李紅艷

(西藏民族學(xué)院醫(yī)學(xué)院; 高原環(huán)境與疾病相關(guān)基因研究實(shí)驗(yàn)室， 咸陽(yáng) 712082)

核糖體蛋白的主要作用在于參與核糖體聚集和蛋白質(zhì)合成. 另外在細(xì)胞周期[1]、凋亡[2-5]和DNA損傷[6]過(guò)程中一些核糖體蛋白也發(fā)揮著一定作用，與核糖體組分作用無(wú)關(guān)[7]. 研究發(fā)現(xiàn)人類某些疾病的發(fā)生與一些核糖體蛋白表達(dá)異常密切相關(guān)，如Diamond-Blackfan貧血癥[8]，特納綜合征[9]，喪失聽(tīng)力[10]和癌癥[11].S6是真核細(xì)胞中核糖體小亞基中一個(gè)主要的磷酸化蛋白. 在果蠅中利用P轉(zhuǎn)座子插入突變法建立的S6突變系，果蠅致死. 主要原因是引起了果蠅造血細(xì)胞中核的過(guò)度復(fù)制，導(dǎo)致組織過(guò)度生長(zhǎng)所致[12]. 但有關(guān)S6蛋白在果蠅發(fā)育中的具體作用尚不清楚. 本實(shí)驗(yàn)利用RNAi技術(shù)分別在果蠅和果蠅S2細(xì)胞中干擾S6基因表達(dá)，探討S6蛋白對(duì)果蠅發(fā)育的影響及可能機(jī)制. 以期為深入了解S6蛋白在果蠅發(fā)育中的作用提供更多的實(shí)驗(yàn)基礎(chǔ)和理論依據(jù).

1 方法和材料

1.1 S6RNAi轉(zhuǎn)基因果蠅的獲得和培養(yǎng)

以果蠅S6cDNA編碼區(qū)為模板進(jìn)行PCR擴(kuò)增(上游引物：5-caagcggccgctcggtgacgagtggaagg-3；下游引物：5-ccggaattcgcgaatggaggcagaacg-3). 將PCR產(chǎn)物克隆到SympUAST-w載體上，獲得S6-SympUAST-w質(zhì)粒. 將S6-SympUAST-w質(zhì)粒通過(guò)顯微注射法注射到果蠅W1118胚胎中，經(jīng)果蠅篩選和分子鑒定獲取S6RNAi轉(zhuǎn)基因果蠅. 實(shí)驗(yàn)中用到的其他果蠅品系包括act5c-Gal4、Hsp70-Gal4、ey-Gal4、sca-gal4和vg-Gal4. 除特殊說(shuō)明，果蠅培養(yǎng)在18 ℃或25 ℃玉米粉-蔗糖培養(yǎng)基上.

1.2 果蠅體長(zhǎng)測(cè)量

S6RNAi雄蠅與Hsp70-Gal4雌蠅進(jìn)行雜交，在培養(yǎng)基上間隔1 h產(chǎn)卵. 產(chǎn)卵后24 h，將幼蟲在37 ℃熱激30 min. 熱激后間隔24 h隨機(jī)收集幼蟲，每組10只，直到對(duì)照組發(fā)育至蛹期為止. 用帶有CCD攝像頭(萊卡)的MZ16體視顯微鏡對(duì)收集到的幼蟲拍照. 通過(guò)MetaMorph軟件分析圖片，測(cè)量幼蟲體長(zhǎng).

1.3 細(xì)胞凋亡檢測(cè)

在PBS緩沖液中解剖S6RNAi/vg-Gal4三期幼蟲翅膀disc，解剖好的disc立即放到4 ℃果蠅培養(yǎng)基(Gibco)中，使其維持正常的生理環(huán)境. 再將其轉(zhuǎn)移到一個(gè)加有50 μL培養(yǎng)基的載玻片上，用100 μL的培養(yǎng)基清洗1次. 以質(zhì)量濃度為1 μg/mL的吖啶橙(Sigma)室溫染色5 min. 染色后直接用熒光顯微鏡觀察拍照.

1.4 體外轉(zhuǎn)錄及細(xì)胞轉(zhuǎn)染

通過(guò)PCR擴(kuò)增果蠅S6編碼序列(上游引物[含有一個(gè)T7啟動(dòng)子]：5-taatacgactcactatagggagaatgaagctcaacgtttcctat-3；下游引物：5-taatacgactcactatagggagattacttcttgtcgctggagac-3).PCR產(chǎn)物經(jīng)高純度PCR純化試劑盒純化(Roche Molecular Biochemicals). 以純化的產(chǎn)物為模板，用MEGAscript T7體外轉(zhuǎn)錄試劑盒(Ambion)合成單鏈RNAs(ssRNAs). ssRNAs在95 ℃/5 min，65 ℃中退火30 min，然后自然降溫至室溫，即生成雙鏈RNAs(dsRNAs). dsRNAs經(jīng)乙醇沉淀后重懸在合適體積的無(wú)RNase水中. 通過(guò)瓊脂糖凝膠電泳對(duì)dsRNAs進(jìn)行定量，保存在-80 ℃?zhèn)溆?

S2細(xì)胞系(果蠅S2胚胎細(xì)胞)培養(yǎng)在Schneider基本培養(yǎng)基(Gibco)中，補(bǔ)加10%胎牛血清(Gibco)、160 μg/mL青霉素、250 μg/mL鏈霉素和4 mmol/L L-谷氨酸(Sigma). 25 ℃培養(yǎng)在75 cm 的盤里(Corning). 細(xì)胞以1∶10的比例每3天傳1代. 在S2細(xì)胞中對(duì)S6基因進(jìn)行干擾：細(xì)胞以1×106個(gè)/mL的密度培養(yǎng)在35 cm大小培養(yǎng)盤的無(wú)血清培養(yǎng)基里.S6dsRNAs (5 μg/mL)加到1 mL無(wú)血清培養(yǎng)基中，室溫培養(yǎng)1 h. 1 h后，補(bǔ)加2 mL含有10%胎牛血清的正常培養(yǎng)基繼續(xù)培養(yǎng). 分別在處理后的第4和第8天收集經(jīng)過(guò)S6RNAi的細(xì)胞，用作實(shí)驗(yàn)分析. 在相同條件下，GFPdsRNA用作對(duì)照.

1.5 基因表達(dá)檢測(cè)

根據(jù)RNeasy Mini Kit(Qiagen)試劑盒說(shuō)明提取第8天S6RNAi組和GFPRNAi組的細(xì)胞總RNA. 在進(jìn)行RT-PCR之前，提取的RNAs用DNase (TaKaRa)進(jìn)行處理. 純化的RNA用作第一鏈cDNA合成. 用M-MLV反轉(zhuǎn)錄酶(Invitrogen)和oligo-dT引物合成cDNA. 在25 μL反應(yīng)體系中，每個(gè)樣品用1 μL cDNA做模板. PCR反應(yīng)在相同條件下進(jìn)行：95 ℃預(yù)變性5 min；94 ℃，30 s，60 ℃，30 s，72 ℃，45 s，30個(gè)循環(huán)；最后72 ℃延伸8 min. 用到的引物見(jiàn)表1.

表1 RT-PCR中用到的引物Table 1 The primers for RT-PCR

1.6 統(tǒng)計(jì)分析

所有實(shí)驗(yàn)至少重復(fù)3次，通過(guò)Excel軟件對(duì)結(jié)果分析. 數(shù)值以平均值±標(biāo)準(zhǔn)差表示. 顯著性分析用t檢驗(yàn)，P<0.05表示差異顯著.

2 結(jié)果與分析

2.1 RNAi干擾S6果蠅發(fā)育延緩

果蠅的發(fā)育經(jīng)歷4個(gè)時(shí)期：卵、幼蟲(一期、二期和三期幼蟲)、蛹和成蠅. 實(shí)驗(yàn)為了實(shí)現(xiàn)S6基因在果蠅周身及局部器官刪除，分別用了act5c-Gal4和Hsp70-Gal4驅(qū)動(dòng)子驅(qū)動(dòng)S6基因的干擾. 結(jié)果對(duì)照組發(fā)育正常，而S6RNAi/act5c-Gal4果蠅都死在胚胎期.S6RNAi/Hsp70-Gal4從一期到三期幼蟲發(fā)育延遲，在幼蟲期維持10～12 d，體積比較小，生命力不旺盛，死在蛹期之前. 而對(duì)照組在幼蟲期維持6～7 d，正常發(fā)育到蛹期和成蠅，完成一個(gè)正常的生命周期. 在48 h初，S6RNAi/Hsp70-Gal4體長(zhǎng)與對(duì)照組相比沒(méi)有明顯變化；72 h后，比對(duì)照組果蠅發(fā)育顯著延遲(P<0.05)(圖1A，1B).表明S6蛋白對(duì)果蠅早期發(fā)育不可缺少.

圖1 減少S6蛋白表達(dá)阻滯果蠅幼蟲發(fā)育

Figure 1 Decreased expression of S6 protein caused theDrosophilalarvae growth arrest

2.2 RNAi干擾S6果蠅的眼睛、翅膀和剛毛發(fā)育缺陷

與對(duì)照組果蠅比較，用ey-gal4、vg-gal4和sca-gal4驅(qū)動(dòng)子分別在果蠅眼睛、翅膀和剛毛中刪除S6蛋白表達(dá). 果蠅S6RNAi/ey-gal4眼睛明顯變小(圖2A).S6RNAi/vg-gal4翅膀皺縮(圖2B). 另外果蠅S6RNAi/sca-gal4剛毛變短(圖2C). 可見(jiàn)S6蛋白的正常表達(dá)與果蠅器官發(fā)育緊密相關(guān).

2.3 RNAi干擾S6果蠅細(xì)胞增殖與凋亡

果蠅S2細(xì)胞在轉(zhuǎn)染了S6dsRNAs后，與對(duì)照組比(轉(zhuǎn)染了GFPdsRNAs的)，其細(xì)胞生長(zhǎng)受到阻滯、數(shù)目明顯減少(圖3B). 通過(guò)吖啶橙染色發(fā)現(xiàn)，在果蠅S6RNAi/vg-gal4三期幼蟲翅膀disc中有明顯凋亡信號(hào). 暗示S6刪除后導(dǎo)致的果蠅表型變化是由細(xì)胞凋亡引起的(圖3A).

RT-PCR分析表明：果蠅刪除S6基因后，細(xì)胞凋亡相關(guān)基因p53，hid，reaper和dcp-1轉(zhuǎn)錄水平升高.Bcl-2(細(xì)胞凋亡抑制因子)轉(zhuǎn)錄水平降低(圖4A). 細(xì)胞周期相關(guān)基因cdc45，cyclinE，MCM5，MCM3，incenp和cyclinB在S6基因干擾后轉(zhuǎn)錄水平也受到了影響(圖4B).

圖2 減少S6蛋白表達(dá)對(duì)果蠅眼睛(A)、翅膀(B)和剛毛表型(C)的影響

Figure 2 The effect of S6 protein reduction on the eye(A), wing(B) and bristle phenotype(C) in flies

圖3 RNAi干擾S6果蠅翅膀disc(A)和S2細(xì)胞數(shù)目(B)

Figure 3 Wing discs of flies(A) and cell mumber of S2 cells(B) in RNAiS6 Drosophila

(A)S6RNAi后，p53、hid、Dcp-1，Bcl-2和reaper轉(zhuǎn)錄水平的變化；(B)S6RNAi后，MCM5、MCM3、cdc45、cyclinB、cyclinE和incenp轉(zhuǎn)錄水平的變化

圖4 S2細(xì)胞中減少S6表達(dá)對(duì)細(xì)胞凋亡和細(xì)胞周期相關(guān)基因表達(dá)的影響

Figure 4 The effect ofS6-depletion in S2 cells on the expression of the genes related to apoptosis and cell cycle

3 討論

為了探討S6基因在果蠅生長(zhǎng)發(fā)育中的作用，本文用了不同的驅(qū)動(dòng)子使S6基因在果蠅中表達(dá)減少. 由表型分析可見(jiàn)：S6蛋白對(duì)于果蠅正常發(fā)育是必要的，其表達(dá)減少會(huì)引起細(xì)胞增殖受抑和凋亡的發(fā)生. 細(xì)胞增殖與細(xì)胞凋亡之間的平衡決定了細(xì)胞的生長(zhǎng)狀態(tài)[13]. 干擾S6基因表達(dá)引起：細(xì)胞數(shù)目減少、果蠅翅膀disc中有細(xì)胞凋亡以及細(xì)胞周期與凋亡相關(guān)基因的異常表達(dá)，這些結(jié)果一致暗示：S6基因干擾后引起果蠅表型異常是由細(xì)胞凋亡引起的.

P53在細(xì)胞凋亡中主要通過(guò)使線粒體釋放細(xì)胞色素C[14-15]，引起caspase活性升高，最終啟動(dòng)細(xì)胞凋亡[9]. 果蠅中2個(gè)重要凋亡誘導(dǎo)因子hid和reaper[16-17]可直接或間接激活p53,以誘導(dǎo)凋亡發(fā)生[18]. 另外這2個(gè)基因通過(guò)caspase引起凋亡[19-20]. Dcp-1是第一個(gè)被發(fā)現(xiàn)的果蠅ICE/CED-3蛋白激酶caspase家族成員，也在凋亡中有著重要作用[21]. Bcl-2是凋亡的一個(gè)抑制因子[22-24]. 減少Bcl-2表達(dá)可以通過(guò)釋放細(xì)胞色素c激活Dcp-1[15]. 在S6干擾后hid，reaper，Dcp-1和p53轉(zhuǎn)錄水平升高，而Bcl-2轉(zhuǎn)錄水平降低.

細(xì)胞凋亡與細(xì)胞周期密切相關(guān). 過(guò)表達(dá)cyclinE激活caspase誘導(dǎo)造血細(xì)胞凋亡[25]. MCM3和MCM家族的其他成員在基因復(fù)制中有著重要作用[26]. 在細(xì)胞周期中，p53負(fù)調(diào)控MCM5[27]. 而cdc45與MCM5/cdc46相互作用，共同調(diào)控細(xì)胞周期[28]. 另外，cyclin B表達(dá)水平降低會(huì)導(dǎo)致細(xì)胞周期阻滯在G2期，并且是一個(gè)p53依賴的負(fù)調(diào)控過(guò)程[29]. Incenp與Aurora B和survivin相互作用，形成復(fù)合物：Aurora B/incenp/survivin，在染色體分離中起作用[30-32]. 研究發(fā)現(xiàn)，survivin表達(dá)水平升高減少p53表達(dá)，因?yàn)橥ㄟ^(guò)caspases和Survivin調(diào)控了Mdm2分裂，增加了p53降解[33]. 本研究結(jié)果證實(shí)在S6RNAi后MCM5、MCM3、cdc45、cyclinB和incenp表達(dá)減少，而cyclinE和p53增加.

綜上所述，S6干擾后主要是通過(guò)細(xì)胞凋亡和細(xì)胞周期阻滯的方式引起果蠅表型異常的，這將為更深入研究S6的生物學(xué)功能提供理論依據(jù)，同時(shí)也為果蠅的發(fā)育研究提供新的思路.

致謝 感謝清華大學(xué)孫方霖教授以及陳蘇同學(xué)在實(shí)驗(yàn)中的幫助.

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