趙　冉　楊　羊　徐　晨　陸　超　孫　寧　錢睿哲

(復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院生理與病理生理學(xué)系　上?！?00032)

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生物鐘基因Clock對(duì)小鼠誘導(dǎo)性多能干細(xì)胞分化的影響

趙冉楊羊徐晨陸超孫寧錢睿哲△

(復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院生理與病理生理學(xué)系上海200032)

目的研究生物鐘基因Clock對(duì)小鼠誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cell,iPSC)非定向分化的作用。方法將Teto-FUW-OSKM、M2rtTA、PsPAX2 和PMD2.G 4種質(zhì)粒共轉(zhuǎn)染293FT細(xì)胞,收集病毒上清,轉(zhuǎn)染小鼠胚胎成纖維細(xì)胞(mouse embryo fibroblast,MEF)獲得小鼠iPSC,并通過(guò)檢測(cè)多能性基因表達(dá)及其體外分化能力對(duì)小鼠iPSC進(jìn)行鑒定;通過(guò)RT-PCR檢測(cè)小鼠iPSC在非定向分化和擬胚體形成過(guò)程中生物鐘基因的表達(dá),以及生物鐘基因Clock下調(diào)后,與野生型細(xì)胞相比小鼠iPSC多能性基因的表達(dá)和形態(tài)學(xué)改變。結(jié)果通過(guò)慢病毒感染MEF的方式獲得了小鼠iPSC;小鼠iPSC在非定向分化過(guò)程中,生物鐘基因Clock、Per2、Rev-erbα表達(dá)升高;生物鐘基因Clock下調(diào)后,小鼠iPSC分化能力下降,多能性基因Sox2、Oct4、Klf4表達(dá)升高。結(jié)論生物鐘基因Clock對(duì)小鼠iPSC的非定向分化起到重要作用。

生物鐘基因;誘導(dǎo)性多能干細(xì)胞;非定向分化;小鼠

晝夜節(jié)律是自然界普遍存在的一種生物節(jié)律,它使生物體的生理、生化、行為等生命現(xiàn)象表現(xiàn)為近似24 h的節(jié)律性[1]。生物鐘基因Clock作為第一個(gè)被發(fā)現(xiàn)和鑒定的生物鐘基因,是調(diào)節(jié)晝夜節(jié)律的核心分子,參與維持生物體的多項(xiàng)生命活動(dòng)[2-3]。近年來(lái)研究發(fā)現(xiàn),與分化成熟的體細(xì)胞具備生物鐘轉(zhuǎn)錄-翻譯反饋環(huán)路、生物鐘基因呈現(xiàn)節(jié)律性表達(dá)不同,小鼠胚胎干細(xì)胞(embryonic stem cell,ESC)中的生物鐘基因缺乏生物鐘轉(zhuǎn)錄-翻譯反饋環(huán)路,并不呈現(xiàn)節(jié)律性表達(dá);小鼠ESC體外分化后,生物鐘節(jié)律振蕩重新出現(xiàn)[4]。siRNA干擾生物鐘基因Clock或Bmal1表達(dá)可以抑制神經(jīng)干細(xì)胞向神經(jīng)元方向分化[5]。生物鐘基因Timeless促進(jìn)ESC的分化[6]。誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cell,iPSC)由已分化的體細(xì)胞誘導(dǎo)而成,具有與ESC相似的特性。然而生物節(jié)律對(duì)iPSC分化的影響在國(guó)內(nèi)尚無(wú)報(bào)道,因此本研究擬建立穩(wěn)定的小鼠iPSC細(xì)胞系,并在此細(xì)胞系基礎(chǔ)上探討生物鐘基因Clock對(duì)小鼠iPSC(miPSC)非定向分化的影響,為干細(xì)胞治療提供新的思路。

材 料 和 方 法

實(shí)驗(yàn)動(dòng)物及細(xì)胞系來(lái)源孕13天C57BL/6J小鼠(n=8)購(gòu)自于南京模式動(dòng)物所,293 FT細(xì)胞、小鼠E14 ESC均由復(fù)旦大學(xué)上海醫(yī)學(xué)院分子病毒實(shí)驗(yàn)室饋贈(zèng)。

主要試劑質(zhì)粒Teto-FUW-OSKM、PsPAX2、PMD2.G、M2rtTA均購(gòu)自于美國(guó)Addgene公司;質(zhì)粒pWPI-EGFP由復(fù)旦大學(xué)醫(yī)學(xué)分子病毒學(xué)重點(diǎn)實(shí)驗(yàn)室惠贈(zèng);DMEM培養(yǎng)基、KnockoutTMD-MEM、敲除血清替代物(knockout serum replacement,KSR)、含谷氨酰胺非必需氨基酸均購(gòu)自美國(guó)Gibico公司;堿性磷酸酶染色試劑盒購(gòu)自上海斯丹賽生物科技有限公司;逆轉(zhuǎn)錄及PCR試劑盒購(gòu)自日本TOYOBO公司。

小鼠iPS細(xì)胞系的建立質(zhì)粒轉(zhuǎn)染前一天,接種293FT細(xì)胞至10 cm細(xì)胞培養(yǎng)板中。轉(zhuǎn)染前1 h將DMEM培養(yǎng)液換成無(wú)血清DMEM培養(yǎng)液600 μL。按照以下比例配制3種DNA-Hily Max轉(zhuǎn)染液:Teto-FUW-OSKM/M2rtTA/pWPI 20 μg、PsPAX2 15 μg、PMD2.G 5 μg。將上述3種不同的DNA-HilyMax轉(zhuǎn)染混合物分別懸空滴入293FT細(xì)胞培養(yǎng)皿中。將培養(yǎng)皿放于37 ℃培養(yǎng)箱培養(yǎng),48 h后在熒光顯微鏡下觀察慢病毒包裝情況并拍照記錄。然后將病毒上清吸入至15 mL的離心管中,4 ℃ 3 000 r/min(離心半徑13 cm)離心5 min,收集上清,并用0.45 μm的小濾器抽濾,分裝保存。將1×105個(gè)MEF細(xì)胞接種于含有1 mL的DMEM培養(yǎng)液(10% FBS不含雙抗)的六孔板中。各取1 mL抽濾好的Teto-FUW-OSKM-慢病毒、M2rtTA-慢病毒加入上述六孔板中。放于37 ℃,CO2濃度為5%的細(xì)胞培養(yǎng)箱中培養(yǎng)。12 h后換成小鼠iPSC培養(yǎng)液3 mL,并加入強(qiáng)力霉素,使其終濃度為2 μg/mL,繼續(xù)培養(yǎng)并逐日觀察拍照記錄。之后每2天換一次液,12 天后去除強(qiáng)力霉素,直到克隆長(zhǎng)到可挑出擴(kuò)增。

小鼠iPSC的鑒定取狀態(tài)良好的iPSC克隆,Trizol法提取總RNA,RT-PCR檢測(cè)多能性基因Oct4、Sox2、Klf4、c-Myc以及Nanog、Dax-1等的表達(dá),其相應(yīng)的引物如表1所示。

選取生長(zhǎng)狀態(tài)良好的iPSC,棄去培養(yǎng)基,用1×PBS清洗3次;加入4%多聚甲醛固定1～2 min;棄去固定液,用1×PBS清洗2次;吸出PBS,用TBST潤(rùn)洗1次;加入適量的堿性磷酸酶染液,染色10～15 min,吸出AKP染色液,用1×PBS緩沖液潤(rùn)洗一次,最后保存于PBS緩沖液中,顯微鏡下觀察染色結(jié)果。

細(xì)胞免疫熒光染色Oct 4、SSEA-1、Nanog (1∶400)檢測(cè)iPSC多能性的分子標(biāo)記。將細(xì)胞用預(yù)冷的1×PBS緩沖液潤(rùn)洗3次,4%多聚甲醛室溫固定15 min,用預(yù)冷的1×PBS緩沖液潤(rùn)洗3次,再用5% TritonX 100室溫處理細(xì)胞15 min;然后加入3% BSA孵育細(xì)胞,室溫封閉1 h;加入一抗, 4 ℃過(guò)夜或室溫1 h;用PBST清洗細(xì)胞3次,每次約10 min;將熒光二抗按1：1 000進(jìn)行稀釋,加到細(xì)胞上,室溫置1 h;用PBST清洗細(xì)胞3次,每次約10 min;將DAPI按1:1 000稀釋,加到細(xì)胞上,室溫染核5 min;觀察免疫熒光并拍照記錄。

表1　iPSC 標(biāo)志性多能性基因的PCR引物序列

在懸浮細(xì)胞皿中,將適量純化后的iPSC接種于含擬胚體的(embryoid body, EB)完全培養(yǎng)液中,觀察EB的形成并拍照。

生物鐘基因?qū)π∈骾PSC非定向分化的影響將純化的iPSC接種在明膠包被的無(wú)滋養(yǎng)層細(xì)胞的細(xì)胞培養(yǎng)板中進(jìn)行培養(yǎng),分別提取培養(yǎng)0、1、2、3天的細(xì)胞總RNA,通過(guò)Real-time PCR檢測(cè)生物鐘基因的表達(dá)變化。將純化的iPSC接種在懸浮細(xì)胞板中,分別提取培養(yǎng)0、1、3、5、7、9、11天的細(xì)胞總RNA,通過(guò)Real-time PCR檢測(cè)生物鐘基因的表達(dá)變化。生物鐘基因引物如表2所示。

接種5×104個(gè)純化的iPSC于含有1.5 mL培養(yǎng)液的細(xì)胞培養(yǎng)板中。制備siRNA-LipofectamineTM2000轉(zhuǎn)染復(fù)合物:將20 μmol/L的Clock-siRNA 10 μL、Control-SiRNA分別與240 μL的Opti-Medium輕輕混勻,室溫孵育5 min,同時(shí)將5 μL LipofectamineTM2000與245 μL Opti-Medium輕輕混勻,室溫孵育5 min。

表2　生物鐘基因的PCR引物序列

將上述兩種混合液輕輕混勻,室溫孵育20 min。將500 μL siRNA-LipofectamineTM2000轉(zhuǎn)染復(fù)合物懸空滴加到細(xì)胞中,輕輕晃動(dòng)混勻。細(xì)胞培養(yǎng)箱中培養(yǎng),4～6 h后換液,轉(zhuǎn)染48 h后顯微鏡下觀察細(xì)胞形態(tài)變化,并抽提RNA,通過(guò)Real-time PCR檢測(cè)目的基因的干擾效果及多能性基因Sox2、Oct4、Klf4的表達(dá)。Clock-siRNA序列(5′-3′)為GGACAAGUCUACUGUUCUAUAGAACAGUAGA-CUUGUCC。

結(jié)　　　果

慢病毒的包裝與轉(zhuǎn)染效率將慢病毒表達(dá)質(zhì)粒pWPI、Teto-FUW-OSKM、M2rtTA分別與慢病毒包裝質(zhì)粒PsPAX2、PMD2.G共轉(zhuǎn)染293FT細(xì)胞48 h,可見(jiàn)綠色熒光蛋白(green fluorescent protein,GFP)表達(dá)(圖1A、B),72 h后收集病毒,將pWPI-慢病毒感染小鼠MEF,48 h可見(jiàn)GFP表達(dá)(圖1C、D)。

小鼠iPSC的誘導(dǎo)為了研究生物鐘基因與iPSC分化的關(guān)系,我們通過(guò)慢病毒轉(zhuǎn)染方法建立了小鼠iPSC細(xì)胞系。將Teto-FUW-OSKM-慢病毒感染MEF(圖2A)后,第1天MEF細(xì)胞偽足變少,形態(tài)由梭形逐漸變?yōu)閳A形(圖2B);第3天細(xì)胞局部聚集成簇生長(zhǎng),體積變小(圖2C);第7天細(xì)胞局部聚集更加明顯,與邊緣分界逐漸清晰(圖2D);第9～12天細(xì)胞形成小鼠ESC樣克隆,邊界清晰,表面光滑,結(jié)構(gòu)緊密,隆起生長(zhǎng)(圖2E、F)。

小鼠iPSC多能性的鑒定為了確認(rèn)由MEF重編程獲得的iPSC具有多能性,我們采用了堿性磷酸酶染色、RT-PCR和免疫熒光法檢測(cè)多能性基因、擬胚體形成實(shí)驗(yàn)等方法進(jìn)行鑒定。堿性磷酸酶染色顯示小鼠iPSC堿性磷酸酶呈陽(yáng)性(圖3A)。RT-PCR結(jié)果顯示小鼠iPSC表達(dá)內(nèi)源性Nanog、Sox2、Oct4、Dax1、Klf4,且與小鼠ESC相比差異無(wú)統(tǒng)計(jì)學(xué)意義(圖3B)。消化后的iPSC在懸浮培養(yǎng)皿中培養(yǎng)呈三維立體的球形生長(zhǎng),形成擬胚體(圖3C)。細(xì)胞免疫熒光結(jié)果顯示小鼠iPSC表達(dá)ESC的特異性分子標(biāo)記SSEA-1、OCT4、NANOG (圖3D)。

A:293FT cells transfected with plasmid pWPI after 48 h (×4);B:The GFP expression of 293FT cells transfected with plasmid pWPI after 48 h (×4);C:MEF infected with lentivirus after 48 h (×4);D:The GFP expression of MEF infected with lentivirus after 48 h (×4).

圖1慢病毒包裝與轉(zhuǎn)染效率

Fig 1Detection of lentiviral packaging and infection efficiency

iPSC非定向分化過(guò)程中生物鐘基因的表達(dá)RT-PCR結(jié)果顯示小鼠iPSC在自分化過(guò)程中生物鐘基因Clock、Per2、Rev-erbα表達(dá)逐漸升高,且差異有統(tǒng)計(jì)學(xué)意義(圖4A);RT-PCR結(jié)果顯示生物鐘基因Clock、Per2、Rev-erbα在EB形成前3天內(nèi)逐漸升高,且在EB形成后3天時(shí)出現(xiàn)高峰,隨后降低但仍高于EB形成后0天,之后又逐漸升高,且差異具有統(tǒng)計(jì)學(xué)意義(圖4B)。

A:Mouse embryo fibroblast (×4);B-F:The 1st,3rd,7th,9thand 12thday of iPSC,separately (×4).

圖2利用轉(zhuǎn)染Teto-FUW-OSKM慢病毒的方法將小鼠MEF 轉(zhuǎn)化為iPSC

Fig 2Generation of iPSC cell lines into MEF cells in mouse by introducing Teto-FUW-OSKM lentivirus

生物鐘基因Clock對(duì)iPSC多能性的影響為了明確核心生物鐘Clock基因與iPSC非定向分化之間的關(guān)系,我們利用siRNA干擾的方法下調(diào)Clock基因的表達(dá),結(jié)果顯示Clock的表達(dá)下降了約70%(圖5B)。在此基礎(chǔ)上,我們進(jìn)行了miPSC細(xì)胞的非定向分化,結(jié)果顯示對(duì)照組的miPSC克隆邊緣不光滑,有偽足伸出;而實(shí)驗(yàn)組(Clock-siRNA)miPSC克隆仍保持邊緣光滑,極少有偽足伸出(圖5A);與Control-siRNA組相比,Clock-siRNA組48 h后,多能性基因Sox2、Oct4、Klf4表達(dá)升高(P<0.05)(圖5B)。提示Clock基因能夠影響干細(xì)胞的分化。

討　　　論

晝夜節(jié)律是自然界普遍存在的一種生物節(jié)律,它使機(jī)體的行為、生理表現(xiàn)為近似24 h的節(jié)律性,由機(jī)體的內(nèi)源性生物鐘調(diào)控和驅(qū)動(dòng);同時(shí)又受到外界環(huán)境因子的影響[7]。目前,在所有的生物鐘系統(tǒng)中,生物鐘分子基礎(chǔ)模型主要包括了由一組高度保守的“生物鐘基因”組成的轉(zhuǎn)錄-翻譯正負(fù)反饋回路[8-9]。生物鐘基因Clock、Bmal1組成哺乳動(dòng)物的核心生物鐘,參與細(xì)胞分化、增殖、死亡等,調(diào)節(jié)生命體的各種生理活動(dòng)。本課題組前期研究發(fā)現(xiàn),生物節(jié)律紊亂的小鼠血脂水平顯著異常,高脂飲食喂養(yǎng)的apoE-/-小鼠中,生物鐘基因Clock表達(dá)的節(jié)律和幅度發(fā)生改變。因此一旦生物節(jié)律發(fā)生紊亂,將會(huì)導(dǎo)致多種疾病的產(chǎn)生[10-13]。

A:Alkaline phosphatase staining of miPSC (×10);B:RT-PCR analysis of miPSC for pluripotent marker genes expression;C:Embryoid body (×10);D:Immunostaining of miPSC for SSEA1,OCT4 and NANOG expression(×10).iPSC vs. ESC,(1)P=0.004 1 (t=15.49);(2)P=0.005 1 (t=13.98) (n=3).

圖3小鼠iPSC的多能性標(biāo)記基因檢測(cè)

Fig 3Detection of miPSC for pluripotent marker

iPSC于2006年由Yamanaka實(shí)驗(yàn)室通過(guò)外源性表達(dá)Oct4、Sox2、Klf4、c-Myc成功誘導(dǎo)小鼠的體細(xì)胞重編程而成[14],具有與ESC相似的多能性,在體外已被成功地分化為生殖細(xì)胞、心血管細(xì)胞和肝細(xì)胞等多種體細(xì)胞[15-17]。目前,iPSC被廣泛應(yīng)用于再生醫(yī)學(xué)和疾病模型的建立,為臨床治療提供理論基礎(chǔ)。在本實(shí)驗(yàn)中,我們首先利用MEF在體外通過(guò)誘導(dǎo)的方法建立小鼠iPSC細(xì)胞系,其主要特征表現(xiàn)為能夠形成ESC樣克隆,細(xì)胞核較大,核質(zhì)比例高,堿性磷酸酶染色呈陽(yáng)性,表達(dá)內(nèi)源性O(shè)ct4、Sox2、Nanog、SSEA1等多能性標(biāo)記分子。誘導(dǎo)的細(xì)胞在體外能夠進(jìn)行自分化,形成擬胚體,這些特征都說(shuō)明我們成功在體外誘導(dǎo)建立了小鼠iPSC細(xì)胞系。

A:The expression of Clock,Per2,Rev-erbαgene during the miPSC spontaneous differentiation;B:The expression of Clock,Per2,Rev-erbαgene during EB formation.The results suggested that Clock genes have some influence on the process of miPSC differentiation and maturation.A:Each time point vs. its previous time point,(1)P<0.05.Clock P=0.032,0.025,0.041 (t=5.48,6.26,4.77);Per2 P=0.012,0.065,0.010 (t=9.00,3.74,9.84);Rev-erbαP=0.005 4,0.022,0.038 (t=13.53,6.57,4.96);B:EB 1 d,3 d,5 d,7 d,9 d,11 d vs. EB 0 d,(2)P<0.01.Clock P=0.005 2,0.000 5,0.0022.0.0038.0.0003,0.000 3 (t=13.81,43.26,21.41,16.14,60.12,61.76);Per2 P=0.001 5,0.000 1,0.0006,0.001 0,0.000 2,0.000 1 (t=25.66,94.93,41.22,31.55,63.99,82.33);Rev-erbαP=0.003 3,0.000 5,0.001 8,0.001 1,0.000 8,<0.000 1 (t=17.32,43.31,23.30,29.58,34.39,128.5) (n=3).

圖4RT-PCR 檢測(cè)小鼠iPSC自分化及EB形成過(guò)程中生物鐘基因的表達(dá)

Fig 4The expression of circadian Clock genes during the miPSC spontaneous differentiation and EB formation

A:The morphology of control-siRNA iPSC and Clock-SiRNA iPSC(arrows indicate the pseudopods formation);B:Pluripotency genes expressions of miPSC after the suppression of circadian Clock gene expression.Clock-siRNA group vs. control-siRNA group(n=3),(1)P=0.004 1 (t=13.52);(2)P=0.011 (t=9.65);(3)P=0.045 7 (t=4.52);(4)P=0.011 (t=9.48) .

圖5生物鐘基因Clock下調(diào)48 h后miPSC形態(tài)和多能性基因的表達(dá)情況

Fig 5Morphological change and pluripotency genes expressions of miPSC after the suppression of circadian Clock gene for 48 h

生物鐘基因與成體干細(xì)胞之間關(guān)系密切。據(jù)文獻(xiàn)報(bào)道,在培養(yǎng)的人神經(jīng)干細(xì)胞的分化過(guò)程中生物鐘基因表達(dá)譜發(fā)生改變,Bmal1基因出現(xiàn)節(jié)律性表達(dá)[5]。生物鐘基因Per2可通過(guò)影響內(nèi)皮祖細(xì)胞的分化,導(dǎo)致缺血性心臟病小鼠新生血管形成減少[18]。Clock、Per2基因缺失可抑制人間充質(zhì)干細(xì)胞向脂肪細(xì)胞的分化[19]。Rev-erbα基因過(guò)表達(dá)促進(jìn)骨間充質(zhì)干細(xì)胞的分化成熟,但在骨生成的最后階段作為負(fù)性調(diào)節(jié)因素發(fā)揮抑制作用[20]。然而,生物鐘基因與ESC之間的關(guān)系卻研究甚少。

Yagita等[4]首次研究了ESC與生物鐘基因的關(guān)系,其結(jié)果表明ESC的生物鐘基因不表達(dá)或呈無(wú)節(jié)律性表達(dá),而分化的ESC表達(dá)生物鐘基因且具有節(jié)律性,如果將成體細(xì)胞重新誘導(dǎo)為iPSC則生物鐘節(jié)律又消失。O′Reilly等[6]發(fā)現(xiàn)生物鐘基因Timeless表達(dá)缺失的ESC中多能性基因表達(dá)水平增加,形成的擬胚體數(shù)目降低、體積減小。根據(jù)這些結(jié)果,我們通過(guò)進(jìn)一步的實(shí)驗(yàn)來(lái)研究生物鐘基因與多能干細(xì)胞的多能性及分化功能的關(guān)系。結(jié)果顯示,iPSC自發(fā)分化過(guò)程中生物鐘基因Clock、Per2、Rev-erbα表達(dá)水平逐漸升高;擬胚體形成過(guò)程中,生物鐘基因Clock、Per2、Rev-erbα表達(dá)水平同樣升高。這些結(jié)果都與Yagita等[4]的結(jié)果相吻合。

目前,生物鐘基因與干細(xì)胞分化之間的關(guān)系尚未明確。事實(shí)上,生物鐘基因在胚胎時(shí)期已經(jīng)有了表達(dá)[21],但是生物鐘節(jié)律的出現(xiàn)和成熟是在出生前幾天[22]。利用條件性敲除生物鐘基因Bmal1后發(fā)現(xiàn),本來(lái)在普通Bmal1-/-小鼠中出現(xiàn)的細(xì)胞衰老表現(xiàn)(如年齡縮短、生殖功能退化和關(guān)節(jié)病等表型)不明顯,而動(dòng)脈粥樣硬化和毛發(fā)生長(zhǎng)等在條件性敲除Bmal1的小鼠中反而有所改善[23]。該結(jié)果提示了生物鐘基因的確在胚胎發(fā)育時(shí)期發(fā)揮了重要作用。

因此,我們希望研究生物鐘基因是否對(duì)干細(xì)胞的分化功能產(chǎn)生了影響。Clock與Bmal1、Per(Per1、Per2、Per3)和 Cry(Cry1、Cry2)是4個(gè)主要的生物鐘核心基因。其中CLOCK與BMAL1形成的異二聚體可以與受生物鐘調(diào)節(jié)基因(包括了Per和Cry)上游的E-box結(jié)合,啟動(dòng)節(jié)律性基因轉(zhuǎn)錄;而Per與Cry表達(dá)升高后,形成的異二聚體可與Clock和Bmal1結(jié)合,反饋性抑制其轉(zhuǎn)錄功能,該調(diào)節(jié)周期近似24 h。單獨(dú)抑制Bmal1或者Clock都可以顯著抑制生物鐘基因及其下游基因的節(jié)律性表達(dá),從而破壞生物鐘調(diào)節(jié)。Per和Cry由于有多個(gè)亞型的存在,同時(shí)抑制幾個(gè)Per (Per1、Per2、Per3)或Cry (Cry1、Cry2)基因亞型的表達(dá)可以達(dá)到類似的效果,但是由于操作上的復(fù)雜性,很少作為研究靶點(diǎn)。如前述,體內(nèi)外實(shí)驗(yàn)均已發(fā)現(xiàn),Clock在細(xì)胞分化、增殖、死亡以及成體干細(xì)胞分化等過(guò)程中起著重要作用,但是其在ESC的分化過(guò)程中是否具有重要作用尚未知。因此我們首先利用RNA干擾的方式建立了生物鐘基因Clock表達(dá)下調(diào)的iPSC細(xì)胞系。結(jié)果表明,與對(duì)照組相比,Clock表達(dá)下調(diào)的小鼠iPSC自分化3天后仍保持邊緣光滑,極少有偽足伸出;同時(shí)多能性基因Sox2、Oct4、Klf4表達(dá)相較于對(duì)照組升高。這些結(jié)果均提示生物鐘基因Clock促進(jìn)了小鼠iPSC的非定向分化。

目前仍未明確生物鐘基因究竟是通過(guò)何種機(jī)制促進(jìn)了干細(xì)胞的分化。根據(jù)已有的文獻(xiàn)報(bào)道,生物節(jié)律對(duì)成體干細(xì)胞分化的影響可能是通過(guò)調(diào)節(jié)細(xì)胞周期及一些信號(hào)通路如Notch、Wnt等發(fā)揮作用。研究發(fā)現(xiàn),生物鐘基因通過(guò)調(diào)節(jié)細(xì)胞周期影響腸道干細(xì)胞(intestinal stem cell,ISC)的自我更新,生物鐘基因Per的存在維持著ISC有絲分裂的節(jié)律性,Per的缺失則使ISC停留在細(xì)胞周期的S期,另一生物鐘基因CYC的缺失使ISC停留在細(xì)胞周期的G1期[24]。Bouchard-Cannon等[25]也提到生物鐘基因通過(guò)參與神經(jīng)祖細(xì)胞的細(xì)胞周期進(jìn)而調(diào)節(jié)神經(jīng)發(fā)生。Li等[26]研究發(fā)現(xiàn),在胚胎發(fā)育過(guò)程中Clock基因表達(dá)與組織分化進(jìn)程相一致,抑制Clock或Timeless基因表達(dá)可以干擾Notch、Wnt信號(hào)通路分子的表達(dá)。而經(jīng)典的Wnt信號(hào)通路相關(guān)基因受到生物鐘基因Bmal1的直接轉(zhuǎn)錄調(diào)控,Wnt信號(hào)通路可恢復(fù)Bmal1-/-小鼠肌細(xì)胞分化的能力,因此Bmal1可能通過(guò)調(diào)節(jié)Wnt信號(hào)通路影響肌細(xì)胞分化[27]。然而,上述機(jī)制是否在多能干細(xì)胞的分化過(guò)程中具有作用有待研究。

綜上所述,本研究發(fā)現(xiàn)生物鐘基因Clock在iPSC非定向分化中發(fā)揮促進(jìn)作用,為iPSC分化機(jī)制研究、臨床藥物篩選和干細(xì)胞治療提供了新的思路和方向。但本實(shí)驗(yàn)仍具有一定的局限性,生物鐘基因Clock是否通過(guò)調(diào)節(jié)細(xì)胞周期及Wnt等信號(hào)通路影響小鼠iPSC分化將是我們下一步研究的目標(biāo)。

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E-mail:rzqian@shmu.edu.cn

The influences of circadian gene Clock on differentiation of induced pluripotent stem cells in mouse

ZHAO Ran, YANG Yang, XU Chen, LU Chao, SUN Ning, QIAN Rui-zhe△

(Department of Physiology and Pathophysiology,School of Basic Medical Sciences,Fudan University,Shanghai 200032,China)

ObjectiveTo study the relationship between circadian gene Clock and induced pluripotent stem cell (iPSC) differentiation in mouse.MethodsTeto-FUW-OSKM,M2rtTA,PsPAX2 and PMD2.G plasmid were transfected into 293FT cells.Then virus-containing supernatants were harvested to infect mouse embryo fibroblast (MEF).The characteristics of mouse iPSC (miPSC) were analyzed by pluripotent marker expression levels and the differentiation capacity in vitro.Real-time PCR was used to detect the expression of circadian Clock gene during spontaneous differentiation and the formation of embryoid body.After reducing the Clock gene expression level of miPSC,the cell morphological changes and the expression of pluripotency genes in miPSC were compared with wild type cells.ResultsWe established miPSC by lentivirus inducing.The expression levels of circadian Clock gene,Per2 and Rev-erbαincreased in miPSC spontaneous differentiation process.After knocking down Clock gene,the differentiation of miPSC ability decreased as well as the expression levels of pluripotency genes increased,such as Sox2,Oct4 and Klf4.ConclusionsThe circadian Clock genes had an important influence on miPSC spontaneous differentiation.

circadian gene Clock;induced pluripotent stem cell;spontaneous differentiation;mouse

R363

Adoi:10.3969/j.issn.1672-8467.2016.05.001

2016-03-28;編輯:張秀峰)

*This work was supported by the National Science Foundation Fostering Talents in Basic Research of China (J1210041) and the National Natural Science Foundation of China (81570771).

國(guó)家基礎(chǔ)科學(xué)人才培養(yǎng)基金(J1210041);國(guó)家自然科學(xué)基金(81570771)