苯通過(guò)激活線粒體凋亡通路對(duì)小鼠骨髓細(xì)胞凋亡的誘導(dǎo)作用及其機(jī)制

于光艷,宋祥福,趙淑華,劉曉梅,孫志偉,2

(1.吉林大學(xué)公共衛(wèi)生學(xué)院勞動(dòng)衛(wèi)生與環(huán)境衛(wèi)生教研室,吉林長(zhǎng)春 130021;2.首都醫(yī)科大學(xué)公共衛(wèi)生學(xué)院衛(wèi)生毒理學(xué)教研室,北京 100069)

苯通過(guò)激活線粒體凋亡通路對(duì)小鼠骨髓細(xì)胞凋亡的誘導(dǎo)作用及其機(jī)制

于光艷1,宋祥福1,趙淑華1,劉曉梅1,孫志偉1,2

(1.吉林大學(xué)公共衛(wèi)生學(xué)院勞動(dòng)衛(wèi)生與環(huán)境衛(wèi)生教研室,吉林長(zhǎng)春 130021;2.首都醫(yī)科大學(xué)公共衛(wèi)生學(xué)院衛(wèi)生毒理學(xué)教研室,北京 100069)

目的:建立小鼠吸入氣態(tài)苯染毒模型,探討苯對(duì)骨髓細(xì)胞凋亡的誘導(dǎo)作用和機(jī)制,為苯骨髓毒作用機(jī)制的研究提供實(shí)驗(yàn)依據(jù)。方法:將24只雄性小鼠隨機(jī)分為對(duì)照組和低、中、高劑量苯染毒組(染毒劑量分別為400、800和1 600 mg·m－3),每組6只。各劑量苯染毒組小鼠進(jìn)行靜式染毒,每天2 h,連續(xù)染毒15 d后將各組小鼠全部處死,HE染色后光鏡下觀察各組小鼠骨髓細(xì)胞的病理學(xué)改變,流式細(xì)胞儀測(cè)定各組小鼠骨髓細(xì)胞凋亡率及線粒體膜電位,免疫組織化學(xué)法測(cè)定各組小鼠線粒體凋亡途徑中相關(guān)基因蛋白的表達(dá)。結(jié)果:低、中和高劑量苯染毒組小鼠骨髓邊緣及中央處細(xì)胞數(shù)明顯減少,高劑量苯染毒組小鼠骨髓還伴有大量的血竇擴(kuò)張。中和高劑量苯染毒組細(xì)胞凋亡率明顯高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(Ρ＜0.01),且高劑量苯染毒組與低和中劑量苯染毒組比較差異也有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05)。低、中和高劑量苯染毒組小鼠骨髓細(xì)胞線粒體膜電位隨著染毒劑量的增加明顯降低,中和高劑量苯染毒組與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05或P＜0.01)。不同劑量苯染毒組Bax、CytC及中、高劑量苯染毒組Caspases-9、Caspases-3陽(yáng)性細(xì)胞數(shù)隨染毒劑量的增加顯著升高,與對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05);而各劑量苯染毒組Bcl-2陽(yáng)性細(xì)胞數(shù)則明顯低于對(duì)照組(Ρ＜0.05),且中和高劑量苯染毒組與低劑量苯染毒組比較差異也有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05)。結(jié)論:一定劑量的苯可以誘導(dǎo)小鼠骨髓細(xì)胞發(fā)生凋亡,促進(jìn)線粒體凋亡相關(guān)基因蛋白的表達(dá)。通過(guò)線粒體凋亡通路誘導(dǎo)的細(xì)胞凋亡可能是苯骨髓毒性的重要機(jī)制之一。

苯;細(xì)胞凋亡;線粒體膜電位;凋亡基因蛋白

苯是一種在工農(nóng)業(yè)生產(chǎn)及日常生活中應(yīng)用很廣泛的有機(jī)溶劑,普遍存在于環(huán)境中。流行病學(xué)和動(dòng)物實(shí)驗(yàn)研究[1-5]表明:長(zhǎng)期接觸苯會(huì)引起血液系統(tǒng)方面的改變,導(dǎo)致白細(xì)胞減少和骨髓抑制,發(fā)生白血病和惡性淋巴瘤的危險(xiǎn)性升高。盡管國(guó)內(nèi)外專家學(xué)者對(duì)苯的毒作用機(jī)制已經(jīng)進(jìn)行了多方面的研究和探討,但其致血液毒性的機(jī)制迄今尚未完全闡明。近年來(lái)研究[6-9]顯示:苯可以誘導(dǎo)造血細(xì)胞發(fā)生凋亡,且凋亡相關(guān)基因的差異表達(dá)與苯血液毒性的發(fā)生、發(fā)展有密切關(guān)聯(lián)。如果對(duì)苯誘導(dǎo)的細(xì)胞凋亡相關(guān)基因及調(diào)控凋亡的信號(hào)轉(zhuǎn)導(dǎo)通路進(jìn)行研究,可為闡明苯中毒的發(fā)病機(jī)制提供重要依據(jù)[5]。本研究通過(guò)建立小鼠吸入氣態(tài)苯染毒模型,采用細(xì)胞生物學(xué)、免疫學(xué)等實(shí)驗(yàn)方法,探討細(xì)胞凋亡相關(guān)信號(hào)轉(zhuǎn)導(dǎo)通路在苯誘導(dǎo)的小鼠骨髓細(xì)胞凋亡中的作用,為闡明苯中毒的發(fā)病機(jī)制和預(yù)防白血病的發(fā)生及降低其發(fā)病率提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物與分組24只普通級(jí)封閉群昆明系雄性小鼠由吉林大學(xué)實(shí)驗(yàn)動(dòng)物部提供,合格證號(hào): 10-1023,體質(zhì)量(13±1)g。按照隨機(jī)分組原則,參照小鼠吸入苯的半數(shù)致死濃度(LC50) [(45 000±935)mg·m－3·2 h－1],分為對(duì)照組和低、中、高劑量苯染毒組(染毒劑量為400、800 和1 600 mg·m－3),進(jìn)行靜式染毒,每天2 h。連續(xù)染毒15 d后將小鼠全部處死,取骨髓,測(cè)定相關(guān)指標(biāo)。

1.2 主要試劑與儀器苯(分析純,天津市致遠(yuǎn)化學(xué)試劑有限公司),Bax、Bcl-2、CytC、Caspase-3 和Caspase-9多克隆抗體(美國(guó)Santa Cruz公司),二氨基聯(lián)苯胺顯色試劑盒(北京中杉金橋生物技術(shù)有限公司),SP超敏試劑盒(福州邁新生物技術(shù)有限公司),碘化丙啶(PI)、RNA酶和Rhodamine123(美國(guó)Sigma公司)。56L靜式染毒柜(吉林大學(xué)勞動(dòng)衛(wèi)生與環(huán)境衛(wèi)生教研室提供), FACScan流式細(xì)胞儀(美國(guó)Becton Dickinson公司),光學(xué)顯微鏡(日本Olympus公司)。

1.3骨髓病理切片制備小鼠眼眶取血處死后,取胸骨,放入10%甲醛溶液中固定,乙醇梯度脫水,常規(guī)石蠟包埋,間斷均勻切片5～10張,切片厚度5μm,HE染色,光鏡下觀察骨髓細(xì)胞形態(tài)學(xué)變化。

1.4 骨髓線粒體膜電位測(cè)定采用流式細(xì)胞術(shù)(FCM)檢測(cè)[10]。將小鼠頸椎脫臼處死,取出股骨,剔除肌肉和結(jié)締組織,剪開(kāi)股骨兩端,用1 m L注射器以0.01 mol·L－1PBS沖出骨髓細(xì)胞,濾網(wǎng)過(guò)濾(200目尼龍網(wǎng)),制成單個(gè)骨髓細(xì)胞懸液。加等體積的20 mg·L－1Rhodamine123,至終體積約10 mg·L－1,37℃避光孵育30 min,以PBS清洗脫色,1 500 r·min－1離心10 min,重復(fù)1次,用微量0.01 mol·L－1PBS(與沉淀等體積)混勻細(xì)胞,采用流式細(xì)胞儀(激發(fā)光波長(zhǎng)488 nm)檢測(cè),每個(gè)樣品收集1×104個(gè)細(xì)胞,測(cè)定細(xì)胞相對(duì)熒光強(qiáng)度,以U值表示,并以實(shí)驗(yàn)組U值/對(duì)照組U值×100%表示細(xì)胞線粒體膜電位變化。

1.5 骨髓細(xì)胞凋亡檢測(cè)采用PI標(biāo)記,FCM檢測(cè)細(xì)胞凋亡率[11]。將小鼠頸椎脫臼處死,取出股骨,剔除肌肉和結(jié)締組織,剪開(kāi)股骨兩端,用1 m L注射器以0.01 mol·L－1PBS沖出骨髓細(xì)胞,濾網(wǎng)過(guò)濾(200目尼龍網(wǎng)),制成單個(gè)骨髓細(xì)胞懸液。用0.01 mol·L－1PBS洗2次,加100μL RNase(10 mg·L－1)和100μL PI(5 mg·L－1), 4℃避光孵育30 min,用流式細(xì)胞儀收集1× 104個(gè)細(xì)胞,采用Cellquest軟件分析結(jié)果,結(jié)果以凋亡細(xì)胞的百分率表示。

1.6 CytC、Bcl-2、Bax、Caspases-3和Caspases-9蛋白表達(dá)的檢測(cè)將小鼠頸椎脫臼處死,取胸骨,常規(guī)涂片,自然晾干后,10%甲醛緩沖液固定15 min,風(fēng)干后放于－20℃冰箱保存。采用免疫組織化學(xué)技術(shù)[11]進(jìn)行檢測(cè)。PBS洗3次,每次5 min。滴加1∶10稀釋的正常血清,置濕盒內(nèi)10 min后,每張玻片加1∶50稀釋的第一抗體, 4℃過(guò)夜。PBS洗3次,每次5 min,滴加生物素標(biāo)記的第二抗體,置濕盒內(nèi)30 min。PBS洗3次,每次5 min,滴加1∶20稀釋的第三抗體(卵白素-生物素過(guò)氧化酶復(fù)合物),置濕盒內(nèi)1 h。PBS洗3次,每次5 min,滴加新鮮配制的酶作用底物(DAB),顯色,顯微鏡下觀察染色情況,當(dāng)目的蛋白出現(xiàn)黃色,立即用自來(lái)水沖洗終止顯色反應(yīng)。蘇木精復(fù)染,水洗。置入70%HCl-乙醇中15 s,水洗。弱氨水返藍(lán)15 s,顯微鏡下觀察,當(dāng)胞核呈現(xiàn)藍(lán)染,立即水洗。乙醇梯度脫水,二甲苯透明,中性樹(shù)膠封片。陽(yáng)性細(xì)胞為胞漿(或胞膜、胞核)呈棕黃色染色。顯微鏡下每張切片隨機(jī)觀察5個(gè)視野,每個(gè)視野觀察100個(gè)細(xì)胞,陽(yáng)性細(xì)胞在500個(gè)細(xì)胞中所占的比例為抗體陽(yáng)性表達(dá)率。

1.7 統(tǒng)計(jì)學(xué)分析采用SPSS 14.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)處理。小鼠骨髓細(xì)胞凋亡率、線粒體膜電位和Bax、Caspases-9、Caspases-3、CytC、Bcl-2蛋白表達(dá)水平以±s表示,組間比較采用方差分析和q檢驗(yàn)。

2 結(jié)果

2.1 小鼠骨髓細(xì)胞病理學(xué)低、中和高劑量苯染毒組骨髓邊緣及中央處細(xì)胞數(shù)明顯減少,高劑量苯染毒組還伴有大量的血竇擴(kuò)張。見(jiàn)圖1(插頁(yè)二)。

2.2 各組小鼠骨髓細(xì)胞凋亡率及線粒體膜電位中和高劑量苯染毒組小鼠骨髓細(xì)胞凋亡率明顯高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(Ρ＜0.01),且高劑量苯染毒組與低、中劑量苯染毒組比較差異也有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05);各劑量苯染毒組小鼠骨髓細(xì)胞線粒體膜電位隨著染毒劑量的增加明顯降低,中和高劑量苯染毒組與對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05或P＜0.01)。見(jiàn)表1。

表1 各組小鼠骨髓細(xì)胞凋亡率及線粒體膜電位Tab.1 Apoptotic rates of bone marrow cells and mitochondrial membrane potential(MMP)of mice in various groups(n＝4,±s,η/%)

表1 各組小鼠骨髓細(xì)胞凋亡率及線粒體膜電位Tab.1 Apoptotic rates of bone marrow cells and mitochondrial membrane potential(MMP)of mice in various groups(n＝4,±s,η/%)

?P＜0.05,??P＜0.01 compared with control group;△P＜0.05 compared with low dose of benzene group;＃P＜0.01 compared with middle dose of bezene group.

Group Apoptotic rate MMP Control 4.40±0.60 23.27±3.01 Benzene Low dose 6.53±1.64 19.48±3.34 Middle dose 11.65±2.35??16.30±1.72?High dose 19.73±2.13??△＃15.64±0.27??

2.3 各組小鼠骨髓細(xì)胞中凋亡相關(guān)蛋白的表達(dá)不同劑量苯染毒組小鼠骨髓細(xì)胞中Bax、CytC及中、高劑量苯染毒組小鼠骨髓細(xì)胞中Caspases-9 和Caspases-3陽(yáng)性細(xì)胞數(shù)隨染毒劑量的增加顯著升高,與對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05或P＜0.01),中和高劑量苯染毒組與低劑量苯染毒組比較差異有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05或P＜0.01);而各劑量苯染毒組小鼠骨髓細(xì)胞中Bcl-2陽(yáng)性細(xì)胞數(shù)則明顯低于對(duì)照組(Ρ＜0.01),且中和高劑量組與低劑量組比較差異有統(tǒng)計(jì)學(xué)意義(Ρ＜0.05)。見(jiàn)表2。

3 討論

細(xì)胞凋亡即細(xì)胞程序性死亡,可以由多個(gè)基因參與調(diào)控。細(xì)胞凋亡調(diào)節(jié)紊亂可引起細(xì)胞增生、分化或異常,從而導(dǎo)致疾病或腫瘤的發(fā)生。有研究[12-13]顯示:細(xì)胞凋亡的發(fā)生與多通路的信號(hào)轉(zhuǎn)導(dǎo)及Caspase級(jí)聯(lián)反應(yīng)有關(guān)。當(dāng)細(xì)胞受到外界刺激后,可通過(guò)線粒體膜通道孔(MPTP)的開(kāi)放,使線粒體膜電位下降,誘導(dǎo)Cyt C等細(xì)胞凋亡相關(guān)因子的釋放,激活Caspase-9,繼而活化Caspase-3,啟動(dòng)Caspase級(jí)聯(lián)反應(yīng),引起細(xì)胞凋亡[14-15]。在這一過(guò)程中,Bcl-2家族蛋白對(duì)于MPTP的開(kāi)放和關(guān)閉起著關(guān)鍵的調(diào)節(jié)作用。促凋亡類蛋白Bax等可以通過(guò)與線粒體內(nèi)膜上的腺苷酸轉(zhuǎn)運(yùn)蛋白(ANT)或外膜上的電壓依賴性陰離子通道(VDAC)結(jié)合介導(dǎo)MPTP的開(kāi)放,促進(jìn)Cyt C的釋放;而抗凋亡類蛋白Bcl-2等則通過(guò)與Bax競(jìng)爭(zhēng)性地與ANT結(jié)合或直接阻止Bax與ANT、VDAC的結(jié)合發(fā)揮其抗凋亡效應(yīng)[16-17]。

表2 各組小鼠骨髓細(xì)胞中Bax、Caspases-9、Caspases-3、CytC和Bcl-2陽(yáng)性細(xì)胞數(shù)Tab.2 Number of Bax,Caspases-9,Caspases-3,CytC and Bcl-2 positive cells in bone marrow cells of mice in various groups (n＝5,±s,η/%)

表2 各組小鼠骨髓細(xì)胞中Bax、Caspases-9、Caspases-3、CytC和Bcl-2陽(yáng)性細(xì)胞數(shù)Tab.2 Number of Bax,Caspases-9,Caspases-3,CytC and Bcl-2 positive cells in bone marrow cells of mice in various groups (n＝5,±s,η/%)

?P＜0.05,??P＜0.01 compared with control group;△P＜0.05,△△P＜0.01 compared with low dose of benzene group.

Group Bax Caspases-9 Caspases-3 CytC Bcl-2 Control 13.00±1.41 9.80±1.92 11.20±1.48 10.20±1.92 54.60±1.34 Benzene Low dose 16.80±1.48?11.80±1.30 14.00±2.24 14.40±1.67?42.00±3.39??Middle dose 38.80±4.09?△19.40±3.78?△30.80±4.87??△△29.00±1.87??△△35.80±0.84??△High dose 43.00±3.39?△24.00±2.45??△△39.40±6.19??△△32.80±4.97??△△31.60±3.21??△

本研究結(jié)果表明:一定劑量的苯可以降低線粒體膜電位,誘導(dǎo)骨髓細(xì)胞發(fā)生凋亡。苯作為化學(xué)物質(zhì),對(duì)機(jī)體的刺激被細(xì)胞接受后,細(xì)胞可以將這些信號(hào)進(jìn)行整合,通過(guò)細(xì)胞凋亡信號(hào)與特異性受體結(jié)合成復(fù)合物,激活Caspase,促使其對(duì)特異性底物進(jìn)行降解,導(dǎo)致凋亡抑制失活,引起細(xì)胞凋亡。本研究采用免疫細(xì)胞化學(xué)方法檢測(cè)小鼠骨髓細(xì)胞中凋亡相關(guān)蛋白CytC、Bax、Bcl-2、Caspase-9和Caspase-3蛋白表達(dá)的結(jié)果顯示:一定劑量的苯可以誘導(dǎo)骨髓細(xì)胞凋亡相關(guān)蛋白的表達(dá),使Cyt C、Bax、Caspase-9和Caspase-3陽(yáng)性細(xì)胞數(shù)升高,而B(niǎo)cl-2陽(yáng)性細(xì)胞數(shù)降低。本研究結(jié)果表明線粒體凋亡通路參與了苯誘導(dǎo)的小鼠骨髓細(xì)胞凋亡,這可能是苯引起骨髓毒性的主要機(jī)制之一。

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Induction effect of benzene on apoptosis of mouse bone marrow cells through mitochondrial-dependent apoptosis pathway and its mechanism

YU Guang-yan1,SONG Xiang-fu1,ZHAO Shu-hua1,LIU Xiao-mei1,SUN Zhi-wei1,2
(1.Department of Occupational and Environmental Health,School of Public Health,Jilin University, Changchun 130021,China;2.Department of Hygienic Toxicology,School of Public Health,Capital Medical University,Beijing 100069,China)

ObjectiveTo establish mouse poisoning model by inhaling benzene,and to investigate the induction effect of benzene on the apoptosis of mouse bone marrow cells and its mechanism,and to provide an experimental basis for study on bone marrow toxicity mechanism.Methods24 male mice were randomly divided into four groups (n＝6).The mice in one group were exposed to ambient air(control group)and the mice in the other three groups were exposed to different doses(400,800,1 600 mg·m－3)of benzene(low,middle and high doses of benzenegroups)for 15 d in the respective inhalation chambers.At the end of the experiment,the mice were killed.The bone marrow of the mice was obtained.The pathological changes of the bone marrow cells of the mice in various groups were observed under light microscope with HE staining.The apoptotic rates and mitochondrial membrane potential(MMP)of the mice in various groups were detected by flow cytometry,and the expressions of mitochondrial-deperdent apoptosis related gene proteins were determined with immunohistochemistry method.ResultsThe number of distal and central cells in different doses of benzene groups were significantly reduced,and accompanied by blood sinus expansion in high dose of benzene group.The apoptotic rates of the cells in middle and high doses of benzene groups were obviously higher than that in control group(Ρ＜0.01),and there were also significant differences between high dose group and low,middle doses of benzene groups(Ρ＜0.05).The MMP was significantly decreased with the increasing of benzene doses,and there were significant differences between middle,high doses of benzene groups and control group(Ρ＜0.05).The number of Bax,CytC positive cells in different doses of benzene groups and the number of Caspase-9,Caspase-3 positive cells in middle and high doses of benzene groups were significantly increased compared with control group(Ρ＜0.05);the number of Bcl-2 positive cells in different doses of benzene groups was decreased(Ρ＜0.05),and number of Bcl-2 positive cells in middle and high doses of benzene groups was decreased compared with low dose of benzene group(P＜0.05).ConclusionBenzene with certain dose can induce the apoptosis of mouse bone marrow cells,and promote the expressions of mitochondrial apoptosis related gene proteins.Benzene-induced apoptosis through mitochondrialdependent apoptosis pathway may be an important mechanism of bone marrow toxicity induced by benzene.

benzene;apoptosis;mitochondrial membrane potential;apoptosis gene protein

R114;R363

A

2013-11-09

國(guó)家安監(jiān)總局計(jì)劃項(xiàng)目資助課題(08-067);中央高?；究蒲袠I(yè)務(wù)費(fèi)專項(xiàng)基金資助課題(450060481119)

于光艷(1976－),女,吉林省吉林市人,講師,醫(yī)學(xué)博士,主要從事工業(yè)及環(huán)境毒理學(xué)研究。

孫志偉(Tel:010-83911507,E-mail:zwsun＠hotmail.com)

1671-587Ⅹ(2014)05-0943-04

10.13481/j.1671-587x.20140507