sox2表達量降低將導致mESC向滋養(yǎng)外胚層分化和多倍體細胞的出現(xiàn)[94]。Sox2-/-突變使小鼠早期胚胎不能形成外胚層并引起胚胎死亡[95]，表明Sox2是維持小鼠早期胚胎發(fā)育所必需的轉(zhuǎn)錄因子。Sox2與POU5F1協(xié)同作用，共同阻止ESC向滋養(yǎng)外胚層分化，同時阻止染色體異常[88]。Nanog基因是ESC研究中發(fā)現(xiàn)的另一個多能性主導基因，它不僅對胚胎發(fā)育過程中ICM多能性的調(diào)控起關鍵作用，同時還可維持外胚層細胞多能性和阻止其向原始內(nèi)胚層的分化[96]，可在ESC、EG (Embryonic germ)和EC (Embryonic carcinoma) 等多能性細胞中檢測到[96-97]，是維持mESC、hESC多能性的關鍵轉(zhuǎn)錄因子[98]。研究表明，雖然mESC與hESC在形態(tài)學、表面標志和生長因子方面存在顯著差別，但是它們的Nanog基因都十分保守[97]。

此外，誘導多能干細胞 (Induced pluripotent stem cells，iPS) 的出現(xiàn)及后期“Yamanaka因子”(POU5F1、Sox2、c-Myc、Klf4) 功能的逐漸明朗，為家畜ESC多能性候選基因的選擇提供了新的參考依據(jù)。2006年，Yamanaka采用逆轉(zhuǎn)錄病毒將POU5F1、Sox2、c-Myc、Klf4導入小鼠胚胎成纖維細胞或成年小鼠尾部皮膚成纖維細胞中，建立了與mESC非常相似的iPS細胞[99]。之后，Yamanaka將上述 4個轉(zhuǎn)錄因子導入到人皮膚成纖維細胞中，也成功獲得了iPS細胞[100]。與此同時，Thomson研究小組也報道了成功誘導胎兒成纖維細胞轉(zhuǎn)化為具有hESC基本特征的人iPS細胞，所不同的是他們使用慢病毒作為載體，選擇了POU5F1、Sox2、Nanog、Lin28等4個基因[101]。Park等發(fā)現(xiàn)POU5F1和Sox2在誘導重構為 iPS細胞過程中是必需的，正是這 2個轉(zhuǎn)錄因子維持了人類iPS細胞的多潛能性，而Klf4和c-Myc的作用是改變?nèi)旧|(zhì)的結(jié)構，利于POU5F1和Sox2的結(jié)合，以提高誘導效率[102]，從而進一步明確了iPS多潛能性誘導過程中POU5F1、Sox2的重要作用。Huangfu等采用組蛋白脫乙?；敢种苿瑢OU5F1和Sox2基因?qū)氲饺祟惼つw成纖維細胞中，也成功獲得了iPS細胞[103]。Kim等將OCT4與Sox2或OCT4轉(zhuǎn)錄因子導入到小鼠神經(jīng)干細胞中，成功獲得了iPS細胞[104-105]。iPS相關技術在家畜iPS研究中的應用，為家畜ESC多能性候選基因的選擇提供了參考依據(jù)。研究表明，采用iPS技術，導入包括POU5F1、Sox2、Nanog在內(nèi)的多個基因，可以獲得與 hESC各項生物學特性極為相似的豬多能性干細胞[106-107]，并已有通過iPS細胞獲得嵌合體豬的報道[108]。

目前的家畜ESC研究表明，mESC、hESC多能性分子標志POU5F1、NANOG、SOX2在家畜多能性鑒定中應當被謹慎使用[12]。比如，POU5F1、NANOG、SOX2基因除在牛、豬、山羊囊胚的ICM表達外，在滋養(yǎng)層細胞、內(nèi)胚層細胞中同樣表達[7-8,12,109-110]。值得注意的是，上述多能性分子標志在ICM與滋養(yǎng)層細胞中的定位存在差別，Pant等研究發(fā)現(xiàn)：NANOG與POU5F1在牛ICM與滋養(yǎng)層細胞的核仁中表達，而NANOG除在ICM核仁表達外，在ICM核質(zhì)中也有所表達[70]。近期研究表明POU5F1、NANOG的 mRNA及其編碼的蛋白在山羊、綿羊ICM表達，而在囊胚滋養(yǎng)層細胞中mRNA表達量顯著降低[9,111]；Hall等通過對11 d豬胚胎外胚層與滋養(yǎng)層細胞對比研究，發(fā)現(xiàn)POU5F1、Nanog、SOX2、FGFR1在外胚層細胞中的表達是特異的[112]；且牛、豬 ICM與外胚層細胞發(fā)生明顯分化前，POU5F1、NANOG、SOX2的表達量發(fā)生明顯變化[70,113-114]。上述相關研究成果為家畜ESC多能性標識的選擇提供了一定依據(jù)。此外，值得注意的是，家畜胚胎POU5F1、NANOG、SOX2的基因表達量還受到外源環(huán)境的調(diào)控，比如Chio等研究發(fā)現(xiàn)，與體外胚胎相比，馬體內(nèi)胚胎 ICM 中POU5F1、SOX2、NANOG的表達量顯著高于滋養(yǎng)層細胞[115]；另外，胚胎體外培養(yǎng)過程中，培養(yǎng)液的選擇 (KSOM 與 SOF) 同樣會對囊胚中上述相關候選基因的表達量產(chǎn)生一定調(diào)控作用[116]。因此，上述候選基因雖然能夠作為家畜ESC多能性分子標識使用，但仍需進一步加以確認，因此與小鼠Nanog相區(qū)別，家畜ESC的NANOG基因則用大寫斜體字母表示。

3 結(jié)語

雖然家畜ESC研究已開展了20多年，但目前尚無實質(zhì)性突破。因此比對 mESC、hESC多能性分子調(diào)控網(wǎng)絡的差異，借鑒mESC、rESC的成功經(jīng)驗，研究家畜ES樣細胞生物學特性，對獲得生殖系傳遞能力的家畜ESC具有十分重要的指導意義；此外，隨著mESC研究的逐步深入，一系列與mESC生物學特性極為相似的細胞 (如FAB-SC、EpiSC細胞) 逐漸被發(fā)現(xiàn)，研究表明，這些類型的細胞可以通過簡單的培養(yǎng)條件的轉(zhuǎn)換從而具備mESC多能性的生物學特性[117-118]，因此對比家畜 ES樣細胞與FAB-SC、EpiSC的生物學特性差異，將會為家畜ES樣細胞向多能性方向的轉(zhuǎn)變提供新的技術途徑；另外，借助iPS研究成果，研究家畜iPS細胞生物學特性及多能性維持調(diào)控途徑，將會對分離、培養(yǎng)、鑒定具備生殖系傳遞能力的家畜 ESC具有十分重要的指導意義。

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Pluripotency candidate signaling network and transcription factors in domesticated ungulates: a review

Yuncheng Zhao1,2, Bo Chen1,2, Chuan Zhou1,2, Xiuhua Zhang1,2, and Juncheng Huang1,2

1Key Laboratory of Animal Biotechnology of Xinjiang,Urumqi830000,China
2Key Laboratory of Livestock Reproduction & Biotechnology of MOA,Xinjiang Academy of Animal Science,Urumqi830000,China

Received:July 26, 2010;Accepted:October 20, 2010

Supported by:National High Technology Research and Development Program of China (863 Program) (No. 2008aa101005), High Technology Research and Development Program of Xinjiang Uighur Autonomous Region (No. 200711104), Youth Research Fund of Animal Science Academy in Xinjiang Uighur Autonomous Region (Nos. 2008QJ01, 2010QJ006).

Corresponding author:Juncheng Huang. E-mail: hjc@sina.com

國家高技術研究發(fā)展計劃 (863計劃) (No. 2008aa101005)，新疆維吾爾自治區(qū)高技術研究發(fā)展計劃項目 (No. 200711104)，新疆畜牧科學院青年科研基金 (Nos. 2008QJ01, 2010QJ006) 資助。