文 勝,劉錦輝,張永勤,肖亞梅
(湖南師范大學(xué)生命科學(xué)學(xué)院,教育部蛋白質(zhì)化學(xué)和魚類發(fā)育生物學(xué)重點(diǎn)實(shí)驗(yàn)室,中國長沙 410081)
色素細(xì)胞是動物體內(nèi)含有生物色素的一類特化細(xì)胞,通過其色素系統(tǒng)選擇性地吸收特定波長的光而產(chǎn)生顏色,從而形成動物體的體色.體色在保護(hù)個體免受天敵或紫外線傷害、信息交流和生理調(diào)節(jié)等方面有著重要生物學(xué)作用.本文概述了動物色素細(xì)胞譜系來源和組成,色素干細(xì)胞分化發(fā)育的分子調(diào)控相關(guān)研究進(jìn)展.
根據(jù)所含色素的不同,動物色素細(xì)胞主要有黑色素細(xì)胞、黃色素細(xì)胞、紅色素細(xì)胞、虹彩細(xì)胞等4 種基本類型(圖1,彩圖見封三).黑色素細(xì)胞(melanophore)內(nèi)含大量由酪氨酸、多巴胺等氧化聚合在體內(nèi)合成的黑色素,能夠吸收特定波長的入射光而使身體呈現(xiàn)黑色.紅色素細(xì)胞(erythrophore)和黃色素細(xì)胞(xanthophore)形態(tài)非常相似,都含有胡蘿卜素與蝶啶的色素體.黃色素細(xì)胞和紅色素細(xì)胞與動物的黃、橙、紅體色有關(guān),一般認(rèn)為黃色素細(xì)胞擁有大量帶黃色的蝶酸色素,而紅色素細(xì)胞則含有較多呈紅色和橙色的類胡蘿卜素.虹彩細(xì)胞(iridophore)也稱為鳥糞素細(xì)胞(guanophore)或白色素細(xì)胞(leucophore),其呈色物質(zhì)主要是與水結(jié)合成晶體形式的鳥嘌呤,通過反射特定波長的光表現(xiàn)為白色、藍(lán)色和紫紅3 種色彩結(jié)晶體.上述幾種色素細(xì)胞類型的數(shù)量和分布、反光結(jié)晶體的能力強(qiáng)弱等,決定了動物體的顏色和斑紋[1-4].黑色素細(xì)胞是動物體內(nèi)存在最廣泛且研究得最多的色素細(xì)胞類型,而其他幾種非黑色素細(xì)胞則主要在體色鮮艷的低等脊椎動物,如魚類、兩棲類中較為常見.也正是由于與高等羊膜動物在色素細(xì)胞類型方面的差異,色彩斑斕的魚類已成為色素細(xì)胞譜系研究的重要材料.
圖1 魚類4 種色素細(xì)胞的顯微觀察(A:紅色素細(xì)胞(E)和虹彩細(xì)胞(I);B:黑色素細(xì)胞(M)和黃色素細(xì)胞(X))Fig.1 Four types of pigment cells in fish(A:Erythrocyte(E)and Iridocyte(I);B:Melanocyte(M)and Xanthophore(X))
對于色素細(xì)胞譜系的研究,主要集中于探究色素細(xì)胞是怎樣起源、遷移和分化,最后形成一定形狀的體紋.人們想知道色素細(xì)胞是怎樣按照特定的時序到達(dá)特定的部位形成特定的器官,這涉及到發(fā)育生物學(xué)的根本問題.神經(jīng)嵴則是胚胎發(fā)育中短暫出現(xiàn)的過渡性結(jié)構(gòu),是由背部外胚層分化的位于神經(jīng)管和表皮之間的細(xì)胞帶.色素細(xì)胞的前體細(xì)胞稱為色素胚或色素母細(xì)胞(chromatoblast),是在胚胎發(fā)生時期,由神經(jīng)嵴細(xì)胞發(fā)展而來的.色素母細(xì)胞具有分化為成黑色素細(xì)胞(melanoblast)、成黃色素細(xì)胞(xanthoblast)和成虹彩細(xì)胞(iridoblast)等色素干細(xì)胞的發(fā)育潛能.在斑馬魚中,黑色素細(xì)胞譜系的特化約在受精后24 h 就開始發(fā)生;孵化出膜后第3 天的斑馬魚胚胎中,就已經(jīng)觀察到黑色素細(xì)胞、黃色素細(xì)胞與虹彩細(xì)胞;出膜2~4 周后,幼體黑色素細(xì)胞由成體黑色素細(xì)胞取代[5].斑馬魚胚胎發(fā)育期出現(xiàn)的黑色素細(xì)胞一般認(rèn)為都是由神經(jīng)嵴細(xì)胞直接分化形成,出膜后的黑色素細(xì)胞則是由黑色素干細(xì)胞(melanocyte stem cell)發(fā)育而來[6].
色素細(xì)胞的數(shù)目和組成直接由色素干細(xì)胞控制.色素干細(xì)胞的最早出現(xiàn)時間是從神經(jīng)嵴發(fā)生色素細(xì)胞譜系的特化開始,隨后它們發(fā)生分化、增殖、遷移,最終定位于表皮基底膜之下或者毛發(fā)著生的毛囊龕中[6].成體色素細(xì)胞的更新依賴于真皮干細(xì)胞.早在1954年,Goodrich 等就發(fā)現(xiàn),在魚類黃色素細(xì)胞局部受損的區(qū)域,原本并不存在的黑色素細(xì)胞在此出現(xiàn),這也就暗示了受損組織中有色素干細(xì)胞存在[7].但由于缺乏對黑色素細(xì)胞的標(biāo)記方法,未能區(qū)分是原本存在的黑色素細(xì)胞的遷移還是非色素化的前體細(xì)胞的分化導(dǎo)致新的黑色素細(xì)胞的產(chǎn)生.在斑馬魚中已經(jīng)證實(shí),大多數(shù)再生鰭條的黑色素細(xì)胞來源于非色素化的前體細(xì)胞[8].人們從小鼠和人類均分離得到了皮膚干細(xì)胞,并在體外進(jìn)行培養(yǎng)[9-10].其中Li 等人獲得的人類真皮干細(xì)胞不僅能夠表達(dá)神經(jīng)嵴的標(biāo)簽基因NGFRp75 和nestin,而且還能表達(dá)在胚胎干細(xì)胞中高表達(dá)的OCT4;這些真皮干細(xì)胞雖然不表達(dá)黑色素細(xì)胞的標(biāo)簽基因,但卻具有分化產(chǎn)生有功能的黑色素細(xì)胞的潛能,這與人類胚胎干細(xì)胞向黑色素干細(xì)胞的分化條件是相同的[10].目前很多研究已經(jīng)證實(shí),在動物的真皮、毛囊中存在黑色素干細(xì)胞(melanocyte stem cell,MSC)[11-12].Lin 等2013年首次在鳥類皮膚下方的圓筒形羽囊底部精確找到黑色素干細(xì)胞,并成功揭示出色素干細(xì)胞導(dǎo)致鳥類羽毛擁有獨(dú)特而又復(fù)雜的黑白圖案并隨著個體的生長保持動態(tài)平衡[13].
人類膚色和發(fā)色主要是由兩種黑色素(黑色或褐色的真黑素Eumelanin 和紅色的棕黑素Pheomelanin)的含量不同所導(dǎo)致的.基于臨床研究的需要,目前對黑色素干細(xì)胞的研究較為深入,而有關(guān)黃色素干細(xì)胞等幾種非黑色素干細(xì)胞的研究資料則十分有限.研究顯示,斑馬魚胚胎成黑色素細(xì)胞與成虹彩細(xì)胞的標(biāo)簽基因有顯著性的重疊,但是與成黃色素細(xì)胞的不同.該結(jié)果表明黑色素細(xì)胞與虹彩細(xì)胞可能來源于共同的前體細(xì)胞,而黃色素細(xì)胞則可能來自于不同的發(fā)育路徑[14].同時Lister 等人也證實(shí),在斑馬魚突變體中,成黑色素細(xì)胞能夠改變原來的細(xì)胞譜系命運(yùn)轉(zhuǎn)變成為成虹彩細(xì)胞[15].然而,利用譜系特異的轉(zhuǎn)座子對斑馬魚早期胚胎進(jìn)行標(biāo)記發(fā)現(xiàn),來自于真皮干細(xì)胞的mFSCs(the same melanocyte-producing founding stem cells)具有分化為黑色素干細(xì)胞的發(fā)育潛能,同時成體黃色素細(xì)胞可能和黑色素細(xì)胞一樣來自于相同的前體干細(xì)胞mFSCs;而虹彩細(xì)胞則可能由另一種前體細(xì)胞iFSCs(iridophores arise from a distinct founding stem cell)產(chǎn)生(如圖2所示)[8].目前對于色素干細(xì)胞的分化發(fā)育途徑還沒有一致性的結(jié)論.
圖2 成體色素細(xì)胞發(fā)育路徑Fig.2 Development of pigment cells in adult animal
神經(jīng)嵴細(xì)胞分化產(chǎn)生色素細(xì)胞以及成體體色模式的發(fā)育和維持,涉及到非常嚴(yán)格而復(fù)雜的分子調(diào)控.在大規(guī)模ENU 誘導(dǎo)突變和篩選過程中,發(fā)現(xiàn)了大量的體色突變斑馬魚品系,這些突變體正在成為研究人類體色多樣性和體色失常的強(qiáng)有力工具.這些突變的基因,功能涉及黑色素細(xì)胞的早期決定(如sox10,mitf)、遷移(如kit)、黑色素的合成(如tyr,dct)等等[14,16-18].
Mitf(microphthalmia transcription factor)基因是黑色素細(xì)胞譜系中已知最早的特異性的標(biāo)簽基因[19-20].在哺乳動物中MITF 至少有6 個異構(gòu)體[21],其中MitfA,MitfD,MitfH 是視網(wǎng)膜色素上皮(retinal pigment epithelium,RPE)發(fā)育必須的,而MitfM 只在黑色素細(xì)胞發(fā)育過程中起作用[22-23].人類mitf 基因突變可以導(dǎo)致Waardenburg 綜合癥,引起皮膚黑色素細(xì)胞缺失和皮膚斑駁樣色素減退、聽覺神經(jīng)性耳聾[24].mitfa 和mitfb是魚類中分離到的mitf 基因的兩個亞型[15,25].青鳉中也獲得了mitfa 和mitfb 的同源基因[26].mitfa 基因?qū)︳~類體色的黑色素細(xì)胞發(fā)育起著重要調(diào)控作用,但不影響眼色素的發(fā)育;而mitfb 基因則只參與眼色素上皮細(xì)胞的發(fā)育[25-27].
MITF 在生物體內(nèi)能調(diào)控黑色素合成關(guān)鍵限制酶——酪氨酸酶基因(tyr)的表達(dá),影響黑色素細(xì)胞的分化[28].在mitfavc7/ BRAFV600E 斑馬魚的黑色素瘤模型中,通過溫敏控制MITF 表達(dá)水平,發(fā)現(xiàn)低劑量MITF能誘導(dǎo)黑色素瘤;若完全抑制MITF 表達(dá),則導(dǎo)致黑色素瘤的消退[29].MITF 通過影響tbx2、cdk2、p16 和p21等細(xì)胞周期調(diào)控基因,控制黑色素細(xì)胞的生長[22,30-31].mitf 基因的表達(dá)受到CREB、SOX10 和PAX3 等信號通路的調(diào)控(圖3),SOX10 通過結(jié)合到mitf 的調(diào)節(jié)序列中,激活mitf 的轉(zhuǎn)錄,是黑素瘤細(xì)胞維持正常發(fā)育和生存潛勢的主要調(diào)控基因[32].研究表明,Ednrb 信號通路對神經(jīng)嵴來源的黑色素前體細(xì)胞的增殖、存活以及黑色素細(xì)胞的分化是必須的,G 蛋白偶聯(lián)Ednrd 受體及其配體Et3 與胚胎色素細(xì)胞及幼體色素細(xì)胞向成體色素細(xì)胞的轉(zhuǎn)變有關(guān)[33-34].
圖3 mitf 基因?qū)谏丶?xì)胞發(fā)育的調(diào)控機(jī)制Fig.3 The mechanism of mitf gene in the development of melanocyte
黃色素細(xì)胞譜系和虹彩細(xì)胞譜系的標(biāo)簽基因已經(jīng)成功獲得了鑒定.Odenthal 在斑馬魚中篩選了多個與黃色素細(xì)胞的形成和遷徙相關(guān)的基因[35].除了較早所知的成黃色素細(xì)胞的標(biāo)簽基因csf1(colony stimulating factor-1)[36],人們又發(fā)現(xiàn)pax 家族中的pax7 在黃色素細(xì)胞譜系中特異性表達(dá),認(rèn)為pax3 與pax7 共同調(diào)控由神經(jīng)嵴向黃色細(xì)胞的發(fā)育[37].Lopes 報道了一個虹彩細(xì)胞缺失的斑馬魚突變體Shady(shd),經(jīng)鑒定,shd 的白細(xì)胞酪氨酸激酶Ltk(leukocyte tyrosine kinase)同源基因發(fā)生了突變[38].ltk 基因在成黑色素細(xì)胞和成黃色素細(xì)胞中不表達(dá),可以作為虹彩細(xì)胞的前體細(xì)胞的基因標(biāo)簽.此外,foxd3 可能作為黑色素細(xì)胞譜系與虹彩細(xì)胞譜系之間的轉(zhuǎn)換開關(guān),與mitf 基因共同參與了對黑色素細(xì)胞譜系和虹彩細(xì)胞譜系的分化發(fā)育調(diào)控[14].
目前,對色素細(xì)胞譜系,尤其是黑色素細(xì)胞譜系的定位、分化發(fā)育及其相關(guān)基因調(diào)控機(jī)制已經(jīng)有了較深入的了解.魚類及其他含有多種色素細(xì)胞類型的低等脊椎動物的色素表型較羊膜動物復(fù)雜,涉及3 種或更多色素細(xì)胞的有序組合,紅/黃色素細(xì)胞譜系、虹彩細(xì)胞譜系的分化調(diào)控及其與黑色素細(xì)胞譜系的發(fā)育關(guān)聯(lián)尚待進(jìn)一步研究.
Ohta 等利用Yamanaka 誘導(dǎo)體系的SOX2、OCT3/4、KLF4 和c-MYC,成功將人皮膚成纖維細(xì)胞誘導(dǎo)為iPS細(xì)胞.同時通過添加Wnt3a,SCF 和ET-3 對獲得的iPS 細(xì)胞進(jìn)一步誘導(dǎo)分化后,檢測到TYR、TYRP1 等黑色素細(xì)胞標(biāo)記分子的表達(dá),并且觀察到培養(yǎng)細(xì)胞中黑色素體的存在,表明從iPS 細(xì)胞誘導(dǎo)產(chǎn)生了黑色素細(xì)胞[39].通過體外培養(yǎng)iPS 細(xì)胞產(chǎn)生色素細(xì)胞這一體外誘導(dǎo)分化體系的建立,一方面為再生組織或器官提供臨床材料來源,另一方面可以作為研究色素細(xì)胞分化發(fā)育機(jī)制的體外模型.
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