腦血紅蛋白表達(dá)增高可減輕α-突觸核蛋白引起的MES23.5細(xì)胞凋亡

楊巍巍 李旭冉 李 昕 李旭穎 于 順,3*

(1.首都醫(yī)科大學(xué)宣武醫(yī)院神經(jīng)生物學(xué)研究室，北京 100053；2.帕金森病研究北京重點(diǎn)實(shí)驗(yàn)室，北京 100053；3.北京腦重大疾病研究院帕金森病研究所，北京 100053)

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· 基礎(chǔ)研究 ·

腦血紅蛋白表達(dá)增高可減輕α-突觸核蛋白引起的MES23.5細(xì)胞凋亡

楊巍巍1,2李旭冉1,2李 昕1,2李旭穎1,2于 順1,2,3*

(1.首都醫(yī)科大學(xué)宣武醫(yī)院神經(jīng)生物學(xué)研究室，北京 100053；2.帕金森病研究北京重點(diǎn)實(shí)驗(yàn)室，北京 100053；3.北京腦重大疾病研究院帕金森病研究所，北京 100053)

目的 觀察腦血紅蛋白(neuronal hemoglobin，nHb)在α-突觸核蛋白(α-synuclein，α-syn)引起的細(xì)胞凋亡中的作用。方法 在MES23.5細(xì)胞中瞬時(shí)轉(zhuǎn)染空載(myc)和nHb質(zhì)粒，部分轉(zhuǎn)染細(xì)胞外加α-syn，免疫熒光化學(xué)方法鑒定基因表達(dá)情況。Western blotting法檢測(cè)α-syn與nHb表達(dá)水平。JC-1檢測(cè)各組細(xì)胞線粒體膜電勢(shì)水平，TUNEL染色和MTT法檢測(cè)各組細(xì)胞死亡情況。結(jié)果 過(guò)表達(dá)nHb可與α-syn在胞質(zhì)及線粒體均形成復(fù)合物，并顯著降低游離α-syn在胞質(zhì)及線粒體的水平，同時(shí)，nHb過(guò)表達(dá)可顯著逆轉(zhuǎn)α-syn所致線粒體膜電勢(shì)下降及細(xì)胞凋亡。結(jié)論 nHb表達(dá)增高可緩解α-syn致細(xì)胞損傷。

α-突觸核蛋白；血紅蛋白；線粒體；凋亡

帕金森病(Parkinson’s disease，PD)是一種復(fù)雜的神經(jīng)退行性疾病，以運(yùn)動(dòng)障礙為主要臨床特征。目前，PD發(fā)病機(jī)制尚不明確，但研究[1-3]顯示遺傳因素與PD發(fā)生發(fā)展密切相關(guān)。

路易體的主要成分是α-突觸核蛋白(α-synuclein，α-syn)，其被認(rèn)為是與散發(fā)性和遺傳性PD發(fā)生發(fā)展緊密相關(guān)的蛋白[4-5]。近來(lái)文獻(xiàn)[6-8]報(bào)道α-syn可以與其他蛋白如超氧化物歧化酶1(superoxide dismutase 1, SOD1)、 促微管聚集蛋白(tubulin polymerization promoting proteins, TPPP)及3,4-二羥基苯乙醛(3,4-dihydroxyphenylacetaldehyde, DOPAL)等發(fā)生相互作用從而參與了α-syn的病理機(jī)制。同時(shí)，文獻(xiàn)[9]顯示α-syn可引起線粒體損傷。

神經(jīng)球蛋白屬于氧結(jié)合蛋白家族，包括腦紅蛋白，腦血紅蛋白(neuronal hemoglobin，nHb)，肌紅蛋白，這些蛋白被普遍認(rèn)為參與神經(jīng)系統(tǒng)興奮性、氧自由基清除及調(diào)節(jié)細(xì)胞存活[10]。但這些蛋白最主要的作用是維持線粒體正常生理功能[11-18]。神經(jīng)球蛋白同時(shí)也參與了神經(jīng)退行性病變的發(fā)生與發(fā)展。例如，在PD患者腦內(nèi)發(fā)現(xiàn)nHb異常過(guò)表達(dá)[19]。但是nHb是否緩解α-syn所致的線粒體損傷及細(xì)胞凋亡尚未見報(bào)道。本文主要探討nHb與α-syn的關(guān)系及nHb對(duì)MES23.5細(xì)胞的保護(hù)作用，可能有助于為PD的發(fā)病機(jī)制及藥物作用靶點(diǎn)研究提供新思路。

1 材料和方法

1.1 儀器與試劑

DMEM/F12培養(yǎng)基、胎牛血清和胰蛋白酶(美國(guó)Gibco公司)；actin抗體(美國(guó)Sigma公司)；鼠抗人Hb單克隆抗體(美國(guó)Abcam公司)；myc抗體(美國(guó)Clontech公司)；鼠抗人α-syn單克隆抗體3D5由本室制備；生物素化的鼠單克隆抗體3D5(北京康為世紀(jì)生物科技有限公司)；4-硝基磷酸二鈉鹽(美國(guó)Sigma公司)；辣根過(guò)氧化物酶(horseradish peroxidase,HRP)標(biāo)記山羊抗小鼠IgG多克隆抗體(北京中杉金橋生物技術(shù)有限公司)；ELISA 96孔酶標(biāo)板(美國(guó)Corning公司)；酶標(biāo)儀(瑞士Tecan公司)；細(xì)胞凋亡檢測(cè)試劑盒(美國(guó)Roche公司)；MTT(北京欣經(jīng)科生物公司)；Lipofectamine 2000 (德國(guó)Qiagen公司)；HRP標(biāo)記的二抗(美國(guó)KPL公司)；人源nHb質(zhì)粒(pCMV-myc-nHb) 及其空載對(duì)照(pCMV-myc) 均由于順教授實(shí)驗(yàn)室構(gòu)建，其他試劑為分析純。

1.2 方法

1)細(xì)胞培養(yǎng)及轉(zhuǎn)染：MES23.5細(xì)胞采用含5%(體積分?jǐn)?shù))胎牛血清和Sato’s添加液、100 U/mL青霉素和100 U/mL鏈霉素的DMEM/F12培養(yǎng)基，置于37 ℃，5%(體積分?jǐn)?shù))CO2培養(yǎng)箱中培養(yǎng)。質(zhì)粒轉(zhuǎn)染按照Lipofectamine2000操作說(shuō)明書進(jìn)行。

2) MTT方法檢測(cè)細(xì)胞活力：分組為正常對(duì)照組(Con)，外加α-syn處理組(10mol/L，1、3、6 h)空載對(duì)照組(myc)，空載對(duì)照組外加α-syn(10mol/L，1、3、6 h)單純過(guò)表達(dá)nHb組，過(guò)表達(dá)nHb外加α-syn處理組(10mol/L，1、3、6 h)

上述各組MES23.5細(xì)胞按1×105個(gè)/孔接種于96孔板，每組細(xì)胞接種8個(gè)復(fù)孔。接種后24 h，向各孔加入MTT(5 g/L) 20 mL，37 ℃，5%(體積分?jǐn)?shù)) CO2培養(yǎng)箱繼續(xù)培養(yǎng)4 h。后每孔加入100 mL二甲基亞砜，振蕩10 min。將孔板置于酶標(biāo)儀中，取550 nm波長(zhǎng)處的吸光度值統(tǒng)計(jì)分析。實(shí)驗(yàn)至少重復(fù)3次進(jìn)行統(tǒng)計(jì)學(xué)分析。

3) Western blotting分析：實(shí)驗(yàn)分組為正常對(duì)照組(Con)，外加α-syn處理組(10mol/L，6 h)空載對(duì)照組(myc)，空載對(duì)照組外加α-syn(10mol/L，6 h)單純過(guò)表達(dá)nHb組，過(guò)表達(dá)nHb外加α-syn處理組(10mol/L，6 h)。將上述每組細(xì)胞按2×108個(gè)接種于每個(gè)大培養(yǎng)皿中，每組細(xì)胞接種3個(gè)大皿。抽提上述各組細(xì)胞胞質(zhì)、線粒體及胞膜蛋白，選用BCA法測(cè)定蛋白濃度，SDS-PAGE電泳，半濕轉(zhuǎn)法將蛋白轉(zhuǎn)移至PVDF膜，10%(質(zhì)量分?jǐn)?shù))脫脂牛奶封閉1 h，分別入抗3D5單抗 (1∶5 000)，Hb(1∶1 000)，actin (1∶1 000)，VDAC(1∶1 000)，calnexin(1∶1 000)室溫孵育3 h。后入相應(yīng)HRP標(biāo)記的羊抗兔(或鼠)免疫球蛋白抗體中(1∶5 000)，室溫孵育1 h。加入化學(xué)發(fā)光液，于暗室化學(xué)發(fā)光及顯影、定影。實(shí)驗(yàn)至少重復(fù)3次進(jìn)行統(tǒng)計(jì)學(xué)分析。

4)nHb檢測(cè)：抽提上述各組細(xì)胞胞質(zhì)、線粒體及胞膜蛋白，利用Western blotting分別檢測(cè)上述各細(xì)胞組分中的nHb，利用肌動(dòng)蛋白(actin)、電壓依賴的選擇性陰離子通道蛋白(voltage-dependent anion-selective channel, VDAC)、鈣連接蛋白(calnexin)分別作為胞質(zhì)、線粒體、膜組分的內(nèi)參。以nHb條帶灰度值比不同內(nèi)參的灰度值作為標(biāo)準(zhǔn)化后不同組細(xì)胞各亞細(xì)胞組分nHb的水平。實(shí)驗(yàn)至少重復(fù)3次進(jìn)行統(tǒng)計(jì)學(xué)分析。

5) 免疫細(xì)胞化學(xué)染色：上述各組細(xì)胞培養(yǎng)24 h后進(jìn)行myc或者nHb質(zhì)粒轉(zhuǎn)染，轉(zhuǎn)染24 h后做免疫熒光染色，1 ×PBS沖洗2次，加入4%(質(zhì)量分?jǐn)?shù))多聚甲醛，室溫固定20 min，10%(體積分?jǐn)?shù))山羊血清室溫孵育1 h封閉，加入抗myc(1∶500)及3D5一抗(1∶1 000)，室溫避光孵育3 h，然后與Alexa Fluor標(biāo)記山羊抗小鼠(或兔)熒光二抗(1∶5 000)室溫孵育1 h。加入4,6-二氨基-2-苯基吲哚(DAPI)于37 ℃避光孵育5 min復(fù)染細(xì)胞核，甘油封片后用共聚焦掃描顯微鏡觀察。

6) 與血紅蛋白結(jié)合的α-syn的ELISA檢測(cè): 實(shí)驗(yàn)分組為正常對(duì)照組(Con)，外加α-syn處理組 (10mol/L，6 h)空載對(duì)照組(myc)，空載對(duì)照組外加α-syn(10mol/L，6 h)單純過(guò)表達(dá)nHb組，過(guò)表達(dá)nHb外加α-syn處理組(10mol/L，6 h)。將上述每組細(xì)胞按2×108個(gè)接種于每個(gè)大培養(yǎng)皿中，每組細(xì)胞接種3個(gè)大皿。抽提每組細(xì)胞胞質(zhì)、線粒體、胞膜組分，BCA定量后進(jìn)行ELISA檢測(cè)。用質(zhì)量濃度為2 mg/L 血紅蛋白單抗包被96孔酶標(biāo)板，2.5%(質(zhì)量分?jǐn)?shù))明膠封閉2 h。加入各組細(xì)胞樣品100L(0.1g/L)，37 ℃孵育2 h，再加入生物素標(biāo)記3D5抗體 (終質(zhì)量濃度1 mg/L)，37 ℃孵育2 h。向各孔加入100 mL堿性磷酸酶標(biāo)記的親和素 (1∶5 000)，37 ℃孵育1 h。最后加入100 mL對(duì)硝基酚磷酸顯色液 (pNPP)，37 ℃顯色30 min。405 nm處測(cè)定吸光度值。實(shí)驗(yàn)至少重復(fù)3次進(jìn)行統(tǒng)計(jì)學(xué)分析。

7)與血紅蛋白結(jié)合的α-syn免疫共沉淀檢測(cè)：實(shí)驗(yàn)所用細(xì)胞樣本數(shù)同上，將上述細(xì)胞組織勻漿20 mg/L與7 mg/L的抗人紅蛋白抗體于4 ℃孵育過(guò)夜，然后與protein G 反應(yīng)，12 000 g離心5 min，棄上清，0.01 mol/L PBS緩沖液500L洗滌沉淀3次，棄上清，將沉淀用3D5抗體進(jìn)行Western blotting檢測(cè)，以分析各組細(xì)胞不同亞細(xì)胞部位與血紅蛋白結(jié)合的α-syn的量。實(shí)驗(yàn)至少重復(fù)3次進(jìn)行統(tǒng)計(jì)學(xué)分析。

8) TUNEL染色：實(shí)驗(yàn)分組為正常對(duì)照組(Con)，外加α-syn處理組(10mol/L，6 h)空載對(duì)照組(myc)，空載對(duì)照組外加α-syn(10mol/L，6 h)單純過(guò)表達(dá)nHb組，過(guò)表達(dá)nHb外加α-syn處理組(10mol/L，6 h)。將上述每組細(xì)胞按1×103個(gè)接種于每個(gè)confocal培養(yǎng)皿中，每組細(xì)胞接種3個(gè)confocal皿。將上述各組細(xì)胞用1×PBS沖洗3次，后用4%(質(zhì)量分?jǐn)?shù))多聚甲醛，室溫固定20 min，5g/mL蛋白酶K處理細(xì)胞5 min，PBST沖洗3次，加入TdT酶反應(yīng)液，37 ℃孵育1 h，避光。DAPI復(fù)染細(xì)胞核，甘油封片后用共聚焦掃描顯微鏡觀察。實(shí)驗(yàn)至少重復(fù)3次進(jìn)行統(tǒng)計(jì)學(xué)分析。

9) MES23.5細(xì)胞線粒體膜電勢(shì)檢測(cè)：JC-1是一種廣泛應(yīng)用于檢測(cè)線粒體膜電勢(shì)(ΔΨm)的染料。在ΔΨm 增高時(shí)，JC-1形成聚合物(J-aggregates)并聚集在線粒體基質(zhì)中，可以產(chǎn)生紅色熒光；在線粒體受損膜電位下降時(shí)，JC-1呈單體形式存在且不聚集在線粒體基質(zhì)中，可以產(chǎn)生綠色熒光。通過(guò)紅綠熒光強(qiáng)度的比值則可以判斷線粒體膜電位的變化。MES23.5細(xì)胞轉(zhuǎn)染myc/nHb質(zhì)粒24 h后外加α-syn孵育1、3、6 h。收集上述各組細(xì)胞，以F12培養(yǎng)基稀釋JC-1至工作質(zhì)量濃度10g/mL，37 ℃，5%(體積分?jǐn)?shù)) CO2培養(yǎng)箱內(nèi)孵育10 min后，利用流式細(xì)胞儀進(jìn)行檢測(cè)。實(shí)驗(yàn)至少重復(fù)3次進(jìn)行統(tǒng)計(jì)學(xué)分析。

1.3 統(tǒng)計(jì)學(xué)方法

2 結(jié)果

2.1 在MES23.5細(xì)胞中nHb-α-syn復(fù)合物形成可減少線粒體游離nHb

Western blotting及免疫熒光結(jié)果顯示，MES23.5細(xì)胞中均沒有檢測(cè)到內(nèi)源性α-syn信號(hào)(圖1A、B)。然而，在α-syn處理組細(xì)胞胞質(zhì)、線粒體及胞膜組分中，均檢測(cè)到外源性α-syn存在(圖1A、B)。過(guò)表達(dá)nHb后，在細(xì)胞胞質(zhì)及線粒體中nHb顯著增高(P<0.05)；而在α-syn處理組中，胞質(zhì)以及線粒體中游離nHb分別較未處理組降低顯著(P<0.05)，詳見圖1A；而Co-IP及ELISA結(jié)果顯示，細(xì)胞過(guò)表達(dá)nHb后，nHb-α-syn復(fù)合物在胞質(zhì)及線粒體組分顯著增高(P<0.05)，詳見圖1C。

2.2 nHb可緩解α-syn所致MES23.5細(xì)胞線粒體膜電勢(shì)降低

JC-1染色結(jié)果顯示，α-syn可顯著降低MES23.5細(xì)胞線粒體膜電勢(shì)(P<0.05，圖2A、B)。然而，過(guò)表達(dá)nHb后，MES23.5細(xì)胞線粒體膜電勢(shì)較α-syn處理組增高顯著(P<0.05)，詳見圖2A、B。

2.3 nHb可緩解α-syn所致MES23.5細(xì)胞凋亡

TUNEL及MTT結(jié)果顯示，α-syn可使細(xì)胞凋亡數(shù)目增加并使細(xì)胞活力降低(P<0.05)，而nHb過(guò)表達(dá)則明顯緩解α-syn所致細(xì)胞凋亡，并增加細(xì)胞活力(P<0.05)，詳見圖3A～C。

3 討論

本文在MES23.5多巴胺能神經(jīng)細(xì)胞中外加人源性重組α-syn純蛋白，在線粒體及胞質(zhì)組分可明顯觀察到α-syn表達(dá)顯著增高。這是由于α-syn加入細(xì)胞培養(yǎng)基后，可以通過(guò)被動(dòng)擴(kuò)散迅速進(jìn)入到MES23.5細(xì)胞中[20]。但是在胞質(zhì)及線粒體組分均發(fā)現(xiàn)游離nHb在α-syn細(xì)胞處理組降低顯著。同時(shí)，筆者還觀察到α-syn積聚可顯著增加線粒體中nHb-α-syn復(fù)合物。原因目前尚不明確，但有可能是由于MES23.5細(xì)胞內(nèi)源性α-syn極低，所以導(dǎo)致內(nèi)源性nHb-α-syn復(fù)合物處于較低水平，而當(dāng)添加外源性α-syn后，線粒體內(nèi)α-syn濃度迅速增高并與nHb結(jié)合，因此，顯示出較高的nHb-α-syn復(fù)合物形成，并在一定觀察時(shí)間內(nèi)沒有降低到基線水平。更重要的是，nHb-α-syn復(fù)合物的形成可顯著降低線粒體內(nèi)游離nHb。

文獻(xiàn)[12]顯示，內(nèi)源性nHb主要定位于線粒體，并且在減輕魚藤酮致細(xì)胞線粒體損傷中起關(guān)鍵作用。線粒體nHb可提高線粒體膜電勢(shì)并且維持線粒體ATP產(chǎn)量[21]。筆者進(jìn)而觀察了α-syn積聚所致線粒體nHb減低是否能使細(xì)胞線粒體膜電勢(shì)降低并增加細(xì)胞凋亡。結(jié)果顯示，加入外源性α-syn可使線粒體游離nHb降低并同時(shí)伴隨線粒體膜電勢(shì)降低。而過(guò)表達(dá)nHb可顯著緩解α-syn積聚所致的線粒體損傷。由于線粒體介導(dǎo)的凋亡起始于線粒體膜電勢(shì)降低，筆者進(jìn)一步觀察了nHb抵抗α-syn致細(xì)胞凋亡的保護(hù)作用。結(jié)果表明，nHb可明顯減輕α-syn致細(xì)胞凋亡及細(xì)胞活力降低。以上結(jié)果提示nHb可能通過(guò)維持線粒體膜電勢(shì)進(jìn)而抵抗α-syn所介導(dǎo)的細(xì)胞凋亡。

圖1 在MES23.5細(xì)胞中nHb-α-syn復(fù)合物形成可減少線粒體游離nHb

Fig.1 Reduction of free mitochondrial nHb levels by formation of nHb-a-syn complex in MES23.5 cells

A：The amount of free nHb, myc, α-syn expressed indicated by Western blotting analysis of the protein samples before IP. Actin, VDAC, Calnexin were detected as a cytosol, mitochondria and membrane loading control, respectively.*P<0.05vs1 group (n=6);#P<0.05vs1, 3, 5 group, respectively (n=6);B： Cells were transfected with a myc or myc/nHb vector for 24 h with or without α-syn (10mol/L) treatment for 6 h. At 24 h after transfection, MES23.5 cells fixed and stained with myc and α-syn antibody, followed by a mouse Alexa 488- or rabbit Alexa 594-conjugated secondary antibody. The nucleus was counterstained by DAPI. Bar=50 μm;C： α-syn immunoprecipitated by an Hb antibody was detected in cytosolic- and mitochondrial-fraction in 2, 4, 6 lanes (upper panel). Levels of nHb-α-syn complex were detected in cytosol, mitochondria and membrane by ELISA (lower panel).*P<0.05vs1 group (n=6);#P<0.05vs2 or 4 group (n=6); Data were expressed as the mean±SD; Tukey’s multiple comparisons test after ANOVA; Hb-Ab: hemoglobin antibody; Cyto-nHb-α-syn: neuronal hemoglobin-α-syn complex in cytosolic fraction; M-nHb-α-syn: neuronal hemoglobin-a-syn complex in membrane fraction; Mt-nHb-α-syn: neuronal hemoglobin-α-syn complex in mitochondrial fraction; 1: control group; 2: α-syn (10mol/L) treatment for 6 h group; 3: myc vector transfection for 24 h group; 4: myc vector transfection for 24 h with α-syn (10mol/L) treatment for 6 h group; 5: myc/nHb vector transfection for 24 h group; 6: myc/nHb vector transfection for 24 h with α-syn (10mol/L) treatment for 6 h group，α-syn：α-synuclein； nHb：neuronal hemoglobin;VDAC:voltage dependent anion-selective channel.

圖2 nHb可緩解α-syn致細(xì)胞線粒體膜電勢(shì)降低

Fig.2 nHb alleviated α-syn induced reduction of mitochondrial membrane potential

MES 23.5 cells were transfected with a myc or myc/nHb vector for 24 h with or without α-syn (10mol/L) treatment for indicated times. At 24 h after transfection, MES23.5 cells were stained by JC-1 and detected the mitochondrial membrane potential. A： Representative images showed the J-aggregate (FL 2) and J-monomer (FL 1) fluorescence intensity. B： Statistical results showed the ratios of J-aggregate/J-monomer fluorescence intensity. Data were expressed as the mean ±SD. Tukey’s multiple comparisons test after ANOVA.*P<0.05vscontrol group (n=6);#P<0.05vsa-syn treatment groups (n=6); α-syn；α-synuclein; nHb：neuronal hemoglobin.

圖3 nHb可緩解α-syn所致MES23.5細(xì)胞凋亡

Fig.3 nHb alleviated α-syn induced MES23.5 cells apoptosis

A： Cells were transfected with a myc or myc/nHb vector for 24 h with or without α-syn (10mol/L) treatment for 6 h. At 24 h after transfection, MES23.5 cells fixed and stained with TUNEL, the nucleus was counterstained by DAPI. Bar=50 μm; B： Statistical results show the TUNEL positive cells. C： MTT assay of the cell viability in 1 - 6 groups with or without addition of α-syn (10mol/L) for 1, 3, 6 h. Data were expressed as the mean ±SD. Tukey’s multiple comparisons test after ANOVA.*P<0.05vs1 group (n=6);#P<0.05vs2 or 4 group (n=6)； 1: control group; 2: α-syn (10mol/L) treatment for 1, 3, 6 h group; 3: myc vector transfection for 24 h group; 4: myc vector transfection for 24 h with α-syn (10mol/L) treatment for 1, 3, 6 h group; 5: myc/nHb vector transfection for 24 h group; 6: myc/nHb vector transfection for 24 h with α-syn (10mol/L) treatment for 1, 3, 6 h group. α-syn：α-synuclein, nHb：neuronal hemoglobin.

綜上所述，在MES23.5細(xì)胞中，nHb與α-syn可形成復(fù)合物，使線粒體游離nHb顯著降低，并緩解α-syn致細(xì)胞凋亡及線粒體膜電勢(shì)降低。這一發(fā)現(xiàn)，可能有助于為PD的發(fā)病機(jī)制及藥物作用靶點(diǎn)研究提供新思路。

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編輯 孫超淵

Expression of neuronal hemoglobin alleviates α-synuclein-induced apoptosis in MES23.5 cells

Yang Weiwei1,2，Li Xuran1,2，Li Xin1,2，Li Xuying1,2，Yu Shun1,2,3*

(1.DepartmentofNeurobiology,XuanwuHospital,CapitalMedicalUniversity,Beijing100053,China；2.KeyLaboratoryofNeurodegenerativeDisease,MinistryofEducation,Beijing100053,China；3.CenterforParkinson’sDisease,BeijingInstituteforBrainDisorders,Beijing100053,China)

Objective To explore the effect of neuronal hemoglobin (nHb) on α-synuclein(α-syn)-induced cell apoptosis. Methods myc vector or nHb was transiently transfected into MES23.5 cells with or without addition of exogenous α-syn. As identification of gene expression by immunofluorescence, Western blotting was used to detect the expression of α-syn and nHb. JC-1 staining was used to detect the mitochondrial membrane potential. The cell injuries were observed by TUNEL staining and MTT assay. Results nHb gene overexpression in MES23.5 cells lead to formation the complexes with α-syn in cytosolic and mitochondrial fraction, and reduced free nHb levels. Meanwhile, nHb could reverse α-syn-induced reduction of mitochondrial membrane potential and cell apoptosis. Conclusion Increase of nHb could alleviate α-syn-induced cell injuries.

α-synuclein (α-syn)；hemoglobin；mitochondria；apoptosis

國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(2011CB504101)，國(guó)家自然科學(xué)基金(81071014, 81371200，81401042)，國(guó)家科技支撐計(jì)劃課題(2012BAI10B03)，首都衛(wèi)生發(fā)展科研專項(xiàng)課題(2011-4001-01)，北京市自然科學(xué)基金(7122035)。 This study was supported by Major State Basic Research Development Program (“973” Program) of China (2011CB504101), National Natural Science Foundation of China (81071014, 81371200, 81401042), National Science and Technology Support Program (2012BAI10B03),The Capital Health Research and Development Special Fund (2011-4001-01), Natural Science Foundation of Beijing (7122035).

時(shí)間：2016-12-14 20∶10

http://www.cnki.net/kcms/detail/11.3662.r.20161214.2010.016.html

10.3969/j.issn.1006-7795.2016.06.016]

R 338

2016-02-14)

*Corresponding author， E-mail：yushun103@163.com