城市不同源霧霾顆粒物健康風(fēng)險(xiǎn)差異評(píng)估比較

蔣錦曉,何建波,陳 彬,李 寧,陳菲菲,單曉棟,唐 娟,張杭君,2*

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城市不同源霧霾顆粒物健康風(fēng)險(xiǎn)差異評(píng)估比較

蔣錦曉1,何建波1,陳 彬1,李 寧1,陳菲菲1,單曉棟1,唐 娟1,張杭君1,2*

(1.杭州師范大學(xué),生命與環(huán)境科學(xué)學(xué)院,浙江 杭州 310016；2.杭州師范大學(xué),生態(tài)系統(tǒng)保護(hù)與恢復(fù)杭州市重點(diǎn)實(shí)驗(yàn)室,浙江 杭州 310016)

通過(guò)研究不同來(lái)源霾顆粒物對(duì)大鼠氣管上皮細(xì)胞(RTE cells)電阻抗變化和細(xì)胞自噬因子的影響,評(píng)價(jià)不同來(lái)源霾顆粒物對(duì)人體健康風(fēng)險(xiǎn)的差異性.分別將RTE暴露于從居民區(qū)(I),高架交通源(II)和化工園區(qū)(III)采集的3種霧霾顆粒物中,統(tǒng)一暴露濃度和時(shí)間分別為100mg/L和24h.通過(guò)電子細(xì)胞基質(zhì)阻抗檢測(cè)(ECIS)細(xì)胞增長(zhǎng)引起的阻抗變化和細(xì)胞電損傷恢復(fù)時(shí)間;通過(guò)蛋白免疫印跡測(cè)定p62, Atg5, Atg7, Beclin1, LC3B和mTOR蛋白表達(dá)量來(lái)分析比較不同來(lái)源霧霾顆粒物對(duì)RTE細(xì)胞自噬的影響.結(jié)果表明,與空白對(duì)照組相比,不同霧霾顆粒物處理組細(xì)胞電損傷恢復(fù)時(shí)間分別延長(zhǎng)了34.6%, 63.2%和78.0%; p62蛋白表達(dá)量差異顯著性下降, Atg5, Atg7, Beclin1, LC3B蛋白表達(dá)量差異顯著性上升.此外, mTOR相關(guān)蛋白表達(dá)量差異顯著性下降, 分別下降了4.38%, 3.34%和2.36%; p-mTOR蛋白表達(dá)量與空白組相比,實(shí)驗(yàn)組I下降24.2%,實(shí)驗(yàn)組II下降37.0%,實(shí)驗(yàn)組III下降60.9%.由以上結(jié)果可知,不同來(lái)源霧霾顆粒物對(duì)RTE細(xì)胞均有一定的毒性損傷作用,能夠減小細(xì)胞增長(zhǎng)速度和削弱細(xì)胞修復(fù)能力,增強(qiáng)細(xì)胞自噬因子蛋白的表達(dá),且化工園區(qū)采集的霧霾顆粒物毒性強(qiáng)于居民區(qū)和高架交通源.不同來(lái)源霧霾顆粒物的細(xì)胞毒性存在明顯差異,基于細(xì)胞電損傷恢復(fù)時(shí)間的測(cè)定以及自噬相關(guān)蛋白的檢測(cè)方法能夠?yàn)殪F霾顆粒物健康風(fēng)險(xiǎn)評(píng)價(jià)提供一種快速的生物學(xué)手段.

霧霾顆粒物；大鼠氣管上皮細(xì)胞；電子細(xì)胞基質(zhì)阻抗檢測(cè)；自噬；健康風(fēng)險(xiǎn)評(píng)價(jià)

近年來(lái),霧霾天氣的持續(xù)存在受到世界范圍的關(guān)注,霧霾顆粒物污染是導(dǎo)致霧霾天氣的主要因素,由于其廣泛的危害而備受各界關(guān)注[1-2].霧霾顆粒物除了能破壞免疫系統(tǒng)增加新生兒的患病幾率之外,還能破壞人體的呼吸系統(tǒng),心血管系統(tǒng)和神經(jīng)系統(tǒng)等[3-5].有研究報(bào)道,霧霾顆粒物極易到達(dá)肺泡,引發(fā)過(guò)敏性鼻炎,哮喘等急性和慢性呼吸系統(tǒng)疾病[6].流行病學(xué)研究證明在高濃度霧霾顆粒物的長(zhǎng)期暴露下,會(huì)使得肺癌,動(dòng)脈硬化等呼吸疾病的發(fā)病率大大增加[7-8].目前,呼吸道上皮細(xì)胞是眾多肺部疾病的重點(diǎn)關(guān)注對(duì)象.評(píng)價(jià)不同來(lái)源霧霾顆粒物在相同濃度下對(duì)呼吸道上皮細(xì)胞的毒性差異以及發(fā)病機(jī)理顯得尤為重要.

霧霾顆粒物的基本特征和監(jiān)測(cè)方法研究較為深入,發(fā)現(xiàn)其危害取決于它的粒徑大小和化學(xué)組分[9].霧霾顆粒物粒徑越小,越容易進(jìn)入人體產(chǎn)生毒害作用.研究報(bào)道,PM1、PM2.5和PM10都能誘導(dǎo)A549細(xì)胞引發(fā)炎癥反應(yīng),PM1因其粒徑小而具有更強(qiáng)的基因毒性[10].另外,不同來(lái)源的霧霾顆粒物含有不同的污染組分,對(duì)細(xì)胞具有不同的毒害作用.研究發(fā)現(xiàn)含有化工園區(qū)采集的霧霾顆粒物具有大量過(guò)渡金屬而誘導(dǎo)A549細(xì)胞產(chǎn)生氧化應(yīng)激[11];交通源采集的霧霾顆粒物中含有大量有機(jī)成分而能誘導(dǎo)肺泡II型上皮細(xì)胞產(chǎn)生內(nèi)質(zhì)網(wǎng)應(yīng)激而加劇細(xì)胞自噬[12].霧霾顆粒物成分復(fù)雜,難以對(duì)所有組分進(jìn)行測(cè)定,因此缺乏不同來(lái)源的霧霾顆粒物組分及毒性的同步研究[9,13].

為了維持細(xì)胞內(nèi)部穩(wěn)態(tài),細(xì)胞自噬扮演了極其重要的角色.自噬一旦啟動(dòng),自噬泡逐漸拉長(zhǎng)形成雙層膜囊泡(自噬體).泛素化Atg7共軛Atg5是自噬前體結(jié)構(gòu)延伸的關(guān)鍵.Atg5和卷曲螺旋蛋白進(jìn)一步相互共軛,使其形成四聚體[14].Atg7的脂質(zhì)化將LC3-I轉(zhuǎn)化為L(zhǎng)C3-II,從而使LC3與自噬囊泡結(jié)合形成自噬小體[14-15].mTOR是一種絲氨酸/蘇氨酸激酶,在調(diào)節(jié)自噬中也起著關(guān)鍵作用[16].Beclin1是酵母ATG6的同源物,是哺乳動(dòng)物參與自噬的特定基因[17].研究表明,Beclin1聯(lián)合凋亡抑制因子Bcl-2蛋白可以抑制自噬.Beclin1基因表達(dá)的上調(diào)可以刺激并引發(fā)哺乳動(dòng)物細(xì)胞中的自噬[18].有研究證明,外源化學(xué)物能夠誘導(dǎo)細(xì)胞自噬,但含有多種外源化學(xué)物的霧霾顆粒物毒害機(jī)制依舊尚未明確[19].

本研究采用RTE細(xì)胞作為研究對(duì)象,將其暴露于3個(gè)不同地點(diǎn)(居民區(qū),高架交通源,化工園區(qū))采集的霧霾顆粒物中.通過(guò)ECIS測(cè)定細(xì)胞增殖以及細(xì)胞損傷恢復(fù)時(shí)間,蛋白印跡免疫法測(cè)定p62,Atg5,Atg7, Beclin1,LC3B和mTOR相關(guān)蛋白表達(dá)量,來(lái)闡明3種不同來(lái)源的霧霾顆粒物在相同濃度暴露下引起RTE細(xì)胞不同程度自噬損傷產(chǎn)生的分子機(jī)理.旨在為評(píng)估城市中不同地點(diǎn)空氣中霧霾顆粒物的健康風(fēng)險(xiǎn)提供科學(xué)依據(jù).

1 材料與方法

1.1 主要試劑與儀器

RTE細(xì)胞株(大鼠氣管上皮細(xì)胞)(北京北納); DEME高糖培養(yǎng)液(美國(guó) Gibco);胰蛋白酶(美國(guó) Gibco);胎牛血清(美國(guó) Gibco);30%丙烯酰胺溶液(美國(guó)Bio-Rad);BCA蛋白濃度測(cè)定試劑盒(上海碧云天);ECL發(fā)光液(美國(guó)GE);電子細(xì)胞基質(zhì)阻抗判斷儀(美國(guó)Applied Biophysics);EDTA(美國(guó),Sigma); YH-1000型大流量粉塵顆粒物采樣器(青島精誠(chéng));玻璃纖維濾膜(美國(guó)Waterman)37℃,5%細(xì)胞培養(yǎng)箱(美國(guó)SHELLAB);生物超凈臺(tái)(北京中科BCM- 1600A);恒溫水浴鍋(白典HHS-21-8);PVDF轉(zhuǎn)印膜(美國(guó)Millipore).

1.2 霧霾顆粒物采集與暴露母液配置

圖1 采樣點(diǎn)位示意

霧霾顆粒物的采樣點(diǎn)設(shè)置在杭州市區(qū)和近郊典型的達(dá)標(biāo)區(qū)域,分別為I居民區(qū),II高架交通源和III化工園區(qū),位置如圖1所示.使用青島精誠(chéng)YH- 1000型大流量粉塵顆粒物采樣器,采樣流量()為30L/min,使用玻璃纖維濾膜(Waterman,美國(guó))收集(濾膜直徑為47mm,孔徑為0.6μm).本次實(shí)驗(yàn)采樣時(shí)間段為2017年12月~2018年4月,非雨天多次采樣,所有采樣點(diǎn)在每天的北京時(shí)間8:00~18:00同時(shí)進(jìn)行采樣,霧霾顆粒物采集后,將吸附于玻璃纖維濾膜的霧霾顆粒物進(jìn)行分組收集和混合,分別密封保存于陰涼,干燥的場(chǎng)所,標(biāo)記為I,II,III,待用.從3組混勻的霧霾顆粒物中選取適量顆粒物,對(duì)其進(jìn)行超聲振蕩洗脫15min,根據(jù)文獻(xiàn)報(bào)道和預(yù)實(shí)驗(yàn)結(jié)果,使用生理鹽水分別配制成100mg/L的暴露液(標(biāo)記為I,II,III),置于-80℃中進(jìn)行儲(chǔ)存.

1.3 細(xì)胞培養(yǎng)與暴露

先前凍存的RTE細(xì)胞株在37℃恒溫水浴鍋中快速融化,再在無(wú)菌條件下轉(zhuǎn)移至含有2mL DMEM高糖培養(yǎng)基無(wú)菌離心管中,在1000rpm轉(zhuǎn)速的離心機(jī)中離心5min.轉(zhuǎn)移沉淀至含有5mL完全培養(yǎng)基的細(xì)胞培養(yǎng)瓶中,置于恒溫37℃,CO2體積分?jǐn)?shù)5%,培養(yǎng)至細(xì)胞存活率達(dá)80%~90%.之后,棄取上清液,再加入2mLPBS進(jìn)行漂洗,加入1mL0.25%酶液對(duì)細(xì)胞進(jìn)行消化至細(xì)胞呈現(xiàn)圓形.再加入2mL完全培養(yǎng)基完成消化后,以1:2比例進(jìn)行傳代接種細(xì)胞.培養(yǎng)方法參照霍婷婷等[20]研究.

細(xì)胞進(jìn)行暴露時(shí),取出配制好的100mg/L霧霾顆粒物暴露母液恢復(fù)至常溫,用微量移液器進(jìn)行充分混勻,在超聲振蕩儀中振蕩5min X 3次,以0.1倍單位體積的比例加入單位體積的細(xì)胞培養(yǎng)液.

1.4 ECIS 檢測(cè)細(xì)胞增殖和電損傷恢復(fù)時(shí)間分析

細(xì)胞粘附發(fā)生在細(xì)胞與培養(yǎng)基底之間,當(dāng)細(xì)胞在工作電極上生長(zhǎng)繁殖,使得阻抗增大,電流值減小.電阻的變化與細(xì)胞數(shù)目具有一定的數(shù)學(xué)關(guān)系,可以根據(jù)測(cè)定的阻抗值得到細(xì)胞數(shù)目[21].

將培育的RTE細(xì)胞使用PBS漂洗之后接種到ECIS八孔電極板培養(yǎng)基上.然后將細(xì)胞層置于含有1mmol/L EDTA的PBS中,在37℃下孵育10min,然后用0.05%(/)胰蛋白酶在1.5mmol/L EDTA存在下消化10min.胰蛋白酶消化結(jié)束后,加入完全培養(yǎng)基,細(xì)胞進(jìn)行重新分布于培養(yǎng)基中.使用血細(xì)胞計(jì)數(shù)板控制每個(gè)培養(yǎng)基細(xì)胞密度為4×105,施加1μA, 4kHz的交流電壓,觀察細(xì)胞阻抗變化.

培養(yǎng)在ECIS八孔培養(yǎng)基中的RTE細(xì)胞匯合率達(dá)80%~90%,給予細(xì)胞電壓5V,頻率40kHz的電信號(hào),持續(xù)30s,電極板上的部分細(xì)胞死亡而脫落,造成電極上出現(xiàn)空白區(qū)域,引起阻抗的變化.當(dāng)周?chē)募?xì)胞重新匯聚到電擊區(qū)域,且鋪滿(mǎn)電極時(shí),阻抗又重新恢復(fù)至電擊創(chuàng)傷前的水平,相隔時(shí)間即為RTE細(xì)胞電損傷恢復(fù)時(shí)間.每組設(shè)置3個(gè)平行樣.

1.5 細(xì)胞自噬相關(guān)蛋白表達(dá)量檢測(cè)

本實(shí)驗(yàn)使用了含有蛋白酶抑制劑的全蛋白提取試劑盒,以及BCA試劑盒測(cè)定蛋白質(zhì)濃度.10%的十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE)處理3h,每個(gè)微凝膠井的總蛋白約為70μg.將甘氨酸溶液恢復(fù)30min至室溫,在將SDS-PAGE轉(zhuǎn)移到使用甲醇處理過(guò)的聚偏氟乙烯膜(PVDF),一起放入甘氨酸中在100V電壓下處理5min,再放入寒冷環(huán)境中處理2h.在室溫下,將PVDF膜放入T-TBS和5%脫脂牛奶中搖晃1h,之后再用T-TBS清洗3次,每次5min.處理之后,將PVDF膜放置在含有3%脫脂奶粉的T-TBS溶液中并加入蛋白相應(yīng)的第一抗體(Anti- MOR antibody,Santa Cruz SC-7488;Anti-LC3B antibody, Santa Cruz SC-292354; Anti-Beclin- 1antibody, Santa Cruz SC-10086; Anti-ATG- 5antibody, SC-133158; Anti-ATG-7antibody, Santa Cruz SC-376212; Anti-p-62antibody, Santa Cruz SC-514790),在4℃環(huán)境中反應(yīng)12h后,取出膜清洗之后將其放入含2%脫脂奶粉的T-TBS中,并且以1:5000比例加入第二抗體,反應(yīng)1h之后,再次使用T-TBS清洗每5min一次,進(jìn)行5次,結(jié)果使用β-Actin基因蛋白表達(dá)量作為內(nèi)參蛋白,重復(fù)以上步驟.1mL增強(qiáng)化學(xué)發(fā)光工作液在轉(zhuǎn)移孵化前處理PVDF膜1min之后,移除工作液.之后在X射線(xiàn)下觀察5~10min,利用軟件Bandsacn5.0進(jìn)行數(shù)據(jù)處理.

2 結(jié)果與分析

2.1 RTE細(xì)胞電損傷恢復(fù)時(shí)間的變化

A為RTE細(xì)胞電損傷過(guò)程示意圖;B為電擊后不同來(lái)源霧霾顆粒物暴露下阻抗變化及電損傷恢復(fù)時(shí)間

由圖2可以看出,與對(duì)照組相比,霧霾顆粒物處理組RTE細(xì)胞電損傷恢復(fù)時(shí)間都顯著性增加(<0.01).在4h時(shí)加5V,40kHz的電擊,阻抗明顯下降,隨后隨著細(xì)胞增長(zhǎng)阻抗逐漸增加.實(shí)驗(yàn)組I電損傷恢復(fù)時(shí)間是空白組的1.35倍,實(shí)驗(yàn)組II是空白組的1.63倍,實(shí)驗(yàn)組III是空白組的1.78倍.也進(jìn)一步說(shuō)明在不同來(lái)源的霧霾顆粒物暴露下RTE細(xì)胞修復(fù)能力不同程度地下降.

2.2 p62蛋白表達(dá)量的變化

由p62蛋白表達(dá)量變化圖(圖3)可知,在霧霾顆粒物暴露下,與對(duì)照組相比,處理組的RTE細(xì)胞內(nèi)p62蛋白表達(dá)量差異顯著性下降(<0.01).在RTE細(xì)胞正常生長(zhǎng)情況下,p62蛋白表達(dá)量是實(shí)驗(yàn)中I暴露下的1.14倍,是實(shí)驗(yàn)組II的1.59倍,是III化學(xué)工業(yè)園區(qū)的2.19倍.

2.3 LC3B蛋白表達(dá)量的變化

在霧霾顆粒物暴露下,與空白處理組相比,RTE細(xì)胞內(nèi)LC3B蛋白表達(dá)量差異顯著性上升(<0.01),實(shí)驗(yàn)組I霧霾顆粒物暴露下LC3B蛋白表達(dá)量上升了4.8倍,實(shí)驗(yàn)組II LC3B蛋白表達(dá)量上升了10.6倍,實(shí)驗(yàn)組II LC3B蛋白表達(dá)量上升了13.6倍(圖4)

2.4 Atg5和Atg7蛋白表達(dá)量的變化

圖5 不同霧霾顆粒物誘導(dǎo)對(duì)RTE細(xì)胞Atg5和Atg7蛋白表達(dá)量的影響

A: Atg5蛋白表達(dá)量; B: Atg7蛋白表達(dá)量

從圖5中可以看出,在霧霾顆粒物暴露下,RTE細(xì)胞內(nèi)Atg5和Atg7蛋白表達(dá)量都差異顯著性增高(<0.01).與對(duì)照組相比,實(shí)驗(yàn)組III的Atg5蛋白表達(dá)量增加了762%,相比于實(shí)驗(yàn)組I增加了32%,相比于實(shí)驗(yàn)組II增加了10%;實(shí)驗(yàn)組III Atg7蛋白表達(dá)量增加了1059%,相比于實(shí)驗(yàn)組I增加了132%,相比于實(shí)驗(yàn)組II增加了32%.

2.5 Beclin1蛋白表達(dá)量的變化

由圖6結(jié)果可知,在霧霾顆粒物暴露下,與對(duì)照組相比,RTE細(xì)胞內(nèi)Beclin1蛋白表達(dá)量差異顯著性升高(<0.01),實(shí)驗(yàn)組I Beclin1蛋白表達(dá)量增加了3.69倍,實(shí)驗(yàn)組II增加了6.08倍,實(shí)驗(yàn)組III增加了7.27倍.

2.6 mTOR相關(guān)蛋白表達(dá)量的變化

圖7為mTOR相關(guān)蛋白表達(dá)量變化圖.從圖中可以看出,在霧霾顆粒物暴露下,RTE細(xì)胞中p-mTOR蛋白表達(dá)量顯著下降(<0.01),并且,mTOR蛋白表達(dá)量也顯著下降(<0.01).與空白組相比,實(shí)驗(yàn)組I p-mTOR蛋白表達(dá)量下降24.2%,實(shí)驗(yàn)組II下降37.0%,實(shí)驗(yàn)組III下降60.9%.對(duì)mTOR蛋白表達(dá)量而言,與空白組相比,實(shí)驗(yàn)組I下降了4.4%,實(shí)驗(yàn)組II下降了3.3%,實(shí)驗(yàn)組III下降了2.3%.

3 討論

根據(jù)文獻(xiàn)[22-23]報(bào)道,霧霾顆粒物通常在10~100mg/L的暴露濃度下,會(huì)對(duì)細(xì)胞產(chǎn)生明顯毒效應(yīng).通常情況下10mg/L的霧霾顆粒物溶液往往需要多次采樣才能配制,由于不同采樣點(diǎn)霧霾顆粒物吸附的污染物存在一定差異,為了便于統(tǒng)一比較,因此,本文全部使用采自于I居民區(qū),II高架交通源,III化工園區(qū)的霧霾顆粒物配制的100mg/L暴露溶液誘導(dǎo)RTE細(xì)胞,進(jìn)行差異性比較.研究結(jié)果表明RTE細(xì)胞在相同濃度(100mg/L)的霧霾顆粒物暴露24h后,與空白組相比,實(shí)驗(yàn)組的細(xì)胞增長(zhǎng)速度下降,細(xì)胞電受損恢復(fù)時(shí)間顯著延長(zhǎng),p62,Beclin1,Atg5,Atg7,LC3B蛋白表達(dá)量都顯著下降,且mTOR蛋白表達(dá)量差異顯著性下降.實(shí)驗(yàn)數(shù)據(jù)表明相同濃度不同來(lái)源的霧霾顆粒物誘導(dǎo)細(xì)胞自噬的程度呈現(xiàn)化學(xué)工業(yè)園區(qū)>高架交通源>居民區(qū)的趨勢(shì),說(shuō)明相同濃度不同來(lái)源的霧霾顆粒物對(duì)細(xì)胞自噬的影響具有差異性.LC3B和Beclin1蛋白表達(dá)量的顯著變化,說(shuō)明霧霾顆粒物能夠誘導(dǎo)細(xì)胞發(fā)生自噬現(xiàn)象,且p62,Atg5,Atg7和mTOR基因都與霧霾顆粒物誘導(dǎo)的自噬扮演重要角色.

霧霾顆粒物因含有不同生物有機(jī)成分和無(wú)機(jī)成分會(huì)導(dǎo)致不同的物理化學(xué)過(guò)程,它的毒效應(yīng)也會(huì)隨著所含組分的不同而具有差異性[24-25].過(guò)渡金屬,VOC和PAH以不同比重存在于霧霾顆粒物中,從而使得霧霾顆粒物具有不同的毒效應(yīng).Zhang等[26]研究表明PM2.5能夠使得GC-2spd細(xì)胞增殖率明顯下降.在5V,40kHz的高壓脈沖下,RTE細(xì)胞產(chǎn)生控制性傷害,從電極上脫落,使得阻抗下降,當(dāng)重新鋪滿(mǎn)電極時(shí)阻抗恢復(fù)[27].本研究結(jié)果顯示,霧霾顆粒物暴露下的RTE細(xì)胞電損傷恢復(fù)時(shí)間明顯延長(zhǎng),說(shuō)明細(xì)胞增長(zhǎng)能力即細(xì)胞的修復(fù)能力下降且呈現(xiàn)實(shí)驗(yàn)組III<實(shí)驗(yàn)組II<實(shí)驗(yàn)組I<空白組的趨勢(shì),電損傷恢復(fù)時(shí)間實(shí)驗(yàn)組III>實(shí)驗(yàn)組II>實(shí)驗(yàn)組I>空白組,說(shuō)明相同濃度下不同來(lái)源的霧霾顆粒物對(duì)RTE細(xì)胞具有不同程度的毒性損傷作用.而細(xì)胞增長(zhǎng)能受多方面調(diào)控,自噬過(guò)程參與調(diào)解了細(xì)胞損傷過(guò)程,實(shí)現(xiàn)細(xì)胞自我保護(hù)作用[28].

細(xì)胞自噬過(guò)程是自噬體與體內(nèi)溶酶體融合形成自噬溶酶體,降解細(xì)胞內(nèi)多余物質(zhì)從而維持細(xì)胞內(nèi)穩(wěn)態(tài)的過(guò)程.目前,在酵母和高等動(dòng)物中,已經(jīng)發(fā)現(xiàn)有超過(guò)30種自噬相關(guān)基因和基因產(chǎn)物受自噬過(guò)程調(diào)控,被稱(chēng)作“Atg”[29].Atg5被譽(yù)為自噬的“核心”,Atg7是一種類(lèi)似泛素化蛋白E1的蛋白,能夠激活A(yù)tg12與Atg5結(jié)合形成Atg12-Atg5復(fù)合物,Atg7能促進(jìn)該復(fù)合物與Atg16結(jié)合使得雙層膜伸長(zhǎng),參與自噬體脂膜擴(kuò)張過(guò)程.有研究報(bào)道,Atg5和Atg7基因的抑制表達(dá)很大程度地減少了HuH7細(xì)胞自噬可能性[30].Kiyono等[30]研究發(fā)現(xiàn)PM2.5能夠明顯地提高A549細(xì)胞中自噬相關(guān)基因Atg5和Beclin1蛋白表達(dá)量,證明了Beclin1在PM2.5誘導(dǎo)的細(xì)胞自噬中扮演著極其重要的角色.在哺乳動(dòng)物自噬過(guò)程中,LC3-I轉(zhuǎn)化為L(zhǎng)C3-II是形成自噬體關(guān)鍵的一步[31],且在此轉(zhuǎn)化過(guò)程中,Atg7起到非常關(guān)鍵的作用.LC3B被認(rèn)為是自噬發(fā)生的標(biāo)志性蛋白,有研究報(bào)道,LC3B在香煙煙霧誘導(dǎo)的細(xì)胞自噬中起關(guān)鍵作用[30].p62是一種泛素結(jié)合蛋白,寡聚化后進(jìn)入內(nèi)質(zhì)網(wǎng)自噬體形成位點(diǎn),與LC3相互作用,使得自噬體脂膜不斷擴(kuò)張,從而不斷擴(kuò)大自噬體體積,因此在自噬過(guò)程中扮演著極其重要的角色[32-33].Su等[34]將Raw264.7cells暴露于20,50,100mg/L PM2.5懸浮液中,發(fā)現(xiàn)p62蛋白表達(dá)量明顯下降,LC3蛋白表達(dá)量明顯上升,從而促進(jìn)細(xì)胞自噬.本研究結(jié)果顯示,RTE細(xì)胞在霧霾顆粒物的暴露下,聚泛素化蛋白p62表達(dá)量下降,Atg5,Atg7,LC3B和Beclin1蛋白表達(dá)量上升,表明Atg5,Atg7,LC3B和Beclin1自噬相關(guān)基因參與介導(dǎo)了霧霾顆粒物誘導(dǎo)的RTE細(xì)胞毒性損傷過(guò) 程.

雷帕霉素靶蛋白質(zhì)(mTOR)是一種非典型絲氨酸/蘇氨酸蛋白激酶,在自噬過(guò)程中控制著自噬體的形成和成熟[35].有研究報(bào)道,mTOR能通過(guò)P13K/ AKT/mTOR路徑誘導(dǎo)細(xì)胞自噬[35].Su等[34]將Raw264.7細(xì)胞暴露在PM2.5中,發(fā)現(xiàn)在此實(shí)驗(yàn)中mTOR蛋白表達(dá)量隨著霧霾顆粒物濃度增加而下降更為明顯,從而誘導(dǎo)細(xì)胞自噬.在本研究中,mTOR蛋白表達(dá)量明顯下降,與空白組mTOR蛋白表達(dá)量相比,實(shí)驗(yàn)組I下降4.38%,實(shí)驗(yàn)組II下降了3.34%,實(shí)驗(yàn)組III下降了2.36%;p-mTOR蛋白表達(dá)量與空白組相比,實(shí)驗(yàn)組下降了24.2%,實(shí)驗(yàn)組II下降了37.0%,實(shí)驗(yàn)組III下降了60.9%.說(shuō)明mTOR對(duì)霧霾顆粒物誘導(dǎo)的RTE細(xì)胞毒性損傷有一定的調(diào)控作用,且相同濃度不同來(lái)源霧霾顆粒物誘導(dǎo)細(xì)胞的自噬具有差異性,呈現(xiàn)化工園區(qū)>高架交通源>居民區(qū)的強(qiáng)度.

在未來(lái)的研究中,將會(huì)針對(duì)細(xì)胞電損傷恢復(fù)時(shí)間與細(xì)胞自噬生物過(guò)程與霧霾顆粒物中化學(xué)污染物組分和濃度的相關(guān)性,進(jìn)一步深入開(kāi)展研究.

4 結(jié)論

4.1 霧霾顆粒物能夠損壞細(xì)胞的修復(fù)能力.

4.2 自噬標(biāo)志蛋白LC3B和Beclin1表達(dá)量明顯上升說(shuō)明霧霾顆粒物能誘導(dǎo)RTE細(xì)胞自噬.

4.3 不同來(lái)源的霧霾顆粒物對(duì)RTE細(xì)胞毒效應(yīng)具有差異性,化工園區(qū)>高架交通源>居民區(qū).

4.4 霧霾顆粒物誘導(dǎo)的細(xì)胞自噬中, p62, Atg5, Atg7, mTOR起到非常關(guān)鍵的作用,上述因子可以作為城市中霧霾顆粒物的健康風(fēng)險(xiǎn)評(píng)價(jià)重要標(biāo)志 物.

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Comparison and assessment of haze particles health risks from different sources in city.

JIANG Jin-xiao1, HE Jian-bo1, CHEN Bin1, LI Ning1, CHEN Fei-fei1, SHAN Xiao-dong1, TANG Juan1, ZHANG Hang-jun1,2*

(1.College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310016, China；2.Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration, Hangzhou Normal University, Hangzhou 310016, China)., 2019,39(1)：379~385

To evaluate the health risks induced by haze particles sampled from three different areas. The present study assessed the effects of haze particles on electrical impedance and autophagy factors of the tracheal epithelial cells. Rat tracheal epithelial (RTE) cells were exposed to haze particles collected from three different areas, including residential area (I), traffic area (II), and chemical industry park (III) in the megacity Hangzhou, China. The particle concentration was treated and designed as 100mg/L and then a period of 24h exposure was given to cells in treatment groups. The electronic impedance and the time needed for the recovery of electricity damage were determined by ECIS (Electric Cell-substrate Impedance Sensing). Western blot was used to analyze the protein expressions of p62, Atg5, Atg7, Beclin1, LC3B and mTOR to compare the effects of different haze particles on RTE cells autophagy. The results showed that the recovery time of cell injury was prolonged by 34.6%, 63.2% and 78.0%, respectively, when compared with the control group. The expression of Atg5, Atg7, Beclin1and LC3B proteins increased, whereas that of p62protein significantly decreased. The expression of mTOR related proteins decreased significantly by 4.38%, 3.34% and 2.36%, respectively. Compared with the control group, the expression of p-mTOR decreased by 24.2% in group I, 37.0% in group II and 60.9% in group III. All the results suggested that haze particles collected from different functional areas could induce various toxic damages on RTE cells such as reduction of growth speed, impairment of revovery ability, and increased authophagy protein expression. The haze particles from chemical industrial areas showed more toxic effects than those from residential areas and traffic areas. The cytotoxicity of haze particles from different sources is obviously different The determination of cell electrical injury recovery time and the detection of autophagy-related proteins can provide a rapid biological method for health risk assessment of haze particles.

haze particles；rat tracheal epithelial cells；electric cell-substrate impedance sensing；autophagy；health risk assessment

X503.1,X513

A

1000-6923(2019)01-0379-07

蔣錦曉(1997-),女,浙江金華人,杭州師范大學(xué)本科生,環(huán)境工程專(zhuān)業(yè).

2018-06-11

杭州市科技發(fā)展計(jì)劃項(xiàng)目(20150533B02);杭州師范大學(xué)本科生創(chuàng)新能力提升工程項(xiàng)目(CX2017106)

* 責(zé)任作者, 教授, 20080099@hznu.edu.cn