逄麗麗，賀琳，宋嬋嬋，連秀麗，劉玥，許頌華，王世鄂,*

（福建醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院1細(xì)胞與發(fā)育工程中心，2人體解剖學(xué)與組織胚胎學(xué)系，福州 350122）

論著

ERα特異性抑制劑MPP降低小鼠2-細(xì)胞胚G1/S期過(guò)渡相關(guān)因子Nanog、cyclin D1和cyclin E水平

逄麗麗1，賀琳2，宋嬋嬋1，連秀麗2，劉玥1，許頌華2，王世鄂1,2*

（福建醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院1細(xì)胞與發(fā)育工程中心，2人體解剖學(xué)與組織胚胎學(xué)系，福州 350122）

目的觀察雌激素受體（ERα）特異性抑制劑甲基哌啶吡唑（methyl-piperidino-pyrazole，MPP）對(duì)小鼠2-細(xì)胞胚G1/S期過(guò)渡相關(guān)因子Nanog、cyclin D1和cyclin E表達(dá)水平的影響，闡明ERα對(duì)小鼠2-細(xì)胞胚G1/S期過(guò)渡的作用，為進(jìn)一步探討ERα在小鼠植入前胚合子基因組激活中的作用機(jī)制提供理論依據(jù)。方法收集昆明雌性小鼠與雄性小鼠交配所得的受精卵，分別置于無(wú)MPP的培養(yǎng)液和含20μmol/L MPP的培養(yǎng)液中培養(yǎng)，獲取體外發(fā)育的2-細(xì)胞胚，采用免疫熒光染色技術(shù)檢測(cè)細(xì)胞內(nèi)Nanog、cyclin D1和cyclin E水平。結(jié)果免疫熒光染色結(jié)果顯示，含20μmol/L MPP培養(yǎng)液培養(yǎng)的受精卵其體外發(fā)育2-細(xì)胞胚內(nèi)Nanog、cyclin D1和cyclin E免疫反應(yīng)性明顯降低。結(jié)論ERα可通過(guò)調(diào)控小鼠2-細(xì)胞胚內(nèi)Nanog、cyclinD1和cyclinE水平，影響細(xì)胞從G1期到S期的過(guò)渡。

雌激素受體α；植入前胚；Nanog；細(xì)胞周期蛋白；小鼠

雌激素受體α（estrogen receptor alpha, ERα）是類固醇激素受體超家族成員之一，可通過(guò)經(jīng)典及非經(jīng)典途徑調(diào)控基因表達(dá)，對(duì)細(xì)胞與組織的增殖、分化和發(fā)育起著重要作用[1]。前期研究發(fā)現(xiàn)，ERα對(duì)小鼠植入前胚體外發(fā)育有一定的作用，經(jīng)ERα特異性抑制劑甲基哌啶吡唑（methyl-piperidino-pyrazole，MPP）處理后，小鼠植入前胚發(fā)育停滯在2-細(xì)胞胚時(shí)期，且合子基因組激活（zygotic gene activation, ZGA）相關(guān)基因MuERV-L mRNA表達(dá)水平下調(diào)[2]。ERα的時(shí)空動(dòng)態(tài)表達(dá)模式也提示，ERα可能調(diào)控小鼠植入前胚第一次卵裂的細(xì)胞周期進(jìn)程，參與ZGA[3]。進(jìn)一步研究發(fā)現(xiàn)，經(jīng)MPP作用后，小鼠2-細(xì)胞胚核呈較小的圓形核，屬于G0/G1期細(xì)胞核象，提示MPP處理后小鼠2-細(xì)胞胚（hCG后42h）發(fā)育阻滯于DNA復(fù)制前期。BrdU標(biāo)記實(shí)驗(yàn)顯示，ERα在小鼠2-細(xì)胞胚S期中的作用與S期啟動(dòng)有關(guān)[4]。但ERα是否通過(guò)調(diào)節(jié)2-細(xì)胞胚G1/S期監(jiān)測(cè)點(diǎn)相關(guān)蛋白的表達(dá)來(lái)影響后續(xù)發(fā)育進(jìn)程尚不明了。

研究表明，參與G1/S期過(guò)渡的關(guān)鍵蛋白主要有cyclinD和cyclinE等。脊椎動(dòng)物G1期的細(xì)胞周期素主要有cyclin D1、cyclin D2和cyclin D3，但它們的含量變化并不依賴于細(xì)胞周期進(jìn)程，而主要依賴于細(xì)胞生長(zhǎng)或生長(zhǎng)促進(jìn)信號(hào)[5]。無(wú)論是在正常細(xì)胞中，還是在腫瘤細(xì)胞中，若cyclin D1蛋白表達(dá)受到抑制，則細(xì)胞周期停滯在G1/S期，無(wú)法順利過(guò)渡到后續(xù)發(fā)育階段[6-8]。G1/S期的另一重要調(diào)控因子Cyclin E的角色則更加復(fù)雜，cyclin D1-細(xì)胞周期依賴性蛋白激酶（CDK）4/6只催化部分pRB的磷酸化和E2F的激活，而cyclin E-CDK2可促使E2F完全激活，并通過(guò)pRB蛋白的磷酸化，對(duì)E2F依賴的基因表達(dá)起主要的協(xié)調(diào)作用[9]。大多哺乳動(dòng)物細(xì)胞中DNA的復(fù)制由S期CDKs啟動(dòng)，cyclin E-CDk2復(fù)合物作為復(fù)制起始的直接調(diào)控者引起S期CDKs抑制蛋白的酶解[10]。此外，胚胎干細(xì)胞轉(zhuǎn)錄因子Nanog對(duì)細(xì)胞周期的推動(dòng)也有重要作用。Nanog能推動(dòng)高分化的小鼠胚胎干細(xì)胞G1/S期過(guò)渡[11]，也可通過(guò)調(diào)控下游的CDK6及CDC25A，推動(dòng)細(xì)胞由G1期進(jìn)入S期[12]。可見(jiàn)，Nanog可作用于細(xì)胞周期關(guān)鍵調(diào)控因子，參與G1/S期的過(guò)渡。本研究通過(guò)免疫熒光染色技術(shù)檢測(cè)MPP處理后小鼠2-細(xì)胞胚中Nanog及細(xì)胞周期蛋白cyclin D1和cyclin E的表達(dá)情況，進(jìn)一步闡明ERα在小鼠植入前胚G1/S期過(guò)渡中的作用機(jī)制。

材料和方法

1 實(shí)驗(yàn)動(dòng)物

昆明（kunming，KM）小鼠購(gòu)自上海斯萊克公司，雌鼠4～6周，雄鼠8周以上，繼續(xù)飼養(yǎng)3～5d調(diào)節(jié)生理周期，使其適應(yīng)環(huán)境供實(shí)驗(yàn)處理。

2 主要試劑和儀器

孕馬血清促性腺激素（PMSG）（寧波激素制藥二廠），人絨毛膜促性腺激素（hCG）（Prospec公司），ERα特異性抑制劑（MPP）（Tocris Bioscience），Nanog兔多克隆抗體（Cell Signaling Technology），cyclin D1鼠單克隆抗體（武漢博士德），cyclin E兔多克隆抗體（Santa Cruz），Alexa Fluor? 488標(biāo)記驢抗鼠二抗、Alexa Fluor? 488標(biāo)記驢抗兔二抗（Life Technology），DAPI（碧云天），倒置顯微鏡（Nikon），水套式二氧化碳培養(yǎng)箱（Thermo）。

3 胚胎的收集和培養(yǎng)

KM雌性小鼠腹腔注射10IU PMSG，46～48h后注射10IU hCG，隨后與KM雄性小鼠1∶1合籠（♀KM×♂KM），次日清晨檢查陰栓判斷是否受精。在注射hCG后27h，從有陰栓雌鼠輸卵管中收集受精卵，經(jīng)M2洗滌，37℃、5% CO2平衡的potassium-supplemented simplex optimized medium (KSOM)漂洗3次，將形態(tài)完好的受精卵移至預(yù)孵育好的培養(yǎng)液滴（其中分別含0μmol/L、20μmol/L的MPP）中，置于37℃、5% CO2飽和濕度的培養(yǎng)箱中連續(xù)培養(yǎng)，每天觀察和記錄植入前胚發(fā)育情況，重復(fù)3次以上，確認(rèn)20μmol/L MPP使1-細(xì)胞胚體外發(fā)育阻滯于2-細(xì)胞期。

4 免疫熒光染色

收集hCG注射后27h的1-細(xì)胞胚，分別置于KSOM和含MPP（20μmol/L）KSOM培養(yǎng)液中培養(yǎng)18h獲取2-細(xì)胞胚，經(jīng)固定、通透、封閉、一抗（Nanog兔多抗1∶800；cyclin D1鼠單抗 1∶400；cyclin E兔多抗1∶3200）4℃孵育過(guò)夜；二抗（Alexa Fluor 488標(biāo)記驢抗兔1∶1000；Alexa Fluor? 488標(biāo)記驢抗鼠1∶1000）孵育、DAPI復(fù)染，倒置熒光顯微鏡觀察、拍照，所得圖片用SmartScape軟件進(jìn)行熒光強(qiáng)度分析。

5 統(tǒng)計(jì)學(xué)處理

利用SmartScape軟件計(jì)算免疫熒光圖片中各組2-細(xì)胞胚內(nèi)熒光灰度值，采用SPSS17.0統(tǒng)計(jì)軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析，檢驗(yàn)方差齊性后運(yùn)用獨(dú)立樣本t檢驗(yàn)進(jìn)行組間比較，均以P＜0.05為差異具有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

圖1 MPP對(duì)2-細(xì)胞胚內(nèi)Nanog影響的免疫熒光檢測(cè)。A，Nanog免疫反應(yīng)性熒光顯微鏡成像；B，Nanog免疫反應(yīng)性統(tǒng)計(jì)學(xué)分析；比例尺，50μm；**，與對(duì)照組比較，P＜0.01。Fig. 1 Immunofuorescent detection of the efect of MPP on Nanog expression in 2-cell embryos. A, fuorescence microscope images of Nanog immunoreactivity; B, statistical analysis of Nanog immunoreactivity; scale bar, 50μm; **, P＜0.01, compared with control

圖2 MPP對(duì)2-細(xì)胞胚內(nèi)cyclin D1影響的免疫熒光檢測(cè)。A，cyclin D1免疫反應(yīng)性熒光顯微鏡成像；B，Nanog免疫反應(yīng)性統(tǒng)計(jì)學(xué)分析；比例尺，50μm；**，與對(duì)照組比較，P＜0.01。Fig. 2 Immunofuorescent detection of the efect of MPP on cyclin D1 expression in 2-cell embryos. A, fuorescence microscope images of cyclin D1 immunoreactivity; B, statistical analysis of cyclin D1 immunoreactivity; scale bar, 50μm; **, P＜0.01, compared with control

圖3 MPP對(duì)2-細(xì)胞胚內(nèi)cyclin E影響的免疫熒光檢測(cè)。A，cyclin E免疫反應(yīng)性熒光顯微鏡成像；B，cyclin E免疫反應(yīng)性統(tǒng)計(jì)學(xué)分析；比例尺，50μm；**，與對(duì)照組比較，P＜0.01。Fig. 3 Immunofuorescent detection of the efect of MPP on cyclin E expression in 2-cell embryos. A, fuorescence microscope images of cyclin E immunoreactivity; B, statistical analysis of cyclin E immunoreactivity; scale bar, 50μm; **, P＜0.01, compared with control

免疫熒光染色觀察顯示，Nanog、cyclin D1和Cyclin E免疫反應(yīng)性在2-細(xì)胞胚的胞質(zhì)和胞核均有分布（圖1A，圖2A，圖3A）。三者在無(wú)MPPKSOM培養(yǎng)的2-細(xì)胞胚內(nèi)核定位更為明顯（圖1A上行，圖2A上行，圖3A上行）；經(jīng)MPP處理后，三者在2-細(xì)胞胚核內(nèi)和細(xì)胞整體的水平均顯著降低（圖1A下行，圖2A下行，圖3A下行，圖1B，圖2B，圖3B）。

討 論

前期研究表明，ERα特異性抑制劑MPP可抑制小鼠合子型標(biāo)志基因的表達(dá)[2]，ERα可能通過(guò)調(diào)節(jié)2-細(xì)胞胚“G1/S期檢測(cè)點(diǎn)”相關(guān)蛋白的功能，影響小鼠2-細(xì)胞胚G1/S期過(guò)渡，進(jìn)而調(diào)控ZGA的發(fā)生，以及后續(xù)的早胚發(fā)育進(jìn)程[4]。Nanog最早被發(fā)現(xiàn)作為轉(zhuǎn)錄因子，對(duì)胚胎干細(xì)胞全能性的維持有重要作用，后期另有大量研究發(fā)現(xiàn)其對(duì)細(xì)胞周期調(diào)控，尤其是G1/S期過(guò)渡亦有重要作用。Nanog表達(dá)水平的改變可影響細(xì)胞周期關(guān)鍵調(diào)節(jié)因子的表達(dá)，從而影響細(xì)胞從G1期到S期的過(guò)渡。有研究發(fā)現(xiàn)，Nanog的C端可結(jié)合于CDK6和CDC25A的調(diào)控區(qū)，從而推動(dòng)人胚胎干細(xì)胞順利由G1期進(jìn)入S期[12]。Nanog可與Oct4、Sox2協(xié)同作用，通過(guò)影響小分子非編碼RNA的表達(dá)，調(diào)控細(xì)胞周期：三者共同結(jié)合于miR-302的啟動(dòng)子區(qū)域，減少G1期細(xì)胞數(shù)，增加S期細(xì)胞數(shù)，促進(jìn)細(xì)胞進(jìn)入S期[13]。Tomonori等人提出CIBZ可通過(guò)PI3K信號(hào)調(diào)控Nanog表達(dá)水平，從而促進(jìn)人胚胎干細(xì)胞G1/S期的進(jìn)程[11]。本研究發(fā)現(xiàn)，經(jīng)ERα特異性抑制劑MPP（20μmol/L）處理后，小鼠植入前胚發(fā)育阻滯在2-細(xì)胞階段， Nanog水平降低，提示ERα可能影響小鼠2-細(xì)胞胚中Nanog表達(dá)。

此外，cyclin D1作為G1/S期特異性周期蛋白，推動(dòng)細(xì)胞周期進(jìn)程，促進(jìn)細(xì)胞的增殖。真核生物細(xì)胞周期調(diào)節(jié)存在“開(kāi)關(guān)”樣的調(diào)控模式，細(xì)胞周期依賴性蛋白激酶（CDKs）作為細(xì)胞周期的“按鈕”持續(xù)存在于細(xì)胞中，其活性受到相應(yīng)細(xì)胞周期蛋白（cyclins）的嚴(yán)格調(diào)控，只有當(dāng)細(xì)胞周期蛋白表達(dá)量到達(dá)一定閾值時(shí)方可激活CDKs，從而推動(dòng)細(xì)胞周期進(jìn)程[14]。Cyclin D1通過(guò)結(jié)合并激活G1期CDK4/6，使G1期的抑制蛋白R(shí)b磷酸化失活，與轉(zhuǎn)錄因子E2F解離，E2F轉(zhuǎn)錄因子啟動(dòng)轉(zhuǎn)錄相關(guān)基因，從而推動(dòng)細(xì)胞周期由G1期進(jìn)入S期[15-17]。本研究顯示，hCG后45h體外正常發(fā)育小鼠2-細(xì)胞胚核增大，大部分呈蠶豆形核象，此時(shí)處于小鼠植入前胚第二輪細(xì)胞周期的G1/S期的關(guān)鍵調(diào)控時(shí)期，核內(nèi)cyclin D1水平正常，從而順利完成CDK4/6激活，使2-細(xì)胞胚順利過(guò)渡到S期、G2期，啟動(dòng)ZGA，克服“2-細(xì)胞阻滯”，完成后續(xù)發(fā)育。而MPP（20μmol/L）處理后，小鼠2-細(xì)胞胚核則呈較小類圓形G0/G1期細(xì)胞核象，cyclin D1明顯減少，2-細(xì)胞胚發(fā)育停滯在第二輪細(xì)胞周期G0/G1期，而無(wú)法完成后續(xù)的植入前胚發(fā)育進(jìn)程。

G1/S期的Start檢驗(yàn)點(diǎn)，是真核生物檢驗(yàn)細(xì)胞是否做好遺傳物質(zhì)復(fù)制的一個(gè)重要檢驗(yàn)點(diǎn)，一旦通過(guò)Start點(diǎn)則標(biāo)志著細(xì)胞周期不可逆地開(kāi)啟[18]。Start檢驗(yàn)點(diǎn)的中心事件是G1/S期周期蛋白cyclin E-CDk2復(fù)合物的激活。動(dòng)物細(xì)胞在Start檢驗(yàn)點(diǎn)進(jìn)入細(xì)胞周期為G1-、G1/S-、S-Cdks復(fù)合物的激活所觸發(fā)，同時(shí)還伴隨E2F依賴的基因表達(dá)激活[19,20]。研究發(fā)現(xiàn)，cyclin E主要在G1末期表達(dá)，在S期表達(dá)量達(dá)高峰，以保證G1/S期的順利過(guò)渡以及S期的維持。Cyclin E-CDk2復(fù)合物參與維持Rb、Cdc6、NPAT以及核仁磷酸蛋白的高磷酸化狀態(tài)，并且在G1/S期磷酸化P27，誘導(dǎo)其降解，促進(jìn)G1/S的過(guò)渡、S期DNA復(fù)制以及細(xì)胞增殖[21,22]。以上研究表明，cyclin E蛋白表達(dá)量的高低對(duì)CDK2的激活，G1-、G1/S-、S-Cdks復(fù)合物的形成，G1期到S期的順利過(guò)渡，以及S期遺傳物質(zhì)復(fù)制的維持等有重要作用。本研究發(fā)現(xiàn)，MPP處理后，小鼠2-細(xì)胞胚cyclin E水平也明顯下降。這些結(jié)果表明，除了Nanog和cyclin D1外，cyclin E也參與ERα在小鼠2-細(xì)胞胚中的調(diào)控作用。

由此可見(jiàn)，ERα可通過(guò)調(diào)控小鼠2-細(xì)胞胚內(nèi)Nanog、cyclin D1和cyclin E蛋白的表達(dá)水平，而影響細(xì)胞從G1期到S期的過(guò)渡。但在肝癌細(xì)胞中，敲低Nanog可導(dǎo)致Cyclin D1水平下調(diào)，阻滯在G0/G1期的細(xì)胞比率增高，而S期與G2期細(xì)胞明顯減少[23]。那么ERα是否通過(guò)影響Nanog的蛋白表達(dá)，從而調(diào)控Cyclin D1、Cyclin E的表達(dá)水平，有待進(jìn)一步研究探討。

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Estrogen receptor α specific antagonist MPP inhibits G1/S transitionrelated factors Nanog, cyclin D1 and cyclin E in mouse 2-cell embryos

Pang Lili1, He Lin2, Song Chanchan1, Lian Xiuli2, Liu Yue1, Xu Songhua2, Wang Shie1,2*(1Cellular and Developmental Engineering Center,2Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350122, China)

ObjectiveTo investigate the efect of estrogen receptor α (ERα) specifc antagonist methyl-piperidino-pyrazole (MPP) on the expression of G1/S transition-related factors Nanog, cyclin D1 and cyclin E in mouse 2-cell embryos, so as to demonstrate the role of ERα in G1/S transition in mouse 2-cell embryos and provide a theoretical basis for further research on the mechanism of the activation of pre-implantation mouse zygotic genome by ERα.MethodsZygotes were collected from Kunming female mice that had been mated with male mice, and cultured in media without MPP and media supplemented with 20mol/L MPP, respectively. The 2-cell embryos were obtained, and the levels of Nanog, cyclin D1 and cyclin E were detected by immunofuorescent staining.ResultsImmunofuorescent staining results showed that the immunoreactivity of Nanog, cyclin D1 and cyclin E in the 2-cell embryos cultured in 20mol/L MPP was obviously lower than in those cultured without MPP treatment.ConclusionERα might afect G1/S transition via regulating the expression of Nanog, cyclin D1 and cyclin E in mouse 2-cell embryos.

Estrogen receptor alpha; pre-implantation embryo; Nanog; cyclin; mouse

R321

A

10.16705/ j. cnki. 1004-1850.2017.02.002

2017-01-17

2017-03-30

國(guó)家自然科學(xué)基金（81170624、81671526）

逄麗麗，女（1980年），漢族，實(shí)驗(yàn)師

*通訊作者(To whom correspondence should be addressed)：shineww@163.com