龔晴麗 李雪 丁高中 凌雨婷 趙文娥 熊喜喜 魯嚴(yán)
210029南京醫(yī)科大學(xué)第一附屬醫(yī)院皮膚科(龔晴麗、李雪、丁高中、凌雨婷、熊喜喜、魯嚴(yán));南京醫(yī)科大學(xué)分析測(cè)試中心(趙文娥)
·論著·
氧化應(yīng)激下黑素細(xì)胞自噬相關(guān)基因差異表達(dá)分析
龔晴麗 李雪 丁高中 凌雨婷 趙文娥 熊喜喜 魯嚴(yán)
210029南京醫(yī)科大學(xué)第一附屬醫(yī)院皮膚科(龔晴麗、李雪、丁高中、凌雨婷、熊喜喜、魯嚴(yán));南京醫(yī)科大學(xué)分析測(cè)試中心(趙文娥)
目的探討過(guò)氧化氫(H2O2)對(duì)黑素細(xì)胞自噬的影響及可能的調(diào)節(jié)機(jī)制。方法取對(duì)數(shù)生長(zhǎng)期健康人黑素細(xì)胞,分為空白對(duì)照組(不予任何處理)、陽(yáng)性對(duì)照組(100 nmol/L西羅莫司處理)和實(shí)驗(yàn)組(體積分?jǐn)?shù)為10?7~10?3的H2O2處理),處理4 h后,使用CCK8法、流式細(xì)胞儀分別檢測(cè)各組黑素細(xì)胞活性及凋亡率。吖啶橙染色檢測(cè)自噬小泡,透射電鏡下觀察自噬小體,Western印跡檢測(cè)自噬特異性蛋白Beclin 1、微管相關(guān)蛋白輕鏈3B(LC3B)。最后采用包含84個(gè)自噬相關(guān)基因的RT2Profiler PCR Array篩選自噬相關(guān)的差異表達(dá)基因。結(jié)果黑素細(xì)胞分別經(jīng)10?3、5 × 10?4、10?4、5 × 10?5、10?5、5 ×10?6、10?6H2O2處理后,細(xì)胞增殖活性及凋亡率與空白對(duì)照組相比差異均有統(tǒng)計(jì)學(xué)意義(F值分別為286.95、301.23,均P<0.05),且隨H2O2體積分?jǐn)?shù)升高,增殖活性降低,凋亡率升高。除5 × 10?6H2O2組分別與10?5、10?6H2O2組間相比細(xì)胞凋亡率差異無(wú)統(tǒng)計(jì)學(xué)意義外,上述各H2O2組間兩兩比較,黑素細(xì)胞增殖活性及凋亡率差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。吖啶橙染色及電鏡觀察發(fā)現(xiàn),10?5H2O2、10?6H2O2和西羅莫司處理的黑素細(xì)胞中有自噬小體形成。Western印跡顯示,10?5H2O2、10?6H2O2和西羅莫司組黑素細(xì)胞Beclin 1表達(dá)量和LC3B?Ⅱ/LC3B?Ⅰ比率均較空白對(duì)照組顯著升高(P<0.05)。RT2 Profiler PCR Array結(jié)果顯示,與空白對(duì)照組相比,10?5H2O2組、10?6H2O2組和西羅莫司組中ATG12、ATG3、ULK1、PIK3CG、PTEN、PIK3C3表達(dá)均顯著上調(diào),EIF2AK3表達(dá)顯著下調(diào);10?5H2O2組和西羅莫司組mTOR表達(dá)顯著下調(diào),ULK2表達(dá)顯著上調(diào);10?6H2O2組mTOR表達(dá)未發(fā)生明顯改變,AMPK、JNK1表達(dá)顯著上調(diào)。結(jié)論體積分?jǐn)?shù)為10?5和10?6的H2O2均能有效誘導(dǎo)黑素細(xì)胞自噬,可能與影響相關(guān)信號(hào)分子表達(dá)相關(guān)。
黑素細(xì)胞;自噬;過(guò)氧化氫;氧化性應(yīng)激;寡核苷酸序列分析;細(xì)胞凋亡
Namazi等[1]提出“黑素細(xì)胞經(jīng)表皮丟失”即:氧化應(yīng)激下線粒體結(jié)構(gòu)功能障礙與自噬能力降低是導(dǎo)致黑素細(xì)胞粘附功能降低及經(jīng)表皮丟失的重要原因[2?4]。適量的自噬被認(rèn)為是重要的細(xì)胞保護(hù)性反應(yīng)[5]。如何調(diào)整氧化應(yīng)激下黑素細(xì)胞的自噬水平從而起到保護(hù)細(xì)胞的作用,需要明確這一過(guò)程中黑素細(xì)胞自噬調(diào)節(jié)的分子機(jī)制。我們應(yīng)用不同體積分?jǐn)?shù)的過(guò)氧化氫(H2O2)模擬氧化應(yīng)激環(huán)境,誘導(dǎo)黑素細(xì)胞發(fā)生自噬,采用PCR陣列技術(shù)(PCR array)篩選相關(guān)差異表達(dá)基因,探討氧化應(yīng)激下黑素細(xì)胞自噬特有的調(diào)節(jié)方式。
Ham F12培養(yǎng)基、胎牛血清(FBS)、胰蛋白酶(美國(guó)Gibco公司),G418、左旋多巴(L?DOPA)、西羅莫司(美國(guó)Sigma公司),山羊抗小鼠IgG、山羊抗兔IgG二抗(上海碧云天生物技術(shù)有限公司),H2O2溶液(南京化學(xué)試劑有限公司),CCK8試劑盒(日本Dojindo公司),膜聯(lián)蛋白V-異硫氰酸熒光素/碘化丙錠(Annexin V?FITC/PI)細(xì)胞凋亡檢測(cè)試劑盒(南京凱基生物技術(shù)有限公司),吖啶橙(美國(guó)Amresco生化試劑公司),鼠抗人Beclin 1抗體、兔抗人微管相關(guān)蛋白1輕鏈3B(LC3B)抗體、鼠抗人3-磷酸甘油醛脫氫酶(GAPDH)抗體(美國(guó) Sigma公司),RT2Profiler PCR Array(德國(guó)Qiagen公司),128C型酶標(biāo)儀(奧地利CliniBio公司),流式細(xì)胞儀(美國(guó)Beckman Coulter有限公司)。
標(biāo)本來(lái)自南京醫(yī)科大學(xué)第一附屬醫(yī)院泌尿外科健康男性包皮環(huán)切術(shù)后的皮膚組織(已征得患者知情同意)。根據(jù)Swope等[6]的方法獲得單細(xì)胞懸液,接種于含常規(guī)黑素細(xì)胞培養(yǎng)基[7]的培養(yǎng)瓶中,置于5%CO2培養(yǎng)箱37℃孵育。24 h后換液去除未貼壁細(xì)胞,并加入G418,48 h后改用不含G418的培養(yǎng)基,當(dāng)細(xì)胞融合至80%~90%時(shí),用2.5 g/L胰酶消化傳代。L-多巴染色鑒定黑素細(xì)胞。
取對(duì)數(shù)生長(zhǎng)期細(xì)胞,分為空白對(duì)照組(不予任何處理)、陽(yáng)性對(duì)照組(100 nmol/L西羅莫司[8]處理)和實(shí)驗(yàn)組[不同體積分?jǐn)?shù)(10?3,5 × 10?4,10?4,5 × 10?5,10?5,5 × 10?6,10?6,5 × 10?7,10?7)H2O2處理],處理 4 h后,檢測(cè)黑素細(xì)胞增殖活性及凋亡率,并篩選出體積分?jǐn)?shù)為10?4、10?5和10?6的H2O2進(jìn)行吖啶橙染色、電鏡觀察及Western印跡實(shí)驗(yàn)。最后,根據(jù)上述實(shí)驗(yàn)結(jié)果確定用體積分?jǐn)?shù)為 10?5和10?6的 H2O2誘導(dǎo)后進(jìn)行PCR array實(shí)驗(yàn),檢測(cè)自噬相關(guān)差異表達(dá)基因。
向已處理好的96孔板中每孔加入100 μl培養(yǎng)基和10 μl CCK8溶液,孵育2 h后,使用酶標(biāo)儀于450 nm波長(zhǎng)處測(cè)定各孔的吸光度(A450值)。每組設(shè)4個(gè)平行孔,實(shí)驗(yàn)重復(fù)3次取均值。
黑素細(xì)胞經(jīng)不同體積分?jǐn)?shù)H2O2處理后,胰酶消化,收集各組細(xì)胞,每組加入500 μl結(jié)合緩沖液、5 μl Annexin?FITC和5 μl PI混勻,室溫避光反應(yīng)5 ~15 min,用流式細(xì)胞儀檢測(cè)凋亡率,將Annexin?FITC單標(biāo)+Annexin?FITC和PI雙標(biāo)細(xì)胞視為凋亡細(xì)胞。實(shí)驗(yàn)重復(fù)3次。
取對(duì)數(shù)生長(zhǎng)期細(xì)胞接種于小培養(yǎng)皿中,細(xì)胞處理同前,磷酸鹽緩沖液(PBS)洗滌后,用4%多聚甲醛室溫下固定細(xì)胞10 min,再加入0.01%吖啶橙溶液,室溫避光孵育10~20 min后,PBS清洗3次,倒置熒光顯微鏡下觀察并攝片。
細(xì)胞處理同上,作用4 h后收集各組黑素細(xì)胞,加入4℃預(yù)冷的3%戊二醛,固定2 h以上,PBS洗滌3次,再加1%鋨酸固定2 h,PBS洗滌3次,經(jīng)脫水后包埋、切片(厚度為80 nm),3%醋酸鈾-枸櫞酸鉛雙染色,置于透射電鏡下觀察自噬小體并攝片。
取對(duì)數(shù)生長(zhǎng)期細(xì)胞,接種于直徑為10 cm的培養(yǎng)皿,細(xì)胞經(jīng)上述處理后,提取各組細(xì)胞總蛋白,經(jīng)十二烷基磺酸鈉-聚丙烯酰胺凝膠(SDS?PAGE)電泳,轉(zhuǎn)移至硝酸纖維素膜上。5%脫脂奶粉封閉1 h,與Beclin 1、LC3B特異性抗體及相應(yīng)二抗作用后,經(jīng)化學(xué)發(fā)光法顯色。用Image J軟件分析圖片,計(jì)算LC3B、Beclin 1與GAPDH的灰度比值。
取對(duì)數(shù)生長(zhǎng)期黑素細(xì)胞接種于T25細(xì)胞培養(yǎng)瓶,細(xì)胞經(jīng)上述處理4 h后,按照試劑盒說(shuō)明操作提取總RNA,反轉(zhuǎn)錄生成cDNA。實(shí)時(shí)PCR采用RT2Profiler PCR Array檢測(cè)自噬相關(guān)的84個(gè)基因,包含5種管家基因即次黃嘌呤磷酸核糖基轉(zhuǎn)移酶、β2微球蛋白、大核糖體蛋白、GAPDH和β肌動(dòng)蛋白。計(jì)算循環(huán)數(shù)閾值(Ct),ΔCt=目的基因Ct-平均管家基因 Ct,ΔΔCt= 實(shí)驗(yàn)組 ΔCt- 對(duì)照組 ΔCt,2?ΔΔCt值即為實(shí)驗(yàn)組目的基因較對(duì)照組相應(yīng)基因表達(dá)的倍數(shù)。
使用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。實(shí)驗(yàn)數(shù)據(jù)均采用±s表示,各組間差異比較采用單因素方差分析法,各處理組與空白對(duì)照組比較采用LSD法,各處理組間兩兩比較采用SNK法,劑量效應(yīng)采用Pearson相關(guān)性分析,P<0.05為差異有統(tǒng)計(jì)學(xué)意義。登陸網(wǎng)站http://www.sabiosciences.com/dataanalysis.php在線分析PCR Array檢測(cè)結(jié)果,差異表達(dá)基因定義為2?ΔΔCt≥ 2的基因。
如表1所示,黑素細(xì)胞經(jīng)10-7~10-3H2O2處理后,細(xì)胞增殖活性與空白對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P<0.05),且隨H2O2體積分?jǐn)?shù)升高而明顯下降(r=-0.979,P<0.001)。100 nmol/L西羅莫司組黑素細(xì)胞增殖活性與空白對(duì)照組、5×10?7H2O2組和10?7H2O2組相比,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P> 0.05),與其他各H2O2組相比差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。除5 × 10?7H2O2與10?7H2O2組間差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.518)外,其他各H2O2組間兩兩比較差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。
分別經(jīng)西羅莫司和H2O2處理4 h后,黑素細(xì)胞凋亡率與空白對(duì)照組相比差異均有統(tǒng)計(jì)學(xué)意義(P<0.05),且隨H2O2體積分?jǐn)?shù)升高而升高(r=0.950,P<0.001)。西羅莫司組與除5 × 10?7H2O2組外的其他各H2O2組相比,黑素細(xì)胞凋亡率差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。除5 × 10?7與10?7H2O2組間、5 × 10?6與10?5或10?6H2O2組間差異無(wú)統(tǒng)計(jì)學(xué)意義外(P>0.05)外,其余各H2O2組間兩兩比較差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)表1。
10?4H2O2組吖啶橙染色未見(jiàn)紅色點(diǎn)狀熒光,西羅莫司、10?5H2O2、10?6H2O23組黑素細(xì)胞內(nèi)紅色熒光強(qiáng)度明顯高于空白對(duì)照組。見(jiàn)圖1。
空白對(duì)照組黑素細(xì)胞內(nèi)自噬小泡少見(jiàn),細(xì)胞膜及核膜結(jié)構(gòu)完整,線粒體及內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)正常。10?4H2O2處理組黑素細(xì)胞中線粒體腫脹,嵴排列紊亂,伴有內(nèi)質(zhì)網(wǎng)擴(kuò)張,未見(jiàn)到自噬小泡。在西羅莫司、10?5H2O2、10?6H2O23個(gè)處理組中發(fā)現(xiàn)黑素細(xì)胞內(nèi)自噬小泡明顯增多,并觀察到線粒體自噬,且線粒體損傷較10?4H2O2組輕,細(xì)胞核結(jié)構(gòu)完整。見(jiàn)圖2。
表1 不同體積分?jǐn)?shù)H2O2對(duì)黑素細(xì)胞增殖活性及凋亡的影響(±s)
表1 不同體積分?jǐn)?shù)H2O2對(duì)黑素細(xì)胞增殖活性及凋亡的影響(±s)
注:n=3。a:與空白對(duì)照組比較,P<0.05;b:與西羅莫司組比較,P<0.05
組別空白對(duì)照組西羅莫司組H2O2組10?3 5 × 10?4 10?4 5 × 10?5 10?5 5 × 10?6 10?6 5 × 10?7 10?7 F值P值增殖活性(A450)0.958±0.041 0.927±0.041凋亡率(%)10.08±1.13b 15.20±1.05a 0.247±0.008ab 0.307±0.026ab 0.388±0.036ab 0.509±0.039ab 0.669±0.032ab 0.748±0.039ab 0.798±0.021ab 0.895±0.014a 0.909±0.022a 286.95<0.05 61.74±1.63ab 53.72±2.30ab 43.76±3.32ab 34.51±3.43ab 22.03±1.47ab 20.85±1.20ab 18.04±3.05ab 12.71±0.58 12.01±1.02b 301.23<0.05
圖1 倒置熒光顯微鏡下觀察自噬小泡(吖啶橙染色×200) 1A:空白對(duì)照組胞質(zhì)中見(jiàn)少量紅色熒光;1B:10?4H2O2組細(xì)胞核及細(xì)胞質(zhì)均呈黃綠色熒光,未見(jiàn)紅色熒光;1C~1E:分別為10?5H2O2組、10?6H2O2組、西羅莫司組,3組細(xì)胞胞質(zhì)內(nèi)可見(jiàn)斑點(diǎn)狀紅色熒光,熒光強(qiáng)度明顯高于空白對(duì)照組 圖2 透射電鏡下觀察自噬小體(黃色箭頭) 2A:空白對(duì)照組(×30 000)自噬小泡少見(jiàn),細(xì)胞膜及核膜結(jié)構(gòu)完整,線粒體及內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)正常;2B:10?4H2O2組(×30 000),線粒體腫脹、嵴排列紊亂,伴有內(nèi)質(zhì)網(wǎng)擴(kuò)張,未見(jiàn)到自噬小泡;2C ~2E:分別為10?5H2O2組(× 30 000)、10?6H2O2組(× 20 000)、西羅莫司組(× 30 000),黑素細(xì)胞內(nèi)自噬小泡明顯增多,可見(jiàn)線粒體自噬,且線粒體損傷較10?4H2O2處理組輕,細(xì)胞核結(jié)構(gòu)完整
黑素細(xì)胞經(jīng)西羅莫司、10?5H2O2和10?6H2O2作用后,Beclin 1/GAPDH灰度比值分別為2.953±0.480、2.048±0.212和2.550±0.281,均顯著高于空白對(duì)照組(0.952 ± 0.110,均P<0.05),而 10?4H2O2組(0.521±0.062)顯著低于空白對(duì)照組(P<0.05);LC3B?Ⅱ/LC3B?Ⅰ比率分別為6.088± 0.064、5.287 ±0.314和6.032±0.082,均顯著高于空白對(duì)照組(2.151 ± 0.106,均P<0.05),而10?4H2O2組(2.396 ±0.144)與空白對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.105),且西羅莫司組和 10?6H2O2組間差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.697)。見(jiàn)圖3。
見(jiàn)表2。與空白對(duì)照組相比,各處理組的自噬相關(guān)基因12(ATG12)、自噬相關(guān)基因3(ATG3)、失調(diào)51樣激酶1(ULK1)、磷脂酰肌醇-3-激酶催化亞單位γ(PIK3CG)、磷脂酰肌醇-3-激酶class 3(PIK3C3)、第10號(hào)染色體缺失的磷酸酶和張力蛋白的同源基因(PTEN)表達(dá)均顯著上調(diào),真核生物翻譯起始因子-2α-激酶-3基因(EIF2AK3)表達(dá)顯著下調(diào);10?5H2O2組和西羅莫司組哺乳動(dòng)物西羅莫司靶蛋白(mTOR)基因顯著下調(diào),失調(diào)51樣激酶2(ULK2)顯著上調(diào);10?6H2O2組mTOR未發(fā)生明顯改變,腺苷酸活化蛋白激酶(AMPK)、c?Jun氨基末端激酶1(JNK1)顯著上調(diào)。
圖3 Western印跡檢測(cè)各處理組Beclin 1與LC3B表達(dá) 1:空白對(duì)照組;2:10?4H2O2組;3:10?5H2O2組;4:10?6H2O2組;5:西羅莫司組。LC3B:微管相關(guān)蛋白1輕鏈3B;GAPDH:3?磷酸甘油醛脫氫酶
表2 H2O2作用后黑素細(xì)胞與空白對(duì)照組相比差異表達(dá)的自噬相關(guān)基因(2?ΔΔCt)a
自噬與凋亡之間存在復(fù)雜的相互作用。在某些特定情況下,自噬能夠抵抗或延緩凋亡,利于細(xì)胞存活;但有些情況下又可與凋亡協(xié)作導(dǎo)致細(xì)胞死亡或在凋亡缺陷時(shí)作為后備機(jī)制誘導(dǎo)細(xì)胞死亡[9]。H2O2濃度、作用時(shí)間及作用的細(xì)胞類(lèi)型不同,細(xì)胞可能發(fā)生自噬、自噬性死亡、凋亡或壞死等[10]。為了誘導(dǎo)氧化應(yīng)激下黑素細(xì)胞發(fā)生自噬,我們首先檢測(cè)不同體積分?jǐn)?shù)H2O2對(duì)黑素細(xì)胞增殖活性及凋亡的影響,結(jié)果顯示,與空白對(duì)照組黑素細(xì)胞相比,10?4~ 10?3H2O2處理的黑素細(xì)胞增殖活性明顯降低,且凋亡率顯著升高;而10?7、5 × 10?7H2O2組黑素細(xì)胞凋亡率與空白對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義,且5 × 10?6與 10?5H2O2組間及5 × 10?6與 10?6H2O2組間細(xì)胞凋亡率差異無(wú)統(tǒng)計(jì)學(xué)意義,僅 10?4、10?5、10?6H2O23組黑素細(xì)胞增殖活性及凋亡率與空白對(duì)照組相比及組間比較均存在統(tǒng)計(jì)學(xué)差異。所以,在后續(xù)自噬實(shí)驗(yàn)中選擇了這3個(gè)濃度。而根據(jù)吖啶橙染色、透射電鏡及Western印跡實(shí)驗(yàn)結(jié)果,在10?5及10?6H2O2這兩組觀察到黑素細(xì)胞發(fā)生自噬,故選擇這兩個(gè)濃度進(jìn)行PCR array實(shí)驗(yàn)。
自噬分子機(jī)制復(fù)雜,涉及30多個(gè)ATG基因、50個(gè)溶酶體水解酶及多條信號(hào)途徑[11]。目前已證實(shí)ULK1/2?ATG13?ATG17復(fù)合物的形成可啟動(dòng)自噬,并受mTOR調(diào)控[12]。當(dāng)mTOR被抑制時(shí),ATG13去磷酸化并和ULK1/2?ATG17復(fù)合物結(jié)合,激活ULK1/2,引發(fā)自噬[13]。已知mTOR的上游受多條信號(hào)通路的調(diào)控,磷脂酰肌醇-3-激酶/絲氨酸-蘇氨酸蛋白激酶(PI3K/AKT)信號(hào)轉(zhuǎn)導(dǎo)通路是經(jīng)典的調(diào)節(jié)自噬的mTOR上游信號(hào)途徑[14]。PTEN是一腫瘤抑制因子,對(duì)PI3K/AKT途徑進(jìn)行負(fù)調(diào)節(jié)[15],從而抑制mTOR激活,促進(jìn)自噬發(fā)生。
本研究顯示,與空白對(duì)照組相比,10?5H2O2組和西羅莫司組PIK3CG、PTEN、ULK1、ULK2均顯著上調(diào)2倍以上,10?5H2O2組和西羅莫司組mTOR顯著下調(diào),而10?6H2O2組mTOR無(wú)明顯改變。由此,我們推測(cè)10?5H2O2和西羅莫司可能主要通過(guò)PTEN負(fù)調(diào)節(jié)PIK3/AKT/mTOR依賴性途徑來(lái)促進(jìn)自噬。而Ueno等[16]證明,在PTEN缺陷自噬受抑制的肝細(xì)胞中,西羅莫司并不能通過(guò)抑制mTOR來(lái)激發(fā)自噬,推測(cè)PTEN可通過(guò)mTOR非依賴性途徑調(diào)節(jié)自噬,10?6H2O2可能通過(guò)以上途徑誘導(dǎo)黑素細(xì)胞發(fā)生自噬。
Egan等[17]在線粒體自噬中觀察到,氧化應(yīng)激作用下線粒體去極化能夠迅速減少細(xì)胞內(nèi)ATP水平,激活A(yù)MPK,使其磷酸化,從而直接促進(jìn)ULK1磷酸化,激活自噬。此外,在饑餓誘導(dǎo)的細(xì)胞自噬和心肌細(xì)胞自噬中觀察到,AMPK還可通過(guò)磷酸化JNK1,使凋亡相關(guān)蛋白BCL2發(fā)生多位點(diǎn)磷酸化,從而抑制BCL2?Beclin 1結(jié)合,Beclin 1得以解離,使得PIK3C3?BECN1多重復(fù)合物活性增加,從而促進(jìn)自噬發(fā)生[18?20]。本研究顯示,與空白對(duì)照組相比,10?6H2O2組AMPK、JNK1顯著上調(diào),BCL2與BECN1的上調(diào)倍數(shù)大于其他兩組;各組PIK3C3均顯著上調(diào)。由此推斷,10?6H2O2誘導(dǎo)的黑素細(xì)胞自噬可能通過(guò)上述途徑進(jìn)行調(diào)節(jié)。此外,10?5H2O2、10?6H2O2和西羅莫司可能均直接刺激PIK3C3活性增加,促進(jìn)自噬。
值得注意的是,EIF2AK3在10?5H2O2、10?6H2O2和西羅莫司組中均出現(xiàn)顯著下調(diào),且下調(diào)倍數(shù)>15。EIF2AK3參與內(nèi)質(zhì)網(wǎng)類(lèi)似激酶?真核細(xì)胞翻譯啟始子2α(PERK?eIF2α)通路介導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)[21],近年研究發(fā)現(xiàn),ERS途徑也是自噬的重要調(diào)控通路[22]。在ERS狀態(tài)下,EIF2AK3通過(guò)自身胞質(zhì)側(cè)羧基端的蛋白激酶催化促進(jìn)自身磷酸化,繼而使eIF2α蛋白51位絲氨酸磷酸化,導(dǎo)致蛋白合成受抑,減輕內(nèi)質(zhì)網(wǎng)負(fù)荷。EIF2AK3下調(diào)和EIF2AK3磷酸化水平增高是同時(shí)進(jìn)行的,這也合理解釋了10?5H2O2、10?6H2O2組 EIF2AK3 基因較空白對(duì)照組顯著下調(diào)的結(jié)果。由此推測(cè),ERS可能參與調(diào)控氧化應(yīng)激誘導(dǎo)的黑素細(xì)胞自噬,通過(guò)EIF2AK3自身磷酸化來(lái)磷酸化eIF2α,進(jìn)而上調(diào)ATG12并促進(jìn)自噬。
綜上所述,10?5H2O2和10?6H2O2模擬的氧化應(yīng)激條件均可誘導(dǎo)黑素細(xì)胞發(fā)生自噬,且不同濃度H2O2的誘導(dǎo)機(jī)制可能不僅涉及共同的調(diào)節(jié)通路,還涉及不同的調(diào)節(jié)方式。
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Differential expression of autophagy?related genes in melanocytes under oxidative stress
Gong Qingli,Li Xue,Ding Gaozhong,Ling Yuting,Zhao Wen′e,Xiong Xixi,Lu Yan
Department of Dermatology,The First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China(Gong QL,Li X,Ding GZ,Ling YT,Xiong XX,Lu Y);Department of Analysis and Testing Center,Nanjing Medical University,Nanjing 210029,China(Zhao WE)
Lu Yan,Email:luyan6289@163.com
ObjectiveTo evaluate the effect of hydrogen peroxide(H2O2) on autophagy in melanocytes,and to explore its possible regulatory mechanisms.MethodsNormal human melanocytes at exponential growth phase were divided into several groups:blank control group receiving no treatment,positive control group treated with 100 nmol/L sirolimus solution,and experiment groups treated with H2O2solution at different volume fractions of 10?7-10?3respectively.After 4?hour treatment,cell counting kit?8(CCK?8)assay and flow cytometry were performed to evaluate the cellular proliferative activity and detect apoptosis of melanocytes respectively.Acridine orange staining was performed to detect autophagosome formation,transmission electron microscopy to observe ultrastructural changes of autophagosomes,and Western blot analysis to measure the expression of autophagy?specific protein Beclin 1 and microtubule?associated protein 1 light chain 3B(LC3B).A total of 84 autophagy?related genes were analyzed by RT2Profiler PCR Array,so as to screen differentially expressed autophagy?related genes.ResultsAfter the treatment with H2O2at different volume fractions of 10?3,5 × 10?4,10?4,5 × 10?5,10?5,5 × 10?6and 10?6,experiment groups showed significantly decreased cellular proliferative activity,but significantly increased apoptosis rate compared with the blank control group(F=286.95,301.23,respectively,bothP< 0.05).With the increase in volume fractions of H2O2,the cellular proliferative activity was significantly gradually decreased(P< 0.05),while the apoptosis rate showed an opposite trend(P< 0.05),except that the 5 ×10?6H2O2group showed no significant differences in the apoptosis rate compared with the 10?5H2O2group and 10?6H2O2group.Acridine orange staining and electron microscopy showed autophagosome formation in the 10?5H2O2group,10?6H2O2group and positive control group.Western blot analysis revealed that Beclin1 expression and LC3B?Ⅱ/LC3B?Ⅰ ratio were significantly higher in the 10?5H2O2group,10?6H2O2group and positive control group than in the blank control group(allP< 0.05).RT2Profiler PCR Array showed significant up?regulation of ATG12,ATG3,ULK1,PIK3CG,PTEN and PIK3C3 genes and significant down?regulation of EIF2AK3 gene in the 10?5H2O2group,10?6H2O2group and positive control group compared with the blank control group.In the 10?5H2O2group and positive control group,the mTOR gene was significantly up?regulated,and the ULK2 gene was significantly down?regulated.The 10?6H2O2group showed no obvious changes in the expression of mTOR gene,but significant up?regulation of AMPK and JNK1 genes.ConclusionH2O2at volume fractions of 10?5and 10?6can induce autophagy in melanocytes,likely by influencing the expression of some related signaling molecules.
Melanocytes;Autophagy;Hydrogen peroxide;Oxidative stress;Oligonucleotide array sequence analysis;Apoptosis
魯嚴(yán),Email:luyan6289@163.com
10.3760/cma.j.issn.0412?4030.2017.08.001
國(guó)家自然科學(xué)基金(81171517);江蘇省“333工程”培養(yǎng)資金(BRA2013279)
Fund programs:National Natural Science Foundation of China(81171517);The“333”P(pán)roject of Jiangsu Province(BRA2013279)
2016?07?12)
(本文編輯:尚淑賢)