劉勝兵，　潘魏巍，　沈忠飛，　徐　營(yíng)，　郭燕君，　王志堅(jiān)，　黃　倩

(嘉興學(xué)院醫(yī)學(xué)院，浙江 嘉興 314001)

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Daxx 抑制K562細(xì)胞向巨核細(xì)胞分化*

劉勝兵△，潘魏巍，沈忠飛，徐營(yíng)，郭燕君，王志堅(jiān)，黃倩

(嘉興學(xué)院醫(yī)學(xué)院，浙江 嘉興 314001)

[摘要]目的： 觀察過(guò)表達(dá)死亡結(jié)構(gòu)域相關(guān)蛋白(Daxx)對(duì)K562細(xì)胞活力和向巨核細(xì)胞分化的影響。方法: 建立穩(wěn)定過(guò)表達(dá)Daxx的K562細(xì)胞，熒光顯微鏡觀察、實(shí)時(shí)熒光定量PCR和Western blot檢測(cè)Daxx的過(guò)表達(dá)效果，CCK-8法檢測(cè)過(guò)表達(dá)后細(xì)胞活力的變化；佛波酯(PMA)誘導(dǎo)K562細(xì)胞向巨核細(xì)胞系分化， Western blot檢測(cè)在K562細(xì)胞向巨核細(xì)胞分化過(guò)程中Daxx和p-ERK的表達(dá)變化,流式細(xì)胞術(shù)檢測(cè)CD41和CD61的表達(dá)變化；PMA處理過(guò)表達(dá)Daxx的K562細(xì)胞，NBT還原實(shí)驗(yàn)檢測(cè)細(xì)胞分化情況，流式細(xì)胞術(shù)檢測(cè)過(guò)表達(dá)Daxx后CD41和CD61的表達(dá)變化，Western blot檢測(cè)p-ERK的蛋白水平。結(jié)果: 建立了穩(wěn)定的過(guò)表達(dá)Daxx的K562細(xì)胞，過(guò)表達(dá)Daxx抑制K562細(xì)胞的活力。PMA誘導(dǎo)K562細(xì)胞向巨核細(xì)胞分化，CD41和CD61表達(dá)水平增高，同時(shí)p-ERK的蛋白水平升高，Daxx表達(dá)水平下降。過(guò)表達(dá)Daxx可以抑制K562細(xì)胞向巨核細(xì)胞分化，CD41和CD61表達(dá)降低，同時(shí)p-ERK的蛋白水平降低。結(jié)論: 過(guò)表達(dá)Daxx可以抑制K562細(xì)胞生長(zhǎng)及向巨核細(xì)胞分化，同時(shí)抑制ERK的磷酸化。

[關(guān)鍵詞]死亡結(jié)構(gòu)域相關(guān)蛋白； K562細(xì)胞； 巨核細(xì)胞分化； 佛波酯

白血病是嚴(yán)重危害人類生命健康的血液系統(tǒng)惡性腫瘤，其生物學(xué)特征表現(xiàn)為細(xì)胞增殖失控、分化成熟受阻、正常凋亡過(guò)程發(fā)生障礙，因而導(dǎo)致分化異常細(xì)胞大量增殖。抑制白血病細(xì)胞增殖、促進(jìn)白血病細(xì)胞分化和凋亡，是治療白血病的基本途徑。死亡結(jié)構(gòu)域相關(guān)蛋白(death domain-associated protein，Daxx)是一種受體胞漿死亡結(jié)構(gòu)域結(jié)合蛋白，在凋亡、轉(zhuǎn)錄調(diào)控、病毒感染等方面發(fā)揮重要作用。Daxx 可以通過(guò)結(jié)合DNA結(jié)合轉(zhuǎn)錄因子、E2A、Ets-1、 Pax3和 Pax5等參與基因表達(dá)的調(diào)控[1-2]，Pax和 Ets轉(zhuǎn)錄因子均和多種類型細(xì)胞的分化活動(dòng)有關(guān)，Daxx可能通過(guò)影響轉(zhuǎn)錄因子來(lái)調(diào)控細(xì)胞分化。Daxx通過(guò)結(jié)合轉(zhuǎn)錄因子E2A，抑制E2A的功能，進(jìn)而抑制肌細(xì)胞生成，參與肌細(xì)胞分化過(guò)程[3]。Daxx基因在白血病細(xì)胞中廣泛表達(dá)，人急性白血病(acute leukemia，AL)、慢性淋巴細(xì)胞白血病(chronic lymphocytic leukemia，CLL)及其它惡性血液病細(xì)胞內(nèi)均可見(jiàn)Daxx表達(dá)[4]。而Daxx在這些血液病中的具體作用機(jī)制，目前研究不多。Daxx在白血病細(xì)胞中的廣泛表達(dá)，是否與白血病細(xì)胞的分化有關(guān)，目前未見(jiàn)相關(guān)報(bào)道。本實(shí)驗(yàn)以K562細(xì)胞為研究對(duì)象，初步研究Daxx與K562細(xì)胞向巨核細(xì)胞分化是否存在相關(guān)性。

材料和方法

1主要試劑

K562細(xì)胞株由嘉興學(xué)院醫(yī)學(xué)院提供；DMEM培養(yǎng)液和新生牛血清購(gòu)于HyClone；Daxx購(gòu)于Sigma；G418購(gòu)自Amresco；兔抗CD41和CD61購(gòu)自BD；辣根過(guò)氧化物酶標(biāo)記山羊抗兔IgG和硝基四氮唑藍(lán)(nitroblue tetrazolium, NBT)購(gòu)自杭州昊鑫生物科技有限公司；RNA逆轉(zhuǎn)錄試劑盒和熒光定量PCR試劑盒購(gòu)自東洋紡生物科技有限公司；蛋白marker 購(gòu)自Thermo；Lipofectamine 2000購(gòu)自Invitrogen。

2主要方法

2.1細(xì)胞培養(yǎng)K562細(xì)胞株由本室保存，常規(guī)傳代培養(yǎng)。采用含有10%胎牛血清的DMEM 培養(yǎng)基培養(yǎng)。

2.2穩(wěn)定過(guò)表達(dá)Daxx的K562細(xì)胞系(Daxx-K562)的建立取對(duì)數(shù)生長(zhǎng)期K562細(xì)胞接種于6孔板中，每孔用50 μL無(wú)血清 DMEM培養(yǎng)基稀釋1 μg GFP-Daxx質(zhì)粒和GFP-空載質(zhì)粒(GFP-Daxx質(zhì)粒和GFP-空載質(zhì)粒由嘉興學(xué)院潘巍巍博士提供);采用50 μL無(wú)血清 DMEN培養(yǎng)基稀釋1 μL Lipofectamine 2000，混勻靜置5 min；將質(zhì)粒與Lipofectamine 2000混合后室溫靜置20 min，分別接種于培養(yǎng)板。置于37℃、5%CO2培養(yǎng)箱中48 h，熒光顯微鏡觀察表達(dá)情況。換液，加G418后繼續(xù)培養(yǎng)。G418篩選2周后，有限稀釋法獲得單個(gè)熒光表達(dá)細(xì)胞，繼續(xù)培養(yǎng)，建立GFP-Daxx-K562細(xì)胞。

2.3實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)TRIzol法提取各組總RNA，按試劑盒步驟逆轉(zhuǎn)錄后，選用10 μL體系進(jìn)行熒光定量PCR，引物序列見(jiàn)表1。反應(yīng)條件：95℃ 5 min; 95℃ 30 s，55℃ 30 s，72℃ 40 s,40個(gè)循環(huán)。

2.4CCK-8法檢測(cè)細(xì)胞活力在96孔板中接種GFP-Daxx過(guò)表達(dá)組細(xì)胞和對(duì)照組細(xì)胞懸液，培養(yǎng)24 h后，每孔加入10 μL CCK-8,37 ℃孵育2 h，酶標(biāo)儀測(cè)定450 nm波長(zhǎng)附近的吸光度。每組4個(gè)復(fù)孔，重復(fù)3次。

2.5流式細(xì)胞術(shù)收集佛波酯(phorbol12-myristate13．a(chǎn)cetate，PMA)處理72 h后的GFP-Daxx過(guò)表達(dá)組和對(duì)照組細(xì)胞，連同PMA未處理組細(xì)胞， PBS 洗2遍，收集10 000個(gè)細(xì)胞，PBS稀釋到50 μL重懸細(xì)胞后加入1 μL PE標(biāo)記CD61抗體或CD41抗體，室溫避光靜置30 min，預(yù)冷PBS洗滌2次后上機(jī)檢測(cè)。

2.6NBT還原實(shí)驗(yàn)取經(jīng)PMA作用72 h后的GFP-Daxx過(guò)表達(dá)組和對(duì)照組細(xì)胞，與2 g/L的NBT溶液混合， 37℃孵育30 min后離心涂片，Giemsa染色，光學(xué)顯微鏡下觀察胞質(zhì)內(nèi)含藍(lán)灰色顆粒的陽(yáng)性細(xì)胞。

2.7Western blot實(shí)驗(yàn)收集細(xì)胞，預(yù)冷PBS 洗滌3次后加入 RIPA 細(xì)胞裂解液，冰上放置 30 min，4℃13 000×g離心30 min，收集上清，BCA法蛋白定量。取30 μg 總蛋白進(jìn)行SDS-PAGE，電轉(zhuǎn)移至PVDF 膜上，5%脫脂奶粉室溫封閉1 h。后加入相應(yīng)I抗4℃孵育過(guò)夜，加 II 抗室溫孵育2 h。最后用ECL顯色試劑盒于暗室曝光顯影。

3統(tǒng)計(jì)學(xué)處理

用SPSS 16.0統(tǒng)計(jì)軟件進(jìn)行分析。數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，多組間比較采用單因素方差分析(one-way ANOVA)，組間兩兩比較采用Bonferroni校正的t檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

1過(guò)表達(dá)Daxx的K562細(xì)胞系的建立

熒光顯微鏡觀察結(jié)果表明，GFP-Daxx在細(xì)胞內(nèi)聚集，并表達(dá)于細(xì)胞核，見(jiàn)圖1。實(shí)時(shí)熒光定量PCR檢測(cè)過(guò)表達(dá)細(xì)胞系Daxx的mRNA水平顯著高于對(duì)照組，Western blot結(jié)果顯示，過(guò)表達(dá)Daxx的K562細(xì)胞中，Daxx蛋白表達(dá)明顯升高，見(jiàn)圖2。

2過(guò)表達(dá)Daxx對(duì)K562細(xì)胞活力的影響

CCK-8實(shí)驗(yàn)結(jié)果顯示，過(guò)表達(dá)Daxx后K562細(xì)胞的活力明顯低于對(duì)照(K562細(xì)胞)組(P<0.01)，見(jiàn)圖3。

Figure 1. Observation under fluorescence microscope for the localization of overexpressed Daxx (green) in the K562 cells.

圖1熒光顯微鏡結(jié)果顯示K562細(xì)胞中過(guò)表達(dá)Daxx呈點(diǎn)狀聚集

Figure 2. The results of real-time PCR (A) and Western blot (B) for determining the overexpression of Daxx in the K562 cells. Mean±SD.n=3.**P<0.01vsK562.

圖2Real-time PCR結(jié)果和Western blot檢測(cè)Daxx的表達(dá)情況

Figure 3. The effect of Daxx overexpression on the viability of K562 cells. Mean±SD.n=3.*P<0.05,**P<0.01vsK562.

圖3Daxx 過(guò)表達(dá)降低K562細(xì)胞活力

3PMA誘導(dǎo)K562細(xì)胞向巨核細(xì)胞分化

流式細(xì)胞術(shù)分析結(jié)果顯示，PMA處理K562細(xì)胞后CD41和CD61的表達(dá)升高，見(jiàn)圖4。Western blot實(shí)驗(yàn)結(jié)果顯示，在PMA誘導(dǎo)K562細(xì)胞分化的過(guò)程中，p-ERK的蛋白水平升高，Daxx的表達(dá)呈下降趨勢(shì)，見(jiàn)圖5。

4過(guò)表達(dá)Daxx對(duì)K562細(xì)胞向巨核細(xì)胞分化的影響

過(guò)表達(dá)Daxx后，PMA誘導(dǎo)GFD-Daxx-K562細(xì)胞向巨核細(xì)胞分化，流式細(xì)胞術(shù)檢測(cè)CD41和CD61的表達(dá)均明顯低于對(duì)照(K562細(xì)胞)組(P<0.01)，見(jiàn)圖6、7。NBT還原實(shí)驗(yàn)結(jié)果顯示，Daxx過(guò)表達(dá)后，分化細(xì)胞數(shù)量與對(duì)照組相比明顯降低(P<0.05)，見(jiàn)圖8。Western blot結(jié)果顯示，過(guò)表達(dá)Daxx后，p-ERK的蛋白水平相對(duì)于對(duì)照組下降，見(jiàn)圖9。

Figure 4.The expression of CD41 (A) and CD61 (B) after induced by PMA in the K562 cells.Mean±SD.n=3.**P<0.01vscontrol.

圖4PMA處理K562細(xì)胞后CD41和CD61的表達(dá)

Figure 5. The expression of p-ERK and Daxx in the PMA-induced K562 cells.Mean±SD.n=3.*P<0.05vs0 h.

圖5PMA處理K562細(xì)胞后 p-ERK和Daxx蛋白水平的變化

討論

Daxx最初發(fā)現(xiàn)它是一種受體胞漿死亡結(jié)構(gòu)域結(jié)合蛋白，之后證實(shí)它是一種定位于細(xì)胞核亞核結(jié)構(gòu)早幼粒細(xì)胞白血病蛋白(promyelocytic leukemia protein，PML)癌基因結(jié)構(gòu)域(PML ocogeneic domains，PODs)的核蛋白。Daxx廣泛分布于哺乳動(dòng)物和人正常組織細(xì)胞及腫瘤細(xì)胞中，在凋亡、轉(zhuǎn)錄調(diào)控、病毒感染等方面發(fā)揮重要作用。作為一種重要的信號(hào)分子，Daxx可以在細(xì)胞質(zhì)介導(dǎo)腫瘤壞死因子受體(tumour necrosis factor receptor，TNFR)超家族成員促凋亡信號(hào)的轉(zhuǎn)導(dǎo)，在細(xì)胞核也可以作為轉(zhuǎn)錄調(diào)控因子, 在轉(zhuǎn)錄調(diào)控中行使轉(zhuǎn)錄抑制或轉(zhuǎn)錄激活雙重功能[5-6]，通過(guò)轉(zhuǎn)位、化學(xué)修飾、染色體調(diào)節(jié)、直接與轉(zhuǎn)錄因子或轉(zhuǎn)錄相關(guān)蛋白相互作用等多種方式發(fā)揮轉(zhuǎn)錄調(diào)控作用。Daxx廣泛分布于哺乳動(dòng)物和人的正常細(xì)胞及腫瘤細(xì)胞，在血液病中廣泛表達(dá)，作為Fas受體胞漿死亡結(jié)構(gòu)域結(jié)合蛋白，Daxx可以經(jīng)Fas-Daxx-ASK1-JNK通路發(fā)揮促凋亡的作用；Daxx也可以發(fā)揮抗凋亡作用，在Daxx基因缺失的小鼠中，其胚胎干細(xì)胞發(fā)生凋亡，不能正常發(fā)育[7]。Daxx 可以結(jié)合轉(zhuǎn)錄相關(guān)蛋白或相關(guān)因子，如E2A、Ets-1、 Pax3和 Pax5等，進(jìn)行轉(zhuǎn)錄激活或轉(zhuǎn)錄抑制的調(diào)控[1-2]。有研究表明，Daxx參與細(xì)胞分化，Daxx通過(guò)結(jié)合轉(zhuǎn)錄因子E2A，抑制E2A的功能，從而抑制關(guān)鍵的肌原性基因表達(dá)和減少肌小管的形成，抑制肌細(xì)胞生成，參與肌細(xì)胞分化過(guò)程[3]；Daxx 可以促進(jìn)卵巢癌細(xì)胞的生長(zhǎng)以及其化療耐藥性[8]，Daxx可以通過(guò)抑制自噬促進(jìn)前列腺癌的發(fā)生[9]。關(guān)于Daxx在血液腫瘤中的作用，相關(guān)研究較少。

Figure 6. The expression of CD41 in PMA-induced GFP-Daxx-K562 cells.Mean±SD.n=3.**P<0.01vsK562.

圖6PMA誘導(dǎo)Daxx過(guò)表達(dá)的K562細(xì)胞CD41的表達(dá)

Figure 7.The expression of CD61 in PMA-induced GFP-Daxx-K562 cells. Mean±SD.n=3.**P<0.01vsK562.

圖7PMA誘導(dǎo)Daxx過(guò)表達(dá)的K562細(xì)胞CD61的表達(dá)

Figure 8.The results of nitroblue tetrazolium (NBT)-reducing test for the differentiation of the K562 cells with Daxx overexpression induced by PMA. The arrowhead pointed the differentiated cell.Mean±SD.n=3.*P<0.05vsK562.

圖8PMA誘導(dǎo)后Daxx過(guò)表達(dá)組和對(duì)照組細(xì)胞的分化情況

人白血病K562細(xì)胞株是慢性粒細(xì)胞白血病的一個(gè)典型的細(xì)胞系，其在體外被相關(guān)藥物誘導(dǎo)后具有向巨核系、單核系或紅系等多個(gè)方向分化潛能。K562細(xì)胞可以作為巨核細(xì)胞系分化模型，PMA是蛋白激酶C(protein kinase C，PKC)的激動(dòng)劑，K562細(xì)胞能被PMA誘導(dǎo)分化成為具有巨核系特征的細(xì)胞[10]。PKC是一類絲/蘇氨酸蛋白激酶，參與多種信號(hào)轉(zhuǎn)導(dǎo)途徑，在細(xì)胞生長(zhǎng)、分化和細(xì)胞因子分泌等過(guò)程中發(fā)揮著重要作用, ERK是經(jīng)典MAP信號(hào)轉(zhuǎn)導(dǎo)分子，介導(dǎo)多種細(xì)胞生物學(xué)過(guò)程，包括細(xì)胞生長(zhǎng)、存活、分化和炎癥反應(yīng)等。PMA誘導(dǎo)K562細(xì)胞向巨核細(xì)胞分化過(guò)程中，ERK信號(hào)通路通常被激活[11]。目前研究表明，影響PMA誘導(dǎo)K562細(xì)胞向巨核細(xì)胞分化的因素很多，KRAB-ZFPs是一類重要的轉(zhuǎn)錄調(diào)控因子，ZNF300是其典型成員，在PMA誘導(dǎo)K562細(xì)胞分化過(guò)程中表達(dá)上調(diào)，參與調(diào)控K562細(xì)胞巨核細(xì)胞分化[12]。電離輻射可以促進(jìn)PMA誘導(dǎo)的K562細(xì)胞巨核細(xì)胞分化，MAPK信號(hào)通路中ERK和P38 MAPK參與此過(guò)程調(diào)控[13]。過(guò)表達(dá)7號(hào)染色體開(kāi)放閱讀框41 (C7ORF41)可以促進(jìn)佛波酯誘導(dǎo)的K562細(xì)胞巨核細(xì)胞分化，p-ERK和JNK的蛋白水平升高[14]。在PMA誘導(dǎo)K562細(xì)胞巨核細(xì)胞分化過(guò)程中，線粒體功能發(fā)生明顯改變，ROS 水平明顯升高，線粒體膜電位下降，呼吸鏈復(fù)合體IV活性降低[15]。而關(guān)于Daxx與K562細(xì)胞巨核細(xì)胞分化的關(guān)聯(lián)研究目前尚未見(jiàn)報(bào)道。

Figure 9.The protein levels of p-ERK in Daxx overexpressed K562 cells after induced by PMA. Mean±SD.n=3.*P<0.05vsK562.

圖9PMA誘導(dǎo)Daxx-K562細(xì)胞p-ERK蛋白水平的變化

為研究Daxx在K562細(xì)胞中的作用，建立穩(wěn)定表達(dá)Daxx的K562細(xì)胞系，CCK8實(shí)驗(yàn)結(jié)果顯示，Daxx過(guò)表達(dá)后，細(xì)胞活力顯著降低。通過(guò)PMA誘導(dǎo)K562細(xì)胞向巨核細(xì)胞分化，發(fā)現(xiàn)在巨核細(xì)胞分化過(guò)程中p-ERK表達(dá)升高，Daxx表達(dá)下降，表明Daxx在K562細(xì)胞向巨核細(xì)胞分化過(guò)程中起抑制作用。PMA誘導(dǎo)Daxx-K562細(xì)胞后發(fā)現(xiàn)分化細(xì)胞數(shù)目降低，細(xì)胞巨核細(xì)胞分化指標(biāo)CD41和CD61的表達(dá)顯著降低，進(jìn)一步表明Daxx參與抑制K562細(xì)胞向巨核細(xì)胞分化。Western blot結(jié)果顯示，Daxx過(guò)表達(dá)后，PMA誘導(dǎo)K562細(xì)胞向巨核細(xì)胞分化，p-ERK的蛋白水平降低，表明Daxx可能通過(guò)下調(diào)p-ERK來(lái)實(shí)現(xiàn)對(duì)K562細(xì)胞向巨核細(xì)胞分化的抑制，但有關(guān)深入的作用機(jī)制，還需要進(jìn)一步的研究。

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(責(zé)任編輯： 陳妙玲， 余小慧)

Role of Daxx in inhibiting megakaryocytic differentiation of K562 cells

LIU Sheng-bing, PAN Wei-wei, SHEN Zhong-fei, XU Ying, GUO Yan-jun, WANG Zhi-jian, HUANG Qian

(SchoolofMedicine,JiaxingUniversity,Jiaxing314001,China.E-mail:ycfbing@163.com)

[KEY WORDS]Death domain-associated protein; K562 cells; Megakaryocytic differentiation; Phorbol 12-myristate 13-acetate

[ABSTRACT]AIM: To examine the effects of death domain-associated protein (Daxx) overexpression on the viability and megakaryocytic differentiation of K562 cells. METHODS: Daxx overexpression in the K562 cells was established. The expression of Daxx was detected by fluorescence microscopy, fluorescence quantitative real-time PCR and Western blot after transfection. CCK-8 assay was used to detect the cell viability after overexpression of Daxx. The expression of CD41 and CD61 in phorbol 12-myristate 13-acetate (PMA) induced K562 cells was detected by flow cytometry. The protein levels of Daxx and p-ERK were determined by Western blot. Nitroblue tetrazolium (NBT)-reducing test was used to assess leukemia cell differentiation in Daxx-overexpressing K562 cells and control cells. The expression of CD41 and CD61 induced by PMA in Daxx-overexpressing K562 cells was analyzed by flow cytometry. The protein levels of Daxx and p-ERK were also examined by Western blot. RESULTS: The stable overexpression of Daxx in the K562 cells was established. The viability was reduced in Daxx-overexpressing K562 cells. The expression of CD41 and CD61 was significantly increased after PMA induction in the K562 cells (P<0.01). The protein expression of Daxx was reduced, but the protein level of p-ERK was increased. The expression of CD41 and CD61 was reduced after PMA induction in Daxx-overexpressing K562 cells (P<0.01). The protein level of p-ERK was also reduced. CONCLUSION: Daxx overexpression inhibits the growth, megakaryocytic differentiation and production of p-ERK in the K562 cells.

[文章編號(hào)]1000- 4718(2016)06- 1004- 07

[收稿日期]2015- 11- 24[修回日期] 2016- 03- 10

*[基金項(xiàng)目]“十二五”浙江省高校藥理學(xué)重點(diǎn)學(xué)科資助項(xiàng)目;嘉興市科技局項(xiàng)目(No. 2015AY23063);嘉興學(xué)院重點(diǎn)SRT項(xiàng)目(No.851715065)

通訊作者△Tel：0573-83643850； E-mail：ycfbing@163.com

[中圖分類號(hào)]R329.21; R730.23

[文獻(xiàn)標(biāo)志碼]A

doi:10.3969/j.issn.1000- 4718.2016.06.007